Fiddes 014164604

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THE UNIVERSITY OF NEW SOUTH WALES

Thesis/Dissertation Sheet
Surname or Family name: Fiddes
First name: Jane Other name/s: Louise Sutton
Abbreviation for degree as given in the University calendar: PhD
School: BABS Faculty: Science
Title: Development of Recombinant Human Monoclonal

Antibodies Suitable for Blood Grouping Using Antibody
Engineering Techniques
Abstract 350 words maximum: (PLEASE TYPE)
Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood
cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens,
monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they
perform as well as or better than the human polyclonal antibody preparations which they have replaced. However,
the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable
reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody
and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC
antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in
RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified
and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines
in the form of RNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to
generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage
particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and
cell based RBC antigens. In addition, a large naive library was biopanned against the same antigens in an attempt to
isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with
respect to size (>10^ clones) and diversity were generated. Biopanning of recombinant libraries resulted in
enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be
specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent
antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human
polyclonal sera in the blood grouping of Duffy a RBC antigen.
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property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
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L
DEVELOPMENT OF RECOMBINANT HUMAN
MONOCLONAL ANTIBODIES SUITABLE FOR BLOOD
GROUPING USING ANTIBODY ENGINEERING
TECHNIQUES
Jane L. Sutton Fiddes
A thesis submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy
School of Biotechnology and Biomolecular Sciences

University of New South Wales
Sydney
Australia
2007
Originality Statement
'I hereby declare that this submission is my own work and to the best of
my knowledge it contains no materials previously published or written
by another person, or substantial proportions of material which have
been accepted for the award of any other degree or diploma at UNSW
or any other educational institution, except where due
acknowledgement is made in the thesis. Any contribution made to the
research by others, with whom I have worked at UNSW or elsewhere,
is explicitly acknowledged in the thesis. I also declare that the
intellectual content of this thesis is the product of my own work, except
to the extent that assistance from others in the project's design and
conception or in style, presentation and linguistic expression is
acknowledged.'
Signed i i A t . S ^
Date
HI
Copyright Statement
'I hereby grant the University of New South Wales or its agents the right to archive and
to make available my thesis or dissertation in whole or part in the University libraries in
all forms of media, now or here after known, subject to the provisions of the Copyright
Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to
use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in
Dissertation Abstract International (this is applicable to doctoral theses only).
I have either used no substantial portions of copyright material in my thesis or I have
obtained permission to use copyright material; where permission has not been granted I
have applied/will apply for a partial restriction of the digital copy of my thesis or
dissertation.'
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Date
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'I certify that the Library deposit digital copy is a direct equivalent of the final officially
approved version of my thesis. No emendation of content has occurred and if there are
any minor variations in formatting, they are the result of the conversion to digital
format.'
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Acknowledgements
UNSW
Thanks to Steve Mahler and Chris Marquis for supervision; David Chin, Dalha Catzel,
Peter Sturgess and Ellen De Leon for technical assistance; and Lucasz Zdanowicz for
co-work on time course flow cytometry experiments.
ARCBS
Thanks to Fang Fang Yuan and Andrew Geczy for supervision; Jenny Bryant for
performing flow cytometry; and Monica Suarez and Sandra Biffin for technical
assistance.
Rest of World
Thanks to Bruce Cornell at AMBRI for the opportunity to live, work and study in
Australia; Marion Reid of the New York Blood Centre for the gift of transfected cells;
Kazimiera Wasniowska of the Ludwik Hirszfeld Institute of Immunology and
Experimental Therapy for the gift of recombinant Duffy antigen; and to Cambridge
Antibody Technology for the Vaughan naïve library.
Home
Many thanks to Rod without whose stellar support this would not have been possible; to
my family in the UK for never losing faith in me; and to Sal for a decade of support.
Dedication
This work is dedicated to the memory of my father and grandfather.
John William George Sutton
March 1945 to January 2004
William George Sutton
March 1922 to 4'^ July 2004
May you both be at peace.
Vll
vili
Table of Contents
Originality Statement iii
Acknowledgements v
Dedication vii
Table of Contents ix
List of Figures xiii
List of Tables xvii
Abstract xix
1 Introduction 1
1.1 A Short History of Blood Typing and Transfusion 1
1.2 Red Blood Cell Surface Structure 10
1.3 Clinical Significance of Red Blood Cell Surface Antigens 16
1.3.1 The Rhesus System 23
1.3.2 The Kell System 24
1.3.3 The Duffy System 26
1.3.4 The Kidd System 28
1.4 Antibodies to Red Blood Cell Surface Antigens 30
1.5 Monoclonal Antibodies 38
1.6 Recombinant Antibodies 42
1.7 Library Construction 44
1.8 Phage Display Technology 47
1.9 Biopanning Strategies 55
1.10 Phage Displayed Antibodies to Blood Group Antigens 59
2 Materials and Methods 61

2.1 Biological Reagents 61
2.1.1 Library Primers 61
2.1.2 PCR Screening Primers 61
2.1.3 E. coli XLlBlue Bacterial Stock 61
2.1.4 E. coli TGI Bacterial Stock 61
2.1.5 Peripheral Blood Mononuclear Cells 62
2.1.6 Helper Phage 62
2.1.7 Recombinant Duffy a (Fy^) Extracellular Domain Fusion Polypeptide 62
2.1.8 Naïve Library 63
2.1.9 pCESl Phagemid Vector 63
2.1.10 Transfected Human Embryonic Kidney 293 Cell Lines 63
2.1.11 Epstein Barr Virus 63
2.2 Bacterial Culture Media 64
2.3 Buffers and Solutions 67
2.4 Equipment 74
2.5 RBC Phenotyping and Cell Culture Methods 75
2.5.1 Indirect Antiglobulin Technique (Saline lAT) 75
2.5.2 Thawing Cells from Liquid Nitrogen 75
2.5.3 EBV Transformation of PBMC 76
2.5.4 Maintenance of Lymphoblastoid Cell Lines 77
2.5.5 Cell Culture Viability Test (Trypan Blue Exclusion) 77
2.5.6 Freezing Cells in Liquid Nitrogen 78
2.5.7 Maintenance of Human Embryonic Kidney 293 Cell Lines 78
2.5.8 Preparation of Samples for Flow Cytometry 79
2.6 Library Construction Methods
2.6.1 RNA Extraction 80
2.6.2 Formaldehyde Agarose (FA) Gel Electrophoresis (for RNA) 80
2.6.3 Reverse Transcription 81
2.6.4 Primary and Secondary Polymerase Chain Reactions (PCR) 81
2.6.5 Agarose Gel Electrophoresis (DNA) 84
2.6.6 Agarose Gel Purification of DNA 84
2.6.7 Nucleic Acid Quantitation 85
2.6.8 Plasmid Extraction 86
2.6.9 Polyacrylamide Gel Electrophoresis of DNA (DNA PAGE) 87
2.6.10 DNA Extraction from Acrylamide Gels 88
2.6.11 SybrGreen Staining of DNA Gels 88
2.6.12 Restriction Digestion of pCES 1 Phagemid Vector for Ligation 88
2.6.13 Purification and Restriction Digestion of PCR Amplified Antibody
Repertoire 91
2.6.14 Purification of Antibody Repertoires from pCESl Single Chain Libraries....
92
2.6.15 Preparation of Samples for Ligation 93
2.6.16 Phenol Chloroform Extraction 94
2.6.17 Ethanol Precipitation 94
2.7 Phage Display and Biopanning Methods 95
2.7.1 Preparation of Electrocompetent Cells 95
2.7.2 Electroporation 96
2.7.3 Glycerol Stocking of Bacteria 96
2.7.4 PCR Screen of Transformants 96
2.7.5 Library Diversity Analysis 97
2.7.6 Preparation of TGI for Phage Rescue 98
2.7.7 Phage Rescue of Library Glycerol Stocks for Biopanning 98
2.7.8 Phage Titration 99
2.7.9 Biopanning of Phage Library Against Solid Phase Antigen 99
2.7.10 Biopanning of Phage Library Against Transfected HEK293 Cells 100
2.7.11 Biopanning of Phage Library Against Whole RBC 101
2.7.12 Preparation of Glycerol Stocks from Biopanning Output 102
2.7.13 Phage Rescue from Biopanning Output Glycerol Stocks 102
2.7.14 Rescue of Phagemid Clones in 96-Well Plates for Phage ELIS A 103
2.7.15 Duffy a (Fy") Phage ELIS A 104
2.7.16 Screening Assays for RBC Antigen Specific Clones 104
3 Antibody Genes from Human Blood Samples 109

3.1 Aim 109
3.2 Introduction 109
3.3 Methods 112
3.3.1 Blood Sample Collection and Storage 112
3.3.2 Indirect Antiglobulin Technique (Saline lAT) 113
3.3.3 Interpretation of Phenocell Panel Results 116
3.3.4 Epstein Barr Viral Transformation 118
3.3.5 Maintenance and Storage of Lymphoblastoid Cell Lines 119
3.3.6 Eukaryotic RNA Extraction 120
3.4 Results 121
3.4.1 Saline Indirect Agglutination Tests 121
3.4.2 Maintenance of Lymphoblastoid Cell Lines, Storage and RNA Extraction...
121
3.4.3 Saline lAT of Cell Culture Supematants 122
3.4.4 Total RNA Extraction 122
3.5 Discussion 142
3.5.1 Saline lATs 142
3.5.2 Generation and Maintenance of LCLs 143
3.5.3 Saline lAT of Cell Culture Supematants 145
3.5.4 RNA Extraction 145
3.5.5 In Summary 146
4 Phage Display Fab Library Construction 149

4.1 Aim 149
4.3 Methods 154
4.3.1 DNA Electrophoresis 154
4.3.2 Reverse Transcription 154
4.3.3 Primary PCR Optimisation 154
4.3.4 Primary PCR Amplification and Purification 154
4.3.5 Secondary PCR Amplification and Purification 155
4.3.6 Trouble Shooting of Ligation Process 155
4.3.7 Preparation of Single Chain Libraries 159
4.3.8 Preparation of Fab Libraries 160
Results 163
4.3.9 Primary PCR Amplification and Purification 163
4.3.10 Secondary PCR Amplification and Purification 167
4.3.11 Purification of pCESl Vector 172
4.3.12 Trouble Shooting of Ligation Process 173
4.3.13 Restriction Digestion and Purification of PCR Insert and pCES 1 Vector 178
4.3.14 Production and Characterisation of Single Chain Libraries 180
4.3.15 Restriction Digestion and Purification of Insert and Vector from Single
Chain Libraries 183
4.3.16 Production and Characterisation of Fab Libraries 187
4.4 Discussion 191
4.4.1 Polymerase Chain Reactions 191
4.4.2 Trouble Shooting of the Transformation Process 192
4.4.3 Library Quality 193
5 Antibody Selection from Immune and Naïve Phage Display Libraries. 195
5.1 Aim 195
5.3 Methods 201
5.3.1 Characterisation of Transfected HEK293 Cells by Flow Cytometry 201
5.3.2 Analysis of Recombinant Duffy a Protein by ELIS A 202
5.3.3 Rescue of Phage Libraries for Biopanning 202
5.3.4 Biopanning of Immune and Naive Libraries Against Recombinant Duffy a
Protein 203
5.3.5 Biopanning of Immune Libraries Against Whole Cells (Transfected
HEK293 and RBCs) 203
5.3.6 Analysis of Duffy a Clones Isolated by Biopanning 203
5.3.7 Development of a Screening Assay for RBC Antigen Specific Clones....204
5.4 Results 206
5.4.1 Characterisation of Transfected HEK293 Cells by Flow Cytometry 206
5.4.2 Analysis of Recombinant Duffy a Protein by ELIS A 215
5.4.3 Rescue of Phage Libraries for Biopanning 216
5.4.4 Biopanning of Immune and Naive Libraries Against Recombinant Duffy a
Protein 217
5.4.5 Biopanning of Immune Libraries Against Whole Cells (Transfected
HEK293 and RBCs) 218
5.4.6 Analysis of Positive Clones Isolated by Biopanning 225
5.4.7 ELISA of Selected Positive Clones 231
5.4.8 Screening Assay for RBC Antigen-Specific Clones 234
5.5 Discussion 237
5.5.1 Characterisation of Transfected Cells by Flow Cytometry 237
5.5.2 Biopanning and Screening 238
6 Discussion 243
Bibliography 253
Appendices 283
Xll
List of Figures
Figure 1.1 Karl Landsteiner (1868-1943), immortalised on the Austrian 1000 Schilling
note (= €70) 2
Figure 1.2 James Blundell, English physician (1790-1878) 3
Figure 1.3 Illustrations of direct transfusion carried out by Dr. J. Roussel in Paris in the
late 1800's 3
Figure 1.4 Landsteiner's ABO blood typing results as reported in his Nobel lecture,
'On individual differences in human blood', December 11, 1930 4
Figure 1.5 Richard Lewisohn and a diagram of his citrate preservation technique for
blood transfusion 6
Figure 1.6 Wartime advertisements to encourage blood donation in the USA and UK. .7
Figure 1.7 ARCBS Headquarters, 1952-1974, at 'Petty's Hotel' York Street, Sydney...8
Figure 1.8 Donors queuing to help after the Granville train disaster of 1977, ARCBS,
Clarence Street, Sydney 9
Figure 1.9 Cross-sectional diagram of the human RBC membrane 10
Figure 1.10 Diagrammatic representation of SDS-PAGE analysis of red cell membrane
ghost membranes 11
Figure 1.11 Model of RBC membrane components carrying blood group antigens 15
Figure 1.12 An example of ABO incompatibility 16
Figure 1.13 Representation of haemolytic disease of the newborn (HDN) and anti-D
prophylaxis 21
Figure 1.14 Incidence of HDN in NSW, Australia between 1964 and 1977 22
Figure 1.15 Diagram of the proposed structure of the Rhesus polypeptide 23
Figure 1.16 Diagrams of the proposed structure of the Kell polypeptide, showing the
Kell antigens 25
Figure 1.17 Diagrams of the proposed structure of the Duffy polypeptide, showing the
Duffy antigens 27
Figure 1.18 Diagram of the proposed structure of the Kidd polypeptide, showing the
Kidd antigens 28
Figure 1.19 Diagrams of the five classes of immunoglobulin 30
Figure 1.20 Diagram of sequence variability in antibody molecules 31
Figure 1.21 Wu and Kabat plot of sequence variability in the variable regions of
antibody molecules 32
Figure 1.22 Generation of antibody diversity from germ line antibody gene 'building
blocks' 33
Figure 1.23 Typical production of IgM and IgG class antibodies in response to antigen
stimulus 35
Figure 1.24 The class switching process resulting in antibodies of identical specificity
but different isotype 36
Figure 1.25 Affinity maturation of antibody genes 37
Figure 1.26 Nobel laureates Georges Kohler (top) and Cesar Milstein who developed
murine hybridoma technology for MAb production alongside a diagram of their
groundbreaking method 39
Figure 1.27 Possible antibody formats used for cloning 42
Figure 1.28 Comparison of antibody library types; synthetic, naive (non-immune) and
immune 44
Figure 1.29 Antibody display systems 46
Figure 1.30 Diagram of Ml 3 bacteriophage 47
Figure 1.31Phagemid vector pCESl 48
Figure 1.32Colony lift screening of antibody library clones 50
Figure 1.33Construction of a phage display antibody library 52
Figure 1.34The phage display biopanning cycle 53
Figure 1.35Phage ELISA 54
Figure 1.36The wide range of approaches to antibody selection from libraries 55
Figure 1.37Ludwik Hirszfeld Institute of Immunology and Experimental Therapy logo.
57
Figure 2.1 Representation of patterns obtained from microplate RBC agglutination
assays 106
Figure 3.1 Cells found in human blood 109
Figure 3.2 An example of EBV transformed B cells Ill
Figure 3.3 Example of separation phases after centriftigation of human blood 112
Figure 3.4 Illustration of direct and indirect agglutination (Coombs test; Coombs et al.,
1946) 114
Figure 3.5 Grading of RBC agglutination reactions 115
Figure 3.6 Denaturing gel electrophoresis showing an example of degraded and intact
RNA 120
Figure 3.7 Saline lAT Results Sheet for donor 1 125
Figure 3.10 Saline lAT Resuks Sheet for donor 4 131
Figure 3.11 Saline I AT Results Sheet for donor 5 133
Figure 3.12 LCL cell growth and viability curve plotted against days after EBV
transformation for donor LCLs 1 and 2 135
Figure 3.13 LCL cell growth and viability curve plotted against days after EBV
transformation for patient LCLs 2, 3 and 4 137
Figure 3.14 Denaturing agarose gel electrophoresis of donor RNA samples extracted
from lymphoblastoid cell lines 140
Figure 3.15 Denaturing agarose gel electrophoresis of patient RNA samples extracted
from lymphoblastoid cell lines 140
Figure 4.1 Plasmid map of pCESl with cloning sites, genes and other features marked.
149
Figure 4.2 Diagram of PCR primers used in library construction (not to scale) 153
Figure 4.3 Site of third restriction digests within pCESl sequence utilised to reduce
vector background upon ligation and transformation 159
Figure 4.4 Agarose gel electrophoresis of donor 4 primary PCR products before
optimisation 164
Figure 4.5 Agarose gel electrophoresis of primary kappa light chain PCR products
using KIB primer and separate donor and patient cDNA samples as template 165
Figure 4.6 Agarose gel electrophoresis of purified primary PCR products 166
Figure 4.7 Agarose gel electrophoresis of donor 4 secondary PCR products before
optimisation 168
Figure 4.8 Agarose gel electrophoresis of donor 4 secondary PCR products from heavy
chain amplification using separate JH primers 169
Figure 4.9 Agarose gel electrophoresis of optimised secondary PCR products from
heavy and kappa light chain, patient and donor amplification 170
lambda light chain, patient and donor amplification 171
Figure 4.11 Agarose gel electrophoresis of purified pCESl at 5, 2 and 1 |iL loading
volumes 172
Figure 4.12 Excision of linearised vector from agarose gel avoiding UV exposure. ..174
Figure 4.13 Effect of purification method on singly digested pCESl vector re-ligation
and transformation 175
Figure 4.14 PAGE molecular weight calibration results for undigested, single and
double digested kappa light chain secondary PGR product 177
Figure 4.15 Agarose gel electrophoresis of digested PGR products 178
Figure 4.16 Agarose gel electrophoresis of purified pGESl single, double and triple
restriction digests 179
Figure 4.17. Agarose gel electrophoresis of PGR screen samples from single chain
libraries; kappa donor, kappa patient, and heavy donor 181
Figure 4.18 Agarose gel electrophoresis of PGR screen samples from single chain
libraries; heavy patient, lambda donor, and lambda patient 182
Figure 4.19 Agarose gel electrophoresis of purified pGESl from single chain library
stocks 184
Figure 4.20 Agarose gel electrophoresis of single and double restriction digests of
pGESl from single chain libraries 185
Figure 4.21 Agarose gel electrophoresis of purified, digested vector and insert from
single chain libraries 186
Figure 4.22 Agarose gel electrophoresis of PGR screen samples from Fab libraries.. 189
Figure 4.23 PAGE analysis of BstOI digested PGR products 190
Figure 5.1 SDS-PAGE analysis of the Fyb/GPA (Duffy b/Glycophorin A) recombinant
protein 195
Figure 5.2 Basic diagram of flow cytometry 198
Figure 5.3 Analysis of blood cell populations by flow cytometry 198
Figure 5.4 Antigen Gomposition Sheet for Phenocell RBG Reagent Panel used in
biopanning 200
Figure 5.5 RBG screening assay formats 205
Figure 5.6 Gharacterisation of transfected HEK293 cells by flow cytometry 207
Figure 5.7 Flow cytometry dot plots (left) and histograms (right) for Duffy a specific
human antiserum binding to HEK cells carrying Duffy a antigen 209
Figure 5.8 Flow cytometry dot plots (left) and histograms (right) for Duffy b specific
human antiserum binding to HEK cells carrying Duffy b antigen 210
Figure 5.9 Flow cytometry dot plots (left) and histograms (right) for Kell (K1 antigen)
specific human anfiserum binding to HEK cells carrying Kell antigen 211
Figure 5.10 Time course flow cytometry data for Duffy a specific human antiserum
binding to cells carrying Fy^ (Duffy a) antigen over a time course of 9 to 25 passages.
212
Figure 5.11 ELISA to test the recognition of Duffy a recombinant protein by specific
human antiserum (anti-Fy^) 215
Figure 5.12 Phage input and output titres for donor (DL) and naïve (NL) libraries over
three (R1 to R3) and four rounds respectively (R1 to R4) 218
Figure 5.13 Phage input and output titres of biopanning against HEK 293 cells over
three rounds 221
Figure 5.14 Phage input and output data for RBG whole cell biopanning over three
rounds ^^^
Figure 5.15 Phage ELISA results for individual clones from second and third round
biopanning of donor and naive libraries 227
Figure 5.16 Repeat phage ELIS A results from third round biopanning of naive library
against Duffy a recombinant antigen 229
Figure 5.17 Photograph of substrate developed phage ELIS A plates from third round
biopanning samples of pooled donor and patient libraries against Duffy a antigen
positive HEK293 or RBC 231
Figure 5.18 ELIS A of positive Duffy a clones isolated from naive and irmnune libraries.
232
Figure 5.19 ELIS A titration of positive and negative Duffy a clones isolated from the
naïve library 234
Figure 5.20 Monitoring by absorbance of RBC adherence to solid phase during ELIS A.
235
List of Tables
Table 1.1 The blood group systems and their symbols 12
Table 1.2 Selected blood group systems and their assigned antigens 13
Table 1.3 Blood type compatibilities within the ABO system 18
Table 1.4 Relative frequency of immune red cell alloantibodies in transfusion recipients
(and some pregnancies) 20
Table 2.1 Peripheral blood mononuclear cells expressing antibodies of interest shown
by ARCBS typing assays to be present donor and patient serum samples 62
Table 2.2 Human embryonic kidney 293 cell lines transfected with human RBC surface
proteins 63
Table 3.1 Rhesus nomenclature 116
Table 3.2 Allelic antigens present in various blood group systems 117
Table 3.3 Collection dates of PBMC samples used in EBV transformation 119
Table 3.4 Saline lAT and NSWBTS database results for donor samples 123
Table 3.5 Cell growth and viability data for donor LCLs 1, 2, 3 and 5 124
Table 3.6 Cell growth and viability data for patient LCLs 2, 3 and 4 139
Table 3.7 Spectrophotometric analysis of purified RNA samples 141
Table 4.1 Primary PCR conditions optimised for PCR product yield and specificity. 156
Table 4.2 Secondary PCR conditions optimised for PCR product yield and specificity.
157
Table 4.3 Gel extraction conditions for Sfil digested pCESl 158
Table 4.4 Cloning strategy for Fab library production 161
Table 4.5 Possible PCR products from library colony screening 162
Table 4.6 PAGE molecular weight calibration results for undigested, single and double
digested kappa light chain secondary PCR product 174
Table 4.7 Total number of transformants obtained for the six single chain libraries and
control samples 180
Table 4.8 Proportion of single chain library clones containing a full length insert 183
Table 4.9 Total number of transformants obtained for the eight Fab libraries, six vector
only negative controls and unmodified pCESl positive control 187
Table 4.10 Proportion of Fab library clones containing a full length insert indicating the
presence of both a light and heavy chain 188
Table 5.1 Characterisation of transfected HEK293 cells by flow cytometry 208
Table 5.2 Flow cytometry results for Duffy a specific human antiserum binding
to293T.Fya.4 HEK293 cells carrying Fy^ (Duffy a) antigen over a time course of 9 to 25
passages 213
Table 5.3 Time course flow cytometry analysis of HEK cells carrying Duffy a antigen.
214
Table 5.4 ELISA to test the recognition of Duffy a recombinant protein by specific
human antiserum (anti-Fy^) 216
Table 5.5 Antibiotic selection of phagemid libraries post helper phage infection 216
Table 5.6 Phage input and output titres for donor and naive libraries over three and four
rounds respectively 217
Table 5.7 Phage input and output titres of biopanning against HEK 293 cells over three
rounds 219
Table 5.8 Phage input and output titres of biopanning against human RBCs over three
rounds 220
Table 5.9 Raw phage ELIS A data results from third round biopanning of pooled donor
and patient libraries against recombinant HEK293 or RBCs carrying the Duffy a antigen.
226
Table 5.10 Raw data from ELIS A of positive Duffy a clones isolated from naive and
immune libraries 233
Table 5.11 Raw data from ELIS A titration of positive and negative Duffy a clones
isolated from the naive library 233
Table 5.12 Raw data for RBC adherence during ELIS A 236
xvni
Abstract
Transfusion medicine is an important part of modem health care and the provision of
reliably phenotyped red blood cells (RBC) is essential for safe and effective blood
transfusions. For identification of many RBC antigens, monoclonal antibodies of either
murine or human origin are available for use in agglutination assays, in which they
perform as well as or better than the human polyclonal antibody preparations which
they have replaced. However, the detection of some blood groups is still reliant on the
use of human polyclonal antisera, which is a less reliable reagent source with respect to
availability, batch to batch variation and bio-safety. The use of recombinant antibody
and phage display technology for the discovery of new monoclonal antibodies with
specificity for some of these RBC antigens has the potential to deliver an economical,
unlimited supply of specific antibody reagents suitable for use in RBC phenotyping.
Samples of human B cells from donors producing useful phenotyping antibodies were
identified and transformed using Epstein Barr virus into lymphocyte cell lines.
Antibody genes were obtained from the cell lines in the form of RNA which was
reverse transcribed, amplified by PCR and cloned into a phagemid vector system to
generate several combinatorial antibody libraries. These antibody libraries were
displayed on the surface of phage particles and subjected to antigen-driven selection by
several rounds of phage display biopanning using soluble and cell based RBC antigens.
In addition a large naive library was biopanned against the same antigens in an attempt
to isolate a wide range of antibodies suitable for blood typing.
Several high quality combinatorial antibody libraries with respect to size (>10^ clones)
and diversity were generated. Biopanning of recombinant libraries resulted in
enrichment of phage antibodies specific for RBC antigens, and several clones were
isolated which were shown to be specific for Duffy a antigen. The isolated antibodies
would be ideal candidates for re-engineering into multivalent antibody molecules
capable of direct agglutination of RBC and as such, have the potential to replace human
polyclonal sera in the identification of Duffy a RBC antigen phenotyping.
Abbreviations
AHTR Acute haemolytic transfusion reaction
AP Alkaline phosphatase
ARCBS Australian red cross blood service
CDR Complementarity determining region
CH CL Antibody regions - constant heavy chain; constant light chain
CIP Calf intestinal alkaline phosphatase
dAb Domain antibody (VH or VL antibody chain)
DHTR Delayed haemolytic transfusion reaction
EBV Epstein Barr virus
ELISA Enzyme linked immunoadsorbent assay
Fab Fragment antibody binding (VHCHI and light chain)
FITC Fluorescein isothiocyanate
Fv Fragment variable (VHVL)
Fy^ Duffy a blood group antigen
Fy^ Duffy b blood group antigen
HDN Haemolytic disease of the newborn
HEK293 Human embryonic kidney cell line; 293 strain.
HRP Horseradish peroxidase
lAT Indirect antiglobulin technique
IgG IgM Immunoglobulin class G or M
Jka Kidd a blood group antigen
Jkb Kidd b blood group antigen
LCL Lymphocyte cell line
Ml 3 Type of bacteriophage used in cloning
MAb Monoclonal antibody
NSWBTS New South Wales blood transfusion service
PAGE Poly acrylamide gel electrophoresis
pCESl Type of phagemid vector used in cloning
PCR Polymerase chain reaction
RBC Red blood cell
Rh Rhesus blood group system
SBBS School of biotechnology and biomolecular sciences
scFV Fragment variable single chain (VHVL);antibody domains are linked
V-Gene Variable antibody gene
VHVL Antibody regions - variable heavy chain; variable light chain
1 Introduction
Since the 1600's, when William Harvey first demonstrated that blood circulates around
the body (Harvey, 1628), the study of blood, blood transfusion and blood compatibility
have been pressing medical interests. Issues involving blood transfusion and
compatibility have in the past resulted in significant morbidity and mortality, and were
thus the focus of early medical research into the scientific basis of blood incompatibility.
The discovery of clinically significant red blood cell (RBC) antigens and their
relationship with haemolytic transfusion reactions led to the characterisation of RBC
surface antigens and their implication in transfusion incompatibility, and highlighted the
need for the discovery of antibodies for use in specific blood phototyping tests in order
to prevent rare transfusion reactions. In this literature review, a brief chronology of
blood typing and transfusions is presented, followed by the application of modem
technologies in antibody engineering, such as the creation of immunoglobulin gene
libraries and isolation of monoclonal antibodies by phage display. These technologies
offer new avenues for the isolation of antibodies against clinically significant RBC
antigens and the development of testing regimes to aid the prevention of transfusion-
related illness.
1.1 A Short History of Blood Typing and Transfusion
The ABO system was the first human blood group system to be identified. The
discovery was made by an Austrian bom medical practitioner Karl Landsteiner
(Landsteiner, 1901), for which he was awarded the Nobel Prize for Medicine in 1930
(Figure 1.1; Landsteiner, 1931). However, Landsteiner was not the first person with an
interest in blood transfusion. In Century England Richard Lower experimented
with animal to animal (Lower, 1665), and animal to human transfusions (Lower, 1667)
as did his French counterpart Jean-Baptiste Denis (Denis, 1667). The latter was the first
person to describe the symptoms of a transfusion reaction and the death of one of his
subjects led to his arrest for murder. Denis was acquitted but, not surprisingly, the
practice of transfusing blood from animals to humans was prohibited by an act of
parliament in both countries before the end of the century.
T A Ü S E N D SCH
AY r k
, 1 I ffr
Xrtv il-««láMItl
T A U S E N D
t
1000
1000
Figure 1.1 Karl Landsteiner (1868-1943), immortalised on the Austrian 1000
Schilling note (= €70).
Landsteiner was recognised for his outstanding contribution to many areas of
science, including blood typing; ® Landsteiner at his microscope, @ Dark field
microscopy of syphilis spirochetes, (D Model of a virus to represent
groundbreaking work in polio research, @ Immunoglobulin model to represent
Landsteiner's contribution to immunology, ® Human blood groups ABO, ® Red
blood cells.
(Figure from Schwarz et al., 2003a)
Figure 1.2 James Blundell, English
physician (1790-1878).
(Figure from Surgenor, 1974)
No further advances in the field were

made until the early 1800's when an
English obstetrician, James Blundell
(Figure 1.2), transfused human blood to
patients suffering haemorrhage after
childbirth (Blundell, 1818). These
transfusions were beneficial to the
patient in about half of the cases due to
the distribution of ABO blood types in
the English population (Beckman, 2004),
which is to be expected considering that Blundell was working without any knowledge
of blood groups. He carried out his transfusions by collecting blood in a syringe and
then injecting it into the patient, realising the importance of avoiding contact with air
and minimising the length of time from collection to administration in order to prevent
coagulation. Other researchers avoided the problems of coagulation by pursuing
methods of direct or 'arm to arm' transfusion (Figure 1.3; Roussel, 1876).
Figure 1.3 Illustrations of direct transfusion carried out by Dr. J. Roussel in Paris
in the late 1800's.
(Figures from (Left) Surgenor, 1974 and (Right) Hagen, 1982)
A medical student, Adolf Creite, in 1869 reported that the protein constituent of serum
had the ability to 'dissolve' (lyse) or 'cluster' (agglutinate) RBCs (Creite, 1869;
Reviewed by Hughes-Jones and Gardner, 2002). In the late 19^^ Century, a German
physiologist, Leonard Landois, continued this work by showing that the RBCs of one
species when combined with serum from another species resulted in clumping or
bursting of the cells and published an extensive paper on the subject (Landois, 1875).
It was this published observation that prompted Landsteiner to investigate whether

differences could be seen between blood from individuals of the same species. He
pursued a very simple plan of investigation allowing interaction of serum and RBCs
from different individuals (Landsteiner, 1901). He observed that in many samples there
was no perceptible alteration in the appearance of the blood but in some, agglutination
occurred. From these experiments he concluded that the blood contained antigens and
antibodies (referred to as iso-agglutinogens and iso-agglutinins by Landsteiner) that
brought about agglutination of cells when different blood types were combined. He
identified three groups; A, B and O based on their reaction to each other (Figure 1.4).
Shortly afterwards, the discovery of the AB blood group was made by some of
Landsteiner's colleagues (von Decastello and Sturli, 1902).
ErY^hrocyUs of
Sentnt Agglutinins
group
of group in serum
O A B AB
o 4,
A ^ —
B a "*"" I' m mm ~ ^
AB —
— — ~
Figure 1.4 Landsteiner's ABO blood typing results as reported in his Nobel
lecture, 'On individual differences in human blood', December 11,1930.
(Figure from Landsteiner, 1931)
Landsteiner went on to discover the MN (Landsteiner and Levine, 1927a) and P
(Landsteiner and Levine, 1927b) systems by immunising rabbits with human RBCs
which resulted in an immune response against the cells. The Rhesus (Rh) system was
discovered in the 1930's by Levine and Stetson (1939) in the case of a human
haemolytic transfusion reaction which they showed to be independent of the known
blood groups at that time (ABO, MN and P) although they did not give the new blood
group a name. The following year, Landsteiner and Weiner (1940) generated another
antibody by immunising rabbits with Rhesus monkey RBCs which appeared to be the
same as Levine and Stetson's antibody, and the blood group was named Rhesus.
However, it transpired eventually that the human and animal 'Rhesus' antibodies did
not recognise the same antigen on human RBCs (Levine et al., 1963) and the
Landsteiner and Wiener antigen was renamed in their honour some 20 years after
Landsteiner's death. It was soon recognised that the Rhesus antigen was responsible for
many of the transfusion reactions occurring with ABO matched blood, and still stands
as the second most important finding in transfusion medicine after discovery of the
ABO system. Subsequently, between 1946 and 1967, many other blood group systems
were discovered.
Landsteiner's discovery of blood groups in 1901 paved the way for the blood
transftision systems as we know them today but there were still many technical hurdles
to overcome. Hektoen suggested in 1907, that blood should be tested for compatibility
before transfusion was attempted (Hektoen, 1907). A similar suggestion was made by
Epstein and Ottenberg (1908) in the following year and Ottenberg appears to be the first
physician to actually make use of blood grouping before performing transfusions
(Ottenberg, 1911; Ottenberg and Kaliski, 1913). Another key discovery was made by
Moreschi in 1908, the antiglobulin reaction (Moreschi, 1908). He showed that red cells
could become sensitised when exposed to non-matching serum resulting in
agglutination when exposed to serum a second time.
The next major discovery was in the area of blood preservation, as transfusions up to
that point had relied on the presence of a donor for the immediate transfer of blood to
the recipient. In 1914 anticoagulants first emerged, starting with sodium citrate,
allowing blood to be stored between collection and transfusion (Figure 1.5). This was
discovered by three independent groups almost simultaneously in Belgium (Hustin,
1914), Argentina (Agote, 1915) and the USA (Lewisohn, 1915). Improvements to the
anticoagulant by the addition of glucose were introduced by Rous and Turner in 1916,
to further protect RBCs during storage and extend storage time to weeks rather than
days (Rous and Turner, 1916).
Figure 1.5 Richard Lewisohn and a diagram of his citrate preservation technique
for blood transfusion.
(Figures from Surgenor, 1974 (right) and Starr, 2002 (left))
These advances allowed the establishment of the first 'blood depot' which occurred in
England in response to a high demand for blood caused by World War I. Its set up was
credited to Dr. Oswald Robertson who stored citrate-preserved blood for subsequent use
in casualty clearing stations (Robertson, 1918). The first public (as opposed to military)
blood bank was opened in the USA at Cook County Hospital, Chicago in 1937. A
laboratory was set up by Bernard Fantus at the hospital, which specialised in testing and
storing donor blood and it was he who coined the term 'blood bank' (Fantus, 1937).
Other blood banks across the United States soon followed with the earliest being set up
in San Francisco, New York, Miami and Cincinnati. The first British blood bank was
set up in the same year in Ipswich followed by several more after the outbreak of World
War II.
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BLOOD TRANSFUSION CAMPAIGN
I H». 12" 1» 2(»"' SUPPORT THE RED CROSS
MATIOMAL CIO WAR RCUEF COMNITnE
Figure 1.6 Wartime advertisements to encourage blood donation in the USA and
UK.
(Figures from Starr, 2002)
During the war, both countries encouraged pubHc participation in blood donation
schemes (Figure 1.6) and the lives of many servicemen and civilians were saved by the
use of blood products. Over a four and a half year period during World War II, the
American Red Cross collected 13,326,242 units of blood (Bishop and Surgenor, 1964).
These blood donation systems were formalised around 1947 by the launch of the
American Association of Blood Banks in the US and the National Blood Transfusion
Service in the UK (two years before the establishment of the National Health Service).
In the meantime, blood storage solutions had been refmed further with the advent of
Acid-Citrate-Dextrose (ACD) as an anti-coagulant (Loutit, et al., 1943) and Citrate-
Phosphate-Dextrose (CPD; Gibson, et al., 1956) both of which are still used today.
Similarly, there was no official blood service operating in Australia before the outbreak
of the Second World War (Cortiula, 2001). Most hospitals maintained their own blood
stocks but shortages were frequent as smaller hospitals didn't have their own stocks and
also relied on the larger institutions. In 1941 blood collection centres were developed
around Australia run by the Australian Army Medical service in conjunction with the
Australian Red Cross. After the war, military involvement in blood collection ceased
and the centres were maintained for civilian purposes by the Australian Red Cross.
During wartime, blood had been collected in wards at Sydney Hospital in the centre of
the city. Once the war was over, the hospital was keen to reclaim its space and the
transfusion service had to find a new home. This was achieved eventually in 1952
when premises were acquired at 1 York Street (formerly Petty's Hotel, Figure 1.7).
Figure 1.7 ARCBS Headquarters, 1952-1974, at 'Petty's Hotel' York Street,

Sydney
(Figure from Cortiula, 2001)
The Australian transfusion service diversified from simple blood collection, testing and
distribution and began pursuing research activities. This research led to the discovery
of a new blood group named 'S' which turned out to be part of the MN system (now
know as the MNS system; Sanger and Race, 1947; Walsh and Montgomery, 1947). The
I960's saw the advent of the Rhesus (D) Antibody Project for the prevention of
Haemolytic disease of the newborn (Section 1.3), and the extension of blood typing to
typing of other tissues which would eventually assist in successful organ transplantation.
This level of expansion in services led to a requirement for larger premises and the
Blood Transfusion Service moved to its current city centre location in Clarence Street in
1974 (Figure 1.8). The present day ARCBS relies on the altruism of approximately
450,000 voluntary blood donors (~3 % of the population) and in the year 2001/2002,
collected 980,000 blood donations (Australian Red Cross Blood Service, 2003).
Figure 1.8 Donors queuing to help after the Granville train disaster of 1977,
ARCBS, Clarence Street, Sydney
(Figure from Cortiula, 2001)
1.2 Red Blood Ceil Surface Structure
I. !
QCO-
iuiii! hi.'.iv (^'^¿¿C'
Figure 1.9 Cross-sectional diagram of the human RBC membrane.

Integral membrane proteins (shown with branched, extracellular carbohydrate
moieties); B3: Band 3, GPA: Glycophorin A, GPC: Glycophorin C.
Anchor proteins; Ank: ankyrin, P4.2: protein 4.2.
Cytoskeletal Proteins; a-Sp: a-Spectrin, ADD: adducin, 4.1: protein 4.1,
TMY: tropomyosin, TMD: tropomodulin, p55: p55 protein.
(Figure from Yawata, 2003)
Blood groups are determined by antigens carried on the RBC surface. They are defined
serologically by reaction with a specific antibody and are all inheritable characters.
Antigens may consist of carbohydrate epitopes on glycoproteins and/or glycolipids, or
peptide antigens on proteins inserted in, or attached to, the cell membrane. For example,
A and B blood group antigens are determined by sugar groups attached to several cell
surface structures including proteins and lipids. Some blood group antigens are also
found on other tissues.
The structure of the RBC membrane has been well characterised (Figure 1.9; Reviewed
by Lux, 1979 and Gratzer, 1981) as the membranes (erythrocyte ghosts) are relatively
simple and easily purified (Dodge, et al., 1963). Analysis of solubilised RBC
membranes by SDS-PAGE (Fairbanks, et al., 1971) has allowed identification of many
protein components. There are ten major proteins and many minor ones (Yawata, 2003)
with the major proteins falling into two main categories; peripheral proteins loosely
associated with the membrane and integral proteins embedded in the membrane itself.
Most blood group antigens are associated with the latter, membrane bound proteins,
which may have several trans-membrane domains. These include the glycophorins (GP)
whose exact biological function has not been determined, and Band 3 glycoprotein (B3)
which is involved in cell skeleton attachment and also has an ion channel domain.
Together, B3 and GP make up over 30 % of the total membrane protein (Fairbanks et al.,
1971; Yawata, 2003).
Sf'fcCTRlNl
ANKYHIN
ANION
EXCHANGE 3— PAS-'. '.GPAi;
PROT F IN J_
1!^ PAS-4. GPA-GPB
r I (GPB,';
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ACT IN V, — ' GPC
G3PD & —
PAS 3. GPO
» ~i!
(ilOBlf^ 9 i
M M
CB PAS
r AiMt'AfjK"
.; Srccx A
.l EMMl i
Figure 1.10 Diagrammatic representation of SDS-PAGE analysis of red ceil

membrane ghost membranes.
Left: Fairbanks et al. Method: homogenous acrylamide gel. (Fairbanks et al.,
1971).
Right: Laemmli Method: gradient acrylamide gel. Gives better resolution
(Laemmli, 1970).
CB: Coomassie Blue Staining (for protein), PAS: periodic acid Schiff staining (for
carbohydrate), S: soluble fraction (erythrocyte cytoplasm obtained from RBC
lysis), M: membrane fraction (SDS/DTT solubilised, washed RBC ghosts).
(Figure from Yawata, 2003)
To date over 600 RBC antigens have been discovered and classified into four categories
(Daniels et al., 2004). The main category is the blood group systems of which there are
29 (Table 1.1) containing 284 antigens (Table 1.2 for examples). These systems consist
of antigens coded for by single or mainly homologous genes and all have been mapped
to specific chromosome locations. Other antigens which do not meet the criteria for
belonging to one of the systems are classified in collections, which consist of closely
related antigens, or into low incidence (<1 % European population) and high incidence
(>99 % European population) antigen series.
Table 1.1 The blood group systems and their symbols

Number System Name System Symbol
001 ABO ABO
002 MNS MNS
003 P PI
004 Rh RH
005 Lutheran LU
006 Kell KEL
007 Lewis LE
008 Duffy FY
009 Kidd JK
010 Diego DI
Oil Yt YT
012 Xg XG
013 Scianna SC
014 Dombrock DO
015 Colton CO
016 Landsteiner-Wiener LW
017 Chido/Rodgers CH/RG
018 H H
019 Kx XK
020 Gerbich GE
021 Cromer CROM
022 Knops KN
023 Indian IN
024 Ok OK
025 Raph RAPH
026 John Milton Hägen JMH
027 I I
028 Globoside GLOB
029 Gill GIL
Table 1.2 Selected blood group systems and their assigned antigens.
*The RH system has a further 30 antigens (026 to 056) in the series.
Antigen Blood Group System
Number ABO RH KEL FY JK
001 A D K Ff Jk^
002 B C K F/ Jk^
003 AB E Kp^ Fy3 Jk3
004 AI C Kp^ Fy4
005 —
E Ku Fy5
006 F Js^ Fy6
007 Ce Js^
008 C^ — -
009 C^ — -
010 V Ul'
011 Kll
012 G K12
013 —
K13
014 — K14
015 —
—
016 - — K16
017 Hro K17
018 Hr K18
019 Hr^ K19
020 VS Km
021 c" Kp^
022 CE K22
pW K23
023
024 K24
025 —etc.* VLAN

Blood group membrane proteins fall into five main groups based on their function;
membrane channels, membrane bound enzymes, structural proteins, receptors and cell
adhesion molecules (Cartron and Colin, 2001). Most blood group antigens have been
assigned to a particular cell surface structure (Anstee, 1990). For example, MN and Ss
antigens are found on the A and B glycophorins; A, B and H antigens mainly on Band 3
glycoprotein; and glycophorins C and D carry the Gerbich antigens. The Kidd
membrane protein has been characterised as a urea transport protein (Olives, et al.,
1995), the Duffy protein is a chemokine receptor (Horuk, et al., 1993) and Colton
antigens are located on a water transport protein (Smith, et al., 1994).
RBC antigen carrier molecules can also be classified into five types depending on their
structure within the membrane (Figure 1.11). These include carbohydrate chains,
membrane proteins with a single transmembrane domain with an extracellular N-
terminus, the same structure with an intracellular N-terminus, membrane proteins with
multiple transmembrane domains and GPI-linked proteins (glycosyl phosphatidyl
inositol: a glycated membrane phospholipid. Reid and Lomas-Francis, 1997).
- N-glycan ,
0-glycan '
O CHO I
Inside
COOH COOH •
GPI-
Single-pass Multipass linked
Figure 1.11 Model of RBC membrane components carrying blood group antigens.
'Single-pass' proteins have just one trans-membrane domain orientated either
with an extracellular C or N terminus. 'Multi-pass' proteins have several trans-
membrane domains resulting in one or both cytoplasmic termini. GPI-linked
proteins are attached to carbohydrate groups on membrane lipids (GPI: glycosyl
phosphatidyl inositol; a glycated membrane phospholipid). Some blood group
systems are not mentioned in this Figure as their antigens are carried by factors
which adsorb on to the RBC surface from the serum (Chido/Rodgers System) or
alternatively the antigen carrier was not known at the time of publishing (Scianna
antigens have now been shown to be located on the erythrocyte membrane-
associated protein, ERMAP, which is a 60-68kDa 'single-pass' glycoprotein with
an extracellular amino terminus; Wagner, et al., 2003).
(Figure from Reid and Lomas-Francis, 1997)
1.3 Clinical Significance of Red Blood Cell Surface Antigens
Type A blood Type B blood
Results in destruction of rbc's ^ haemoiytic transfusion reaction.
Figure 1.12 An example of ABO incompatibility.

Transfusion of Type A blood into a Type B individual (or vice versa) results in
destruction of RBCs due to the presence of naturally occurring anti-A and anti-B
antibodies.
The clinical significance of RBC surface antigens is related to the potency of the natural
or immune antibodies that bind to them, and how commonly the antigen occurs in the
general population (Mollison, et al., 1997, p63). Despite all of the progress made in the
clinical management of blood transfusion, fatalities still occur at a rate estimated to be
between one per 250,000 to one per million transfusions (British Committee for
Standards in Haematology, 2001).
The ABO blood group system is the most clinically significant of all the groups. This is
due to the presence in individuals of naturally occurring anti-A and/or anti-B blood
group antibodies that are strongly reactive at 37 and frequently cause severe
transfusion reactions (Figure 1.12). In fact, most fatal transfusion reactions are due to
the accidental transfusion of ABO incompatible blood (Reid, et al., 2000). These
antibodies are usually of the IgM subclass (Section 1.4) and occur in the absence of any
exposure of the immune system to the red cell antigen via transfusion or pregnancy
(Australian Red Cross Blood Service, 2003). They are not present at birth but develop
during early childhood. Since the A and B red cell antigens consist of carbohydrate
structures (A^-acetyl-D-galactosamine and D-galactose respectively; Watkins, et al.,
1959), it is believed that the naturally occurring antibodies against them are produced
by the body in response to similar substances found within the environment such as
bacteria. Several experiments were performed in the 1950's and 60's which indicated
that anti-B antibody production could be caused by exposure to a particular strain oiE.
coli (Springer and Horton, 1969), and chicks raised in a germ-free environment did not
make anti-B (Springer, et al., 1959). There are naturally occurring antibodies to other
blood group antigens but they are of little clinical importance as they are usually
inactive at 37 °C and therefore unlikely to cause transfusion reactions (referred to as
cold agglutinins; Mollison, et al., 1997, p80).
An individual who is type A (carries the A antigen on their RBCs) will not recognise a
similar antigen as foreign and therefore will not produce anti-A antibodies (Mollison, et
al., 1997, pl23). However, if this same individual is exposed to a 'B-like' antigen, they
will treat it as foreign and generate anti-B antibodies. Thus type A individuals have
potent anti-B serum antibodies and B type individuals have anti-A antibodies.
Individuals of the AB group carry both antigens on their RBCs and therefore have
neither anti-A nor anti-B in their serum. Conversely, O group individuals have neither
A nor B antigen on their RBCs and have both anti-A and anti-B antibodies in their
serum. The absence of AB antibody in AB positive individuals and the absence of AB
antigen in Group O individuals led to them being referred to respectively as 'universal
donors' and 'universal recipients' (Table 1.3). These terms refer only to the recipient
reaction to received RBCs and do not consider reactions due to antibodies present in the
blood plasma (donor to recipient plasma compatibility is the reverse of RBC
compatibility). In addition, as the knowledge of blood groups has expanded to include
other clinically significant antigens, these simplistic terms are less likely to be used.
Table 1.3 Blood type compatibilities within the ABO system.
Table shows the origin of the terms 'Universal Donor' for O type individuals and
'Universal Recipient' for AB type individuals.
^: Compatible blood; Incompatible blood.
Recipient Universal
y i Recipient
Donor RBC
A B AB O
Antigen
A X X
B X y X
AB X X X Universal
Donor
O
These naturally occurring anti-A and anti-B antibodies become critically important in
the event of blood transfusion. Transfusion of incompatible blood results in serum
antibody binding to RBCs (Figure 1.12). This triggers haemostatic reactions including
complement activation and can result in the recipient lapsing into shock and suffering
renal failure due to the destruction of RBCs. These effects can be fatal, even after the
transfusion of a small amount of blood, but modem health practices mean that these
cases are relatively rare. This type of reaction is referred to as an acute haemolytic
transfusion reaction (AHTR) and is usually, although not always, a result of ABO
incompatibility (Australian Red Cross Blood Service, 2003).
In the UK, adverse transfusion reactions are monitored in the SHOT Report (Serious
Hazards of Transfusion; SHOT Steering Group, 2006), a survey in which about half of
UK hospitals participate. Out of more than 3 million issues of blood products from the
Transfusion Services (including RBCs, plasma and platelets) in 2005, 609 adverse
transfusion events were reported. In 5 cases the patient died as a direct result of the
transfusion. A further 32 cases resulted in 'major morbidity' which is defined as
requiring intensive care admission, renal failure, severe haemorrhage, intravascular
haemolysis or a risk of D sensitisation in a female of child-bearing potential. The
remaining cases resulted in minor or no symptoms. The major cause of these incidents
was the transfusion of incompatible blood due to clinical errors. Most reactions were a
result of ABO incompatibility followed by Rhesus and then other blood groups.
Transfusion reactions can also be caused by 'immune' red cell antibodies (referred to as
alloantibodies, from Greek alios meaning other). These occur in response to foreign
RBC exposure either by transfusion or through pregnancy. The most important of these
belong to the Rhesus blood group system but antigens from other systems such as Kell,
Duffy, and Kidd are also implicated (Mollison, et al., 1997, p87-9). All blood donations
are routinely tested for ABO and Rhesus blood group as well as for the presence of
antibodies against other clinically significant blood groups. The main antigen in the
Rhesus group is called 'D' and in blood banking shorthand, cells carrying D antigen are
referred to as Rhesus positive (+) and cells without D are Rhesus negative (-). For
example, an individual referred to as A+ has A antigen and D antigen on their RBCs, an
0 + person has D (no A nor B antigen) and an O- person has no A, B nor D antigen.
As mentioned previously, RBCs carry many antigens in addition to the ABO and
Rhesus groups. Transfusion with ABO and Rhesus cross-matched blood may still result
in the recipient producing antibodies to other antigens on the RBC surface that are not
present on their own RBCs such as Duffy, Kell or Kidd antigens (Table 1.2). These
antibodies are more often of the IgG subclass and as a result, cause a different set of
clinical problems to the naturally occurring IgM antibodies of the ABO system. These
IgG antibodies may occur at a very low level in patient serum and remain undetected in
pre-transfusion testing (Ramsey and Smietana, 1994). Subsequent transfusion with
RBCs bearing this antigen results in a delayed haemolytic transfusion reaction (DHTR)
which occurs 1 to 2 weeks after transfusion and is often not as severe as AHTR.
Table 1.4 Relative frequency of immune red cell alloantibodies in transfusion
recipients (and some pregnancies).
Table shows alloantibodies associated with acute (immediate) haemolytic
transfusion reactions (AHTR) and delayed haemolytic transfusion reactions
(DHTR; Table from Mollison et al., 1997, p89).
Blood Group System Alloantibodies (Excl. ABO. Le, P, MN)
No. of Rh K Fy Jk Other
Data Set Cases (excl. D)
Transfusion
4177 55% 29% 11 % 4% 1%
Recipients
Assoc. with
142 42% 30% 18% 9% 1%
AHTR
Assoc. with
82 34% 15% 16% 33% 2%
DHTR
Another serious clinical problem associated with immune antibodies is haemolytic

disease of the newborn (HDN) which can affect the foetus or neonate. In the UK, 16 %
of women are RhD negative blood group and 10 % of all pregnancies involve a
foetomatemal RhD incompatibility (Chilcott et al., 2003). If the foetus is carrying a
RBC antigen inherited from its father to which the mother has antibodies, then these
antibodies can cross the placenta and destroy foetal RBCs. The subclass of antibody is
significant in that IgG antibodies can cross the placenta whereas IgM antibodies cannot
(van der Meulen, et al., 1980). Thus, it is the RBC antigens that cause the production of
potent IgG antibodies that are most frequently implicated in HDN.
Ant-Rh
• r/no9¥Ci Antilodes
Figure 1.13 Representation of haemolytic disease of the newborn (HDN) and anti-
D prophylaxis.
a) RhD negative blood group mother carrying an RhD positive baby.
b) Mother is exposed to foetal RBCs via transplacental haemorrhage either
during pregnancy or birth.
c) Mother makes anti-D antibodies in response to the foreign D antigen
encountered on the foetal RBCs.
d) Mother remains sensitised to D antigen.
e) Any subsequent RhD positive babies will suffer from HDN as anti-D crosses
the placenta from the maternal circulation and destroys foetal RBCs.
HDN can be prevented by the administration of anti-D antibody to the mother to
remove foetal RBCs from her circulation before she becomes immunised.
(Figure adapted from Archer and Parker, 1982)
Over 90 % of all HDN cases occur when an RhD negative mother gives birth to an RhD
positive infant (Chilcott et aL, 2003). Frequently, the mother is sensitised by exposure
to a foetal (paternally inherited) RBC antigen during pregnancy or birth via
transplacental haemorrhage (TPH), which does not affect the foetus but has a severe
effect on any subsequent pregnancies. This problem can be addressed by the use of
anti-D prophylaxis (prevention) developed in the 1960's (Finn, et al., 1961; Freda, et aL,
1967), where anti-D antibody is administered to the mother to remove any D positive
RBCs from her circulation before they have a chance to provoke an immune response
(Figure 1.13). The incidence of HDN in NSW, AustraHa dropped after the introduction
of the RhD Antibody Project in 1967 (Figure 1.14). Anti-D was harvested from the
plasma of women who had already been immunised via transfusion or pregnancy. In
addition, an immunisation scheme was initiated for immunising male volunteers against
D antigen who subsequently donated plasma (plasmapheresis) from which anti-D was
harvested.
Introduction of Anti-D Prophylaxis
1970 1971
Year
Figure 1.14 Incidence of HDN in NSW, Australia between 1964 and 1977.
Anti-D prophylaxis was introduced in 1967 which saw a decline in the number of
reported cases of HDN.
(Data source Cortiula, 2001, pl41)
As a result of anti-D prophylaxis, the cause of HDN has shifted from anti-D to other red
cell alloantibodies although anti-D remains the commonest cause of death from HDN
(Moise, 2000). In Australia, anti-RhD immunoglobulin is obtained from pooled serum
collected from D negative plasmapheresis donors who have been deliberately
immunised with D positive RBCs (Archer and Parker, 1982) and from individuals
sensitised via pregnancy or incompatible RBC transfusion. It has been shown that
donors who develop a high titre of serum anti-D in response to immunisation are also
likely to respond to other antibodies on the RBCs used for immunisation. In one data
set, out of the recipients who responded to D antigen, 50 % also made anti-K, 20 %
made anti-Fy^ and 20 % made anti-Jk^ (Mollison et al, 1997, p92). Recipients who
didn't respond to D didn't make any other alloantibodies either.
Some of the blood samples used in this project were obtained from plasmapheresis
donors who had also generated antibodies to other RBC antigens during D
immunisation. Other samples were obtained from patients who had undergone a
transfusion reaction. These included antibodies to clinically significant RBC surface
antigens particularly in the Rhesus, Kell, Duffy and Kidd systems including; anti-e,
anti-c, anti-K, anti-Jk^, anti-Fy^ and anti-Fy^.
1.3.1 The Rhesus System
r,
Pii'
I (H t
t i>Uil Ml
Figure 1.15 Diagram of the proposed structure of the Rhesus polypeptide,

(a) Zigzag lines represent palmitoyl groups and open circles represent amino acid
differences in the D and CcEc polypeptides, (b) Three-dimensional representation
showing the twelve transmembrane domains as cylinders.
(Figure from Daniels, 2002, p203)
The Rhesus system is the most complex of all the blood groups having 49 antigens; 001
to 0056 with 7 obsolete numbers (Daniels et al., 2004) and was the fourth system to be
discovered after ABO, MNS and P (Table 1.1). The most clinically significant of the
Rhesus antigens is D, which was also the first to be discovered in the system. It is still
often referred to as the Rhesus antigen as it was originally thought to be the same as an
antigen discovered by Landsteiner and Wiener (1940) who immunised rabbits with
Rhesus monkey RBCs (Section 1.1). Other common antigens in the Rhesus system
include C, c and E, e. The D antigen is carried on the RhD polypeptide whereas C, c, E
and e Rhesus antigens are carried on the very similar (90 % amino acid identity)
RhCcEe polypeptide. Both are -45,000 Da molecular weight and are believed to have
12 trans-membrane domains giving 6 extracellular loops and 7 intracellular domains
including both termini (Figure 1.15; Daniels, 2002, p201-3). The Rhesus protein
associates with another glycoprotein in the RBC membrane and the complex is linked to
the cell skeleton (Nicolas et al., 2003).
The occurrence of D+ individuals is high, at 85 % in the Caucasian population rising to

99 % in Asians (Reid and Lomas-Francis, 1997). It is of considerable clinical
importance as the majority (80 %) of Rh negative individuals will become sensitised
after a single exposure to Rh positive RBCs (Daniels, 2002, p247). This is especially
important, as mentioned previously in the case of females, as it can lead to haemolytic
disease of the newborn (Section 1.3).
1.3.2 The Kell System
The Kell blood group system was the first to be discovered using the indirect
antiglobulin test (Section 3.3.2; Coombs, et al., 1946). It was discovered by the
examination of RBCs from a baby who had suffered HDN and the system name was
taken from the family surname of Kelleher. The antigen was designated 'K' and three
years later the antithetical antigen 'k' was discovered due to an antibody also generated
as a result of HDN (Levine, et al., 1949) which was found to be present in nearly 100 %
of all populations. In 1957, two further Kell antigens were discovered, Kp^ and Kp^
(Allen and Lewis, 1957) and the system now stands at 25 antigens making it the third
most complex group after Rh and MNS.
Other than ABO and Rh system antibodies, anti-K is the most commonly occurring
immune red cell antibody suggesting that Kell antigens are highly immunogenic. It
forms in about 10 % of K negative individuals who are exposed to K positive RBCs and
as such is about 5 to 10 times less antigenic than the D antigen (Daniels, 2002, p299-
300).
(• — •
Figure 1.16 Diagrams of the proposed structure of the Kell polypeptide, showing
the Kell antigens.
Y shaped molecules represent carbohydrate structures.
A: Kell protein with one transmembrane domain is disulphide linked to the XK
protein which belongs to a different blood group. The catalytic domain is shown at
the C terminus as the Kell protein is a zinc endopeptidase.
(Figure from Westhoff and Reid, 2004).
B: Closed circles represent cysteine residues which are involved in folding of the
extracellular domain although the exact structure is not known. Note the absence
of polymorphisms at the C terminus presumably to conserve enzyme function.
All Kell antibodies are considered to be clinically significant as they can cause severe
haemolytic transfusion reactions and HDN (Reid et al., 2000) as shown by the manner
in which the blood system was discovered. HDN due to Kell antibodies appears to be
much more unpredictable as compared to anti-D related HDN in that antibody titre in
the mother's serum doesn't correlate well with the severity of the effect on the foetus.
This may be due to the fact that Kell antigens are very well developed on foetal RBCs
(Southcott et al., 1999), and that the antibodies are inhibiting erythropoiesis rather than
destroying RBCs (Vaughan et al., 1994; Daniels et al., 2003).
The Kell antigens are carried on a -93,000 Da molecular weight glycoprotein (Figure
1.16). Unlike the Rhesus and Duffy polypeptides, it has only one transmembrane
domain. It has a short cytoplasmic N-terminal domain and a long extra cellular domain
on which the Kell epitopes are situated (Lee et al., 1991). The extracellular domain
contains 15 cysteine residues which cause the extracellular domain to fold, although the
exact structure is not known. The extracellular domain is also disulphide linked to
another protein from a different blood group, XK. The Kell protein has been classified
as a zinc endopeptidase and has a conserved zinc binding site at the C-terminus (Marsh
and Redman, 1990).
1.3.3 The Duffy System
The Duffy system was defined by the discovery of an antibody in the serum of a
multiply transfused haemophiliac after whom the system was named (Cutbush et al.,
1950). The antigen was designated Fy^ and the following year the antithetical antigen,
Fy^ was found via the discovery of a different Duffy antibody in the serum of a woman
who had been sensitised via pregnancy (Ikin et al., 1951). A ftirther three Duffy
antigens were discovered in the 1970's (Albrey et al., 1971; Behzad et al., 1973;
Colledge et al., 1973) and the final one in 1987 (Nichols, et al., 1987).
In 1975 the link was made between Fy^ and Fy^ antigens and the infection of red cells
by malaria parasites (Miller et al., 1975). It had long been known (Sanger et al., 1955)
that the majority of the African native population did not carry Fy^ or Fy^ antigens on
their RBCs (Fya-b- phenotype) and this was shown by Miller et al. to confer malarial
resistance. The occurrence of Fy^ antigen in the black population is 10 %, 66 % in
Caucasians, and 99 % in Asians.
Alloimmunisation to Fy^ in Caucasian populations is not high despite its relatively high
occurrence. Fy^ antibody occurs three times less frequently than anti-K despite the fact
that Fy^ antigen occurs approximately seven times more frequently than the K antigen.
Using these data, the 'immunisation potential' of blood group antigens can be estimated
(Giblett, 1961) and reveals that K is - 2 5 times more efficient as an immunogen than Fy'
Anti-Fy'' occurs approximately 20 times less frequently than anti-Fy^ Most Fy^
antibodies can only be detected by the indirect antiglobulin test indicating that they
belong to the IgG subclass ((Mollison et al, 1997, pi91) and occasionally cause
transfusion reactions and mild or severe HDN (Reid et al, 2000).
nh2 S > Fy^/Fy^

L Gly42Asp
Ey-trscoliyiar Pvi
RBC Lipid
Bilayer
InlfACOllulnr
COOH
FyX
A Arg89Cys
Figure 1.17 Diagrams of the proposed structure of the Duffy polypeptide, showing
the Duffy antigens.
Y shaped molecules represent carbohydrate structures.
A: The Duffy protein is predicted to have seven transmembrane domains
(Westhoff and Reid, 2004).
B: Three-dimensional representation showing the seven transmembrane domains
as cylinders. Closed circles represent cysteine residues and dotted lines represent
the likely position of disulphide bonds.
The protein carrying the Duffy antigens is a glycoprotein of 35-43,000 Da molecular

weight (depending on the level of glycosylation) and is believed to have seven trans-
membrane domains (Figure 1.17; Chaudhuri et aL, 1989; Neote et aL, 1994). The
amino terminus of the protein is extracellular and carries the sugar groups while the C-
terminus is intracellular. The Duffy gene has been cloned (Chaudhuri et aL, 1993) and
the extracellular domain expressed as a soluble protein (Wasniowska et al., 2000). In
1993 the Duffy protein was discovered to be a chemokine receptor (Horuk et al., 1993)
and was named DARC (Duffy antigen Receptor for Chemokines). The difference
between the Fy^ and Fy^ glycoprotein is a single amino acid substitution at position 42.
1.3.4 The Kidd System
The Kidd blood group system was discovered by Allen et al. (1951) in the serum of Mrs.
Kidd from whom the system takes its name. This serum antibody caused HDN in Mrs.
Kidd's sixth child and the corresponding antigen name, Jk^ included baby John's
initials. The antithetical antigen, Jk^, was discovered two years later as a result of a
transfusion reaction. (Plaut et al, 1953). A third Kidd antibody was found in the serum
of an individual who did not carry either Jk or jk" on their blood cells, type Jk(a-b-),
(Pinkerton et al., 1959). This antibody was originally referred to as anti-JkaJkb but is
now called anti-Jk3 and defines the third antigen in the Kidd series which is present in
nearly 100 % of all populations.
JkîJk^
ËxlraOoHulâr
RBC Lipid
Bilayer
Intra nelluli^r
COO H
Figure 1.18 Diagram of the proposed structure of the Kidd polypeptide, showing
the Kidd antigens.
The Y shaped molecule represents a carbohydrate structure. The Kidd protein is
predicted to have ten transmembrane domains.
(Figure from Westhoff and Reid, 2004)
No further antigens in the Kidd system have been identified since the 1950's and it
remains among the least complex of groups. However, this simplicity belies its clinical
significance as Kidd antibodies are notoriously difficult to detect and handle. Among
all of the reported cases of delayed transfusion reactions in the 2005 SHOT report,
'Kidd antibodies were the most commonly implicated' (Serious Hazards of Transfusion;
SHOT Steering Group, 2006). Anti-Jk^ has been referred to as 'one of the most
dangerous immune antibodies occurring in human serum' (Reid et al, 2000). Some Jk^
antibodies will not bind to Jk(a+b+) RBCs but only to Jk(a+b-) cells (the latter being
homozygous for, and therefore expressing more of the Jk^ antigen than heterozygous
RBC) and are inclined to lose reactivity in vivo and in vitro (Daniels, 2002, p344-5;
Thompson et al, 1991). In addition, Kidd antibodies occur frequently as a mixture with
other red cell alloantibodies, which makes typing more difficult (Westhoff and Reid,
2004). Kidd antibodies can only be detected by Indirect Antiglobulin Test (Section
3.3.2) and even then results can be variable which may be due to the antigen sites being
clustered and not accessible for antibody binding (Mougey, 1988). The possibility of
the weak binding being due to the copy number of Kidd antigens on the RBC surface
has been ruled out as there are estimated to be 14,000 binding sites (Masouredis et al.,
1980), many more than for the Kell antigen where no such detection problems exist.
Kidd antigens are regarded as relatively non-immunogenic compared to Rhesus, Kell

and Duffy antigens and antibodies against them are not common (Table 1.4). However,
once an individual is sensitised, a second exposure to the antigen tends to result in a
rapid increase in antibody titre resulting in severe DHTR (Westhoff and Reid, 2004).
This is referred to as an anamnestic response and is clinically problematic as the
antibody titre often drops to an undetectable level after the first exposure and is
therefore not taken into account during cross-matching of blood. As a result of this,
antibodies to the Kidd antigen are responsible for at least a third of all cases of DHTR
(Pineda et al., 1999).
Kidd antigens are carried on a 46-60,000 Da molecular weight glycoprotein which is

predicted to have ten membrane spanning domains (Figure 1.18). The difference
between Jk^ and Jk'' polypeptides is a single amino acid substitution on the fourth extra-
cellular loop at position 280 (Olivès et al., 1997). The protein was suspected to be a
urea transport protein as Jk(a-b-) RBCs were observed to be resistant to lysis by 2 M
urea (Heaton and McLoughlin, 1982). This was later confirmed by protein sequencing
(Rousselet et al, 1996) and the gene has been cloned (Olivès et al., 1995). It is believed
that urea transport in RBCs is necessary to maintain osmotic stability as cells pass
through the kidney (Macey and Yousef, 1988).
1.4 Antibodies to Red Blood Cell Surface Antigens
Id) icjA <dimer) le) igM i(>ftn!amet)
Hirtje region
J chain-.
(
Figure 1.19 Diagrams of the five classes of immunoglobulin.

Solid black lines indicate disulphide bonds. Classes are based on differences in
heavy chain structure designated y (IgG), ö (IgD), £ (IgE), a (IgA) and \i (IgM).
Light chains have the same structure in all classes and are either K (kappa) or
(lambda) type (both never occur in the same molecule).
(Figure from Kuby, 1997)
Antibodies are glycoproteins belonging to the immunoglobulin family. They have two
main regions, one involved in antigen binding and the other in effector functions. There
are five classes of immunoglobulin, all containing combinations of two identical heavy
chains and two identical light chains, so called because heavy chains are larger than
light chains. The chains are folded into globular domains from which the protein family
gets its name, with the heavy chain consisting of four domains and the light chains
having two.
S
I
S
Figure 1.20 Diagram of sequence variability in antibody molecules.

The majority of the heavy chain (CH) and half of the light chain (CL) are constant
regions with little variation in amino acid sequence. The remaining regions show
considerable sequence variation between antibody clones and are designated the
variable regions (¥„ and VL). Within these variable regions are three sections
showing even higher variability (hyper variable regions or complementarity
determining regions; CDRs) which are surrounded by four framework regions.
(Figure from Roitt and Delves, 2001, p40)
The five classes are IgG, IgM, IgA, IgD and IgE, all having different roles within the
immune system (Figure 1.19). The immune responses to RBC surface antigens (Section
1.3) mainly involve IgM and IgG antibodies. Having said that, some anti-A antibodies
are of the IgA class (Kunkel and Rockley, 1963) and interestingly, anti-A and B
antibodies in an O type individual are more likely to be of the IgG class whereas A or B
individuals are likely to have more IgM antibodies (Rawson and Abelson, 1960).
Rhesus, Kell, Duffy and Kidd antibodies are predominantly IgG (with some IgM)
whereas Lutheran antibodies frequently contain IgA (Mollison et al., 1997, p200).
The antibody molecules are Y shaped with the two heavy chains covalently linked in
the centre near a flexible hinge region (a linear structure between the tightly folded
globular domains), and electrostatically held together for half of their length (Figure
1.20). The other half is not linked and the flexible hinge allows movement of the two
'arms' of the Y shape. It is to these two arms that the two light chains are linked,
covalently near to the hinge region (ChI to Cl), and electrostatically for their whole
length. The ends of the chains furthest from the hinge (N termini) constitute the two
antigen binding sites. The IgM subclass is unique in that it consists of five Y shaped
units joined together by the J Qoining) chain to form a pentamer having ten antigen
binding sites (Figure 1.19).
a HEAVY OiWNS LO
t HT CHAINS
IEFO FrI ScOftl F fr3 iCOfOl

R"
100
bO r-
IS -J •
?0 40 60 80 100 l?0 J'O /fi
POSio' n
Figure 1.21 Wu and Kabat plot of sequence variability in the variable regions of
antibody molecules.
The sequences from a large number of heavy and light chains were determined
and the variability at each residue was calculated (number of different amino acids
found divided by the frequency of the most common amino acid). The higher the
number, the greater the variability and the hyper variable regions (or CDRs ) are
easily distinguished from the less variable framework regions.
(Figure from Roitt and Delves, 2001, p40)
Heavy-Chain Genes Light-Chain Genes
D jH C Jk C.
80' : 30) (6) 19)
( 70) (5,
•CKEHSKZHS}^' • • "
J - 'ear-'a'-'eeme"; .'K Jk 'CdCdnpemen;
i
Eiuiiucii-.ietet.or! Va'iable region
N ddd t.o' I' " . ' A 4 ^
DNA '•ea'i'anHOfre"!
I
•ij- ; rse drtcti j'l
^ ¿dd'ilon (TdT)
Rearranged D N A Rearranged DNA

^'anstfiDl'or
RNA <,pl • RNA >p!i( ing
i
-lBeEHES-
mRNA mRNA
*'tini>iatior
•
I variable region
Constant region
if iH I
• i-ep: '!»> i."". fnam polypeptide
Figure 1.22 Generation of antibody diversity from germ line antibody gene
'building blocks'.
In heavy chain expression, the first step is the joining of a random D (diversity)
segment to a J (joining) segment. This is brought about by recombination
activating gene products (RAG-1 and RAG-2) which act on recombination signal
sequences between the blocks to loop out the intervening DNA, excise the loop and
rejoin the blocks (Figure 1.24). The process is repeated to join a variable region,
with nucleotide deletions and additions occurring at each join to further increase
diversity. Finally, a constant region is joined to the J segment thus determining
the class of the antibody and the mRNA is translated and transported to the B cell
membrane under the control of the leader sequence (L). A similar process occurs
with the light chain polypeptide to join a variable, joining and constant segment
(either kappa or lambda type) to produce a whole antibody.
(Figure from Schwarz, 2003b)
IgG is the antibody class found at the highest concentration in plasma and can be further
divided into several subclasses based on slight variations in structure. The effector
region (C terminal end) of the molecules is highly conserved within subclasses and is
referred to as the Fc (Fragment crystallisable) and contains the Ch3 and Ch2 domains (ChI
constant heavy). The antigen binding region of the heavy chain consists of a further
constant domain (CHI) and a variable domain (VH: variable heavy). The light chain also
consists of one constant (CL: constant light) and one variable region (VL: variable light).
The variable domains, as the name suggests, are not highly conserved regions. They
can be any one of many sequences and it is this variation which confers the huge
number of antigen recognition sites available in the human repertoire. The sequence
variation is concentrated in three sections of the variable regions called hyper variable
loops or CDRs shown in Figure 1.21. (Wu and Kabat, 1970). The combination of the
heavy and light chain CDRs form the antigen binding site which is identical on both
arms of the molecule.
The theoretical repertoire of human antibody specificities is very large, with estimates
reported from (Abbas et al., 2000, pl43), to 10^^ (taking somatic mutation into
account; Winter and Milstein, 1991), making it impossible for the genome to contain a
gene for each specificity (the entire human genome contains only 3-4 x 10"^ protein
coding genes; International Human Genome Sequencing Consortium, 2001). Instead
the variation is brought about by the shuffling of relatively few 'building blocks'
(Tonegawa, 1983). In heavy chain expression, these consist of 51 VH segments,
approximately 30 D (Diversity) segments and 6 JH (Joining) segments (Cook and
Tomlinson, 1995).
Light chains are constructed from either kappa or lambda antibody genes (VL: ~30
different segments of each) and joining segments (JL~5 different segments of each;
Skerra, 2 0 0 3 ) . Mathematically this allows the production of - 6 0 0 0 different heavy
chains and 300 light chains, which when recombined can make -1.8 million antibodies
(Amaout, 2 0 0 5 ) , referred to as combinatorial diversity. Further variation is introduced
when segments are joined through 'inaccurate' recombination including random
removal of bases or addition of non-coded bases (called N-nucleotides and P-
nucleotides; Abbas et al., 2 0 0 0 , p i 4 3 ) . This 'somatic hypermutation' results in a highly
diverse repertoire, even though not all gene segments are utilised to the same extent
(Ignatovich et al., 1997).
I si injection 2nG njecfion
of aniigen ot antigen
-r
c:
<C
SjgM
0 1 2 3 Weeks 4 5 6 7
Figure 1.23 Typical production of IgM and IgG class antibodies in response to
antigen stimulus.
The IgM response usually precedes the IgG response but IgG class antibodies
usually (but not always) predominate in the long term.
(Figure from Roitt and Delves, 2001, pl93)
Typically the primary immune response of humans to a foreign antigen is the

production of IgM class immunoglobulin (Figure 1.23; Roitt and Delves, 2001, pi 93).
The IgG response follows, which leads to conversion of some antibody producing B
cells into memory cells that persist in the circulation long after the IgM has disappeared.
If the immunised individual is subjected to a second exposure to the antigen then these
memory cells quickly proliferate and secrete large amounts of IgG (an anamnestic or
'brisker and fatter' response according to Roitt). An amnestic response can occur
anytime from weeks to years, or even decades after the first exposure to antigen. The
change from IgM production to IgG is called class switching and results in a different
antibody class (isotype) but the same specificity of antibody (Figure 1.24).
Loop lor mot ion
I 1
Bmi I
ONA 0
(STERILE RNA transcriptB|)
D
i\
—DNA
ONA R
R EEPAIR
/ X ENZYV.
Excison
Class s^vitch
f
mRNA V D J «
Se^TEO fgM
WIBOOY
Figure 1.24 The class switching process resulting in antibodies of identical

specificity but different isotype.
The red circles represent recombination signal sequences which allow the DNA to
form a loop and be excised. This allows the selection of a different heavy chain
constant region sequence for translation and therefore changes the isotype of the
expressed antibody.
(Figure from Roitt and Delves, 2001, pl93)
As well as class switching, the antibody response undergoes a process of affinity

maturation (Berek and Milstein, 1987). During the increase in B cell population (clonal
expansion) that takes place during the second antigen exposure, a higher than normal
rate of point mutations occur in the antibody structure, and insertions and deletions take
place (Figure 1.25; de Wildt et al., 1999). This could result in a change in affinity for
better or worse, but any mutations producing a higher affinity antibody are favoured by
antigen selection and proliferate further. This mechanism of antibody production has
been shown to be similar in both human and murine systems (Abbas et al., 2000, pi32).
Heavy chain Light chain

V regions V regions
K,
CDR2 CDR3 CDR1 CDR2 CDR3 10 M
J:. 28
JH 28
J
28
h -j:r 3 /
Day 7
^ i J? — i hn 36
primary
Jh 4.0
Ji -Lh 33
J.: n
f 0.5
J.j
60
t - -
J? —il 34
f f Ji 4t 0 7
n
f J. u- 04
Day 14
primary » J;
J. i 0 1
f — -
—i> 02
; Secondary
f-
f-
1»
M
It u
If ^ ^
1 J.5
J?
dif
. .
f
r
• . —ij.
09
0.02
1 1
—i
W « J.!
f 003
Tertiary i t - J.; 0 03
11 1 f J » J J I ih 0,03
(D) J
hJucleotide dilferefice
f Mutation causing a nino acid difference
Figure 1.25 Affinity maturation of antibody genes.

Mice were immunised with a hapten at various time points and hybridomas
produced from spleen cells (Section 1.5, Figure 1.26). Hapten specific antibodies
were obtained from the clones and the amino acid sequence of the variable regions
was determined. The results clearly showed that mutations in the antibody genes
increase with time and with repeated immunisation (Primary, Secondary and
Tertiary immunisation), and that mutations occur mainly in the hyper variable
regions. The results also show that there is a concomitant rise in antibody affinity
as indicated by the decreasing antibody dissociation constant (Kd).
(Figure from Abbas et al., 2000, p202 adapted from Berek and Milstein, 1987)
1.5 Monoclonal Antibodies
In one of the most significant discoveries in the history of biotechnology, Kohler and
Milstein (1975) fused murine spleen cells (conferring antibody specificity) with
myeloma cells (conferring immortality of the resulting cell line), to produce a
monoclonal antibody (MAb; Figure 1.26). The significance of this discovery was
recognised by the award of the 1984 Nobel Prize in Medicine along with Niels Jeme
(Cruse and Lewis, 2005, p294). The resulting cells were called hybridomas and
allowed long-term culture of cell lines secreting a single specificity of antibody: a MAb.
These MAbs went on to be developed into an enormous range of applications in basic
research, and in diagnostic and therapeutic technologies with MAbs raised against a
massive number of diverse antigens including human RBCs.
Until around 15 years ago, all blood grouping tests were carried out with polyclonal
antibodies of human or animal origin (Mollison et al., 1997, p242) but now many
monoclonal antibodies are used. Murine and murine/human hybrid monoclonal
antibodies against A and B antigens (Voak et al., 1980; Lowe et al., 1984; Sacks and
Lennox, 1981; Messeter et al., 1984) have replaced polyclonal antibodies in ABO
typing (Mollison et al., 1997, p86; Thompson et al., 1991; Scott and Voak, 1994) and
several antibodies of mouse origin are currently licensed by the US Food and Drug
Administration for use as blood typing reagents including anti-A, AB, B, Le^, Le^, M, N
and PI (United States Food and Drug Administration, 2007). However, some blood
group antibodies cannot be obtained from a murine system, most notably anti-D
(Goossens et al., 1987). Other murine monoclonal antibodies have been raised against
blood group antigens including K (Parsons et al., 1982); Le^ and Le^ (Fraser et al.,
1984); M (Nichols et al., 1985). However, many murine antibodies miss the 'subtle
human polymorphisms' and recognise the carrier molecule rather than the antigen itself
such as reacting with the Rhesus glycoproteins rather than D, C or E antigens (Scott and
Voak, 1994; Siegel, 2002). This led to the development of antibody producing human
cell lines via immortalisation of B cells with Epstein-Barr vims (EBV).
A'^tigen
>
v.
Spleen
Ji
V HGPRT myeioma cells
PEG . / /
, to" - !
, HAT m e d turn
/
v' /
¿.I
«t o t- c
J
B c:e is Hyb-ndcma Myoioma
No growth Growth No growth
Figure 1.26 Nobel laureates Georges Kohler (top) and Cesar Milstein who
developed murine hybridoma technology for MAb production alongside a diagram
of their groundbreaking method.
The key to the success of the technique was selection of only successfully fused cells
by the use of HAT medium (hypoxanthine, aminopterine and thymidine).
Selection occurred due to the use of mutant myeloma cells for fusion that lacked an
enzyme required for survival in HAT medium. Fused cells regained the required
enzyme from their B cell fusion partner and survived in the selection medium
whilst unfused cells died.
(Figures from Cruse and Lewis, 2005, p295-6)
The first human MAbs developed were specific for a synthetic hapten, 4-hydroxy-3,5-
dinitrophenacetic acid (Steinitz, et al., 1977). This was achieved by selecting B cells
that were secreting specific antibody by resetting with antigen coated RBCs, and
transforming them with EBV. The same group went on to produce anti-trinitrophenol
antibody cell lines (Kozbor et al., 1979) and then anti-RhD, chosen for study 'because
of its great practical importance' (Koskimies, 1980). Lymphocytes were obtained from
an Rh negative male who had been hyperimmunised with Rh positive RBCs and all
antibodies produced from the transformation were of the IgG class. Boylston et al.
(1980) used the same technique to produce an IgM class anti-D antibody.
Unfortunately not all EBV produced cell lines are stable in long term culture as
antibody production is often lost during expansion or cloning (Roder et al., 1986). This
can be remedied by ftision of the lymphocyte cell line to mouse or human myeloma
cells (Thompson et al., 1986) and many human or mouse/human monoclonal antibodies
against RBC antigens are available as diagnostic reagents. These include anti-C, c, E, e,
G (Thompson et al., 1990); Jk", Jk^ (Thompson et al., 1991); H, K, Le' and Le^
(Mollison et al., 1997, p86) and a human monoclonal IgM antibody against Rh(D) has
been in use world-wide as a grouping reagent for many years (Thompson et al., 1992).
There have been many studies comparing the performance of monoclonal and
polyclonal reagents in blood typing assays (Kemper and Sazama, 1994; McGowan et al.,
1989; Messeter et al, 1984; Lowe et al., 1984; Sacks and Lennox, 1981) and, on the
whole, monoclonal antibodies work as well as traditional polyclonal antisera, although
there have been some anomalies (Beck et al., 1993; Scott and Voak, 1994). Monoclonal
antibodies have several advantages over polyclonal reagents (Mollison et al., 1997,
p246; Spivey, 1991) as follows:
-Better quality reagents that give stronger reactions in shorter reaction times.
-Stable and well defined reagents.
-Economical to produce in large scale.
-Unlimited supply of identical material.
-No contaminating antibodies to cause false positive results.
-Free from viruses (e.g. Hepatitis and HIV).
-Obviate the need for plasmapheresis donors.
However disadvantages with monoclonal antibodies include;

-Specificity varying with concentration.
-Unexpected reactions (e.g. potent anti-B reacts with group A cells and vice versa; Beck
etal., 1993).
-Potent monoclonal antibodies need to be diluted to avoid unexpected reactions and
these low levels of antibody can result in small agglutinates which cause the strength of
agglutination reaction to be underestimated (can be overcome by blending with a lower
avidity MAb of the same specificity; Scott and Voak, 1994).
-Failure to detect some variants of an antigen; many polyclonals although displaying a
single blood group specificity are actually a collection of antibodies that bind to
different epitopes on the same antigen. Monoclonal antibodies by definition bind to a
single epitope and may not give a positive reaction to the blood group antigen if a
particular individual lacks that epitope.
The latter problem has been overcome in some cases by the use of blended monoclonals
to ensure detection of all epitopes on the antigen. Despite the successful production of
some human monoclonal antibodies to RBC surface antigens, the process is still a
difficult one due to low transformation and fusion efficiencies, slow growth, and
instability of EBV produced clones and heterohybridomas (Siegel and Silberstein, 1994;
Olsson et al., 1983). As a result, more reliable and cost effective methods are being
sought. These new methods employ recombinant DNA technology to utilise human
antibody genes for antibody modification and expression of so called 'man-made' or
recombinant antibodies (Winter and Milstein, 1991).
1.6 Recombinant Antibodies
V^ domain
(••ISkDa) Fab scFv
-55 kDa) i: -28 kDa)
V^ domain
1-15 kDai Bis-scFv Dia body
(bisp©::ifici ibispecific)
(-55 kDa) (-50 kDa)
Fabg Triahody
(bi specific) (trivalent)
(-1 lOkDö) (-75 kDa)
Fab^ Minibody Tetrabody
(tri specific) (bivalent) itetravalent)
(-165 kDa) (-75 kDa) (-100 kDa)
Figure 1.27 Possible antibody formats used for cloning.

Formats include V domains (dAbs), Fabs consisting of light chain plus truncated
heavy chain, Fvs consisting of just the variable regions of both chains, and various
multi valent fragment combinations. The most common formats used in phage
display technology are Fab and scFv.
(Figure from Holliger and Hudson, 2005)
Just as hybridoma technology brought about a revolution in research and healthcare, so

too did gene technology which allowed antibody genes to be altered to fit a particular
purpose. In the first experiments, antibody genes were obtained from hybridoma cell
lines and expressed in mammalian cells as whole antibodies (Oi et al., 1983). However
it didn't take long for antibody fragments to become the focus of interest, as their
smaller size allowed expression in bacterial systems which were experimentally much
easier to use and more cost effective than mammalian systems (Cabilly et al., 1984).
Later work involved direct cloning of genes from lymphocytes of immunised animals.
Ward et al. (1989) expressed just the variable region of an antibody heavy chain (VH),
referred to as 'single domain antibodies' (dAbs), which were shown to have nanomolar
affinities for their respective antigen (lysozyme). One of the major problems associated
with the development of dAbs was the formation of aggregates when using E. coli
expression systems due to the hydrophobic interaction of the natural interface between
heavy and light antibody chains. The production of dAbs has had a recent resurgence as
a result of research into the prevention of hydrophobicity. This is achieved by the
addition of polyethylene glycol to the molecules or by genetic engineering to substitute
hydrophobic residues for ones likely to cause the molecules to be more soluble (Jespers
et al., 2004).
Direct cloning of genes from lymphocytes was also used to express antibody repertoires
as combinatorial libraries (Section 1.7). This recombinant technology allowed the
production of antibodies in many different forms. For example, human MAbs are more
useful as therapeutic reagents than foreign MAbs which can cause unwanted immune
responses, therefore murine antibodies have been 'humanised'. This involves utilising
framework regions from a human MAb and inserting CDRs from a murine MAb to
form a chimeric antibody (Morrison et al., 1984). Other applications are best served by
the absence of the Fc effector region, giving molecules referred to as antibody
fragments, the smallest of which are dAbs as previously mentioned. Other VH only
antibodies have been derived from camelids (camels and llamas) and fish (Wobbegong
and nurse sharks) where they naturally occur without a light chain (Hamers-Casterman
et al., 1993; Streltsov and Nuttall, 2005). However most antibody fragments have both
heavy and light chains and can be expressed separately in bacteria where they assemble
into Fv or Fab fragments. Fab fragments consist of the entire light chain and the
variable region and first constant region of the heavy chain giving a molecule of
-50,000 Da. Chains are disulphide linked as in the whole antibody conferring increased
stability.
Fv fragments contain only the variable regions of the antibody chains and therefore are
about half the size of Fabs. A popular format for antibody fragment expression is single
chain Fv where the variable domains are held together by a polypeptide linker which
can confer greater stability (Huston et al., 1988). Linking of antibody domains has
allowed the production of many diverse fragments including trimers (Pei et al., 1997) or
tetramers (Power et al., 2003), where three or four Fvs are linked together. This
technology has also generated bispecific fragments where two Fabs or Fvs are joined,
and each arm of the molecule has a different specificity (Holliger et al., 1993).
1.7 Library Construction
Pre-B cell Beeil Plasma/memory celi
Synthetic Naive Immune
V-gene Unrearranged Rearranged V-genes Rearranged V-genes

V-gene segments from IgM pool from I g G pool
source
Contents Controlled Uncontrolled Uncontrolled

1
Repertoire Once New repertoire for

construction (Single-pot) every antigen
Depending on library size: Biased for high affinity

Affinity of ^M from standard size repertoire (nM; if antigen Is
antibodies
nM from very large repertoire (10^°) immunogenic)
Specificity Any Originally biased Immunodominant epitopes,

against self biased against self
Figure 1.28 Comparison of antibody library types; synthetic, naïve (non-immune)

and immune.
(Figure from Hoogenboom, 1997)
There are two main types of antibody library which can be constructed; immune and
naive (non-immune), which involve obtaining antibody repertoires from immunised or
non-immunised individuals, respectively. Immune libraries will contain affinity
matured antibodies to the antigen of interest, but are of little use for isolating antibodies
of any other specificity. Naive or 'single pot' libraries are designed to circumvent the
need to generate a new immune library every time a different antibody specificity is
required. Usually, naïve libraries need to be much larger than immune ones in order to
isolate antibodies of a similar affinity but can be used to isolate a wider range of
specificities. For example, de Haard et al. (1999) generated a large naive Fab library
(3.7 X clones) and isolated nanomolar affinity antibodies to six different antigens.
High affinity antibodies have been isolated from immune libraries containing -10^ to
10^ members whereas naïve libraries of a similar size only yielded micromolar affinity
antibodies (reviewed by Aujame et al., 1997). Typically, libraries of 10^ to 10^ clones
yield affinities up to 10 nM but libraries with members give affinities up to 0.1 nM
(Hoogenboom, 2005).
Naive libraries may also be generated from semi-synthetic and synthetic human V genes.
Semi-synthetic libraries utilise a germ line antibody sequence for the framework and
library diversity is created by randomisation of one or more of the CDRs (Hoogenboom
and Winter, 1992; Barbas et al., 1992). Fully-synthetic libraries have been constructed
by assembling PGR generated fragments to form antibody chains (Nissim et al., 1994).
Synthetic libraries have the advantage of not being affected by the immune history of
the B cell donor and can be used to isolate antibodies which are difficult to obtain in
vivo such as those specific for self-proteins or toxic antigens.
Library construction methods used in this project were based on techniques developed
by Hoogenboom (de Haard et al., 1999). Antibody genes from human B cells were
PGR amplified and cloned into a phagemid vector as g3p fusions. The PGR primers
used were designed to give efficient amplification of all commonly used V-gene
segments and products were ligated into pGESl phagemid vector (de Haard et al.;
Figure 1.31). A two step cloning procedure was used to maximise cloning efficiency;
heavy and light chain PGR products were first cloned separately as single chain libraries
and then combined by restriction fragment cloning to form Fab libraries. This is
believed to be a more efficient method of library construction than direct cloning of
digested PGR products. Gloning of Fab fragments rather than scFvs was chosen to
avoid problems with screening. Phage displayed scFv fragments frequently aggregate
to form dimers or higher order multimers making isolation of high affinity antibodies
more difficult. Fabs also tend to be expressed at lower levels on phage than Fvs, further
reducing the likelihood of avidity effects. In this system, secretion of Fab chains is
directed to the cytoplasm where heavy and light chains can pair.
de Haard et al. (1999) aimed to generate a very large, non-immunised library from
which they could isolate antibodies against a wide range of antigens. To this end they
used an IgM specific constant region PGR primer during library construction. This
strategy was intended to isolate a naive antibody library which wasn't biased towards
antigens to which the donor had recently been exposed. Antigen exposure results in
activation and proliferation of B cells most of which will be producing IgG type
antibody. In addition to this, activated B cells contain many more mRNA molecules
than non-activated cells making it more likely that recently generated antibodies would
be highly represented in the resulting library. Thus, targeting IgM antibodies results in
the isolation of a less differentiated antibody repertoire. In contrast, the aim of this
project was the generation of an immune library with the intention of recovering the
original antibody pairings present in the B cell population. For this purpose, an IgG
specific constant region primer was used to select for IgG subclass antibodies rather
than IgM.
Generation of an antibody library containing millions of members necessitates a

'sorting' method to find antibodies of interest. This library screening is achieved by
antibody display systems of which there are three major types; phage display, cell
surface display and ribosome display (Figure 1.29). The most widely used of these
techniques is phage display which was the approach chosen for this project.
Figure 1.29 Antibody display systems.

om A: Phage display. Antibody is
A Phageiiiid displayed by fusion with
bacteriophage coat protein (pIII).
Antibody B: Cell surface display. Antibody
gene is maintained on a plasmid and
B
expressed on the cell surface.
C: Ribosome display. Antibody
mRNA is translated but the
transcript is not released from the
ribosome.
mRNA (Figure from Maynard and Georgiou,
0 « Ribosome
2000).
P^ --Antibody
1.8 Phage Display Technology
Phage display is a powerful technique that allows the selection of a particular molecule
from a library of million or even billions of different proteins or peptides. The
foundation for the technology was laid by Smith (1985) who inserted foreign DNA into
a filamentous phage genome such that the gene product was expressed (or displayed) on
the surface of the phage particle. Parmley and Smith (1988) coined the term 'panning'
for selection of library clones of interest by exposure to immobilised ligand.
Minor coat Wajor coat

./1 protein g6p protein g8p Minor coat
'Displayed'
protein g9p
protein
fused to g3p
N Minor coat
protein g7p
Figure 1.30 Diagram of M13 bacteriophage.

The particle is rod shaped (-6 x 860 nm) and consists of a major coat protein g8p
of which there are -3000 copies per particle and up to 5 copies each of four minor
coat proteins. These proteins form the viral capsid which encloses the single-
stranded circular DNA of the phage genome. Fusion phage can be made by
inserting foreign DNA into the phage genome adjacent to the coat protein gene and
the gene product is co expressed with the viral host protein. Fusions with g8p have
been made which result in the display of many antibodies along the length of the
phage particle. A more common strategy is fusion to g3p protein as shown, which
results in fewer copies of antibody displayed on each particle.
(Figure adapted from Smothers et al., 2002)
Bacteriophages are viral particles which infect bacteria, first described in the early
1900's (Encyclopaedia Britannica, 2006). There are many different types and particular
strains (such as M l 3 and lambda) have proved to be useful tools in molecular biology in
the form of phage or phagemid vectors. Phage consist of a relatively simple genome
(for example, in Ml3 has single stranded circular DNA) surrounded by a coat, or capsid,
consisting of few different proteins. In Ml 3, the most frequently occurring protein
(-3000 copies per particle) is g8p (the product of phage gene 8) referred to as the major
coat protein which makes up the majority of the capsid (Figure 1.30). Four other minor
coat proteins are also present at approximately 5 copies per phage; g3p, g6p, g7p and
g9p which form the ends of the particle.
Aiwtf x/KJf ASCI sta Bsma ifou

1 VL CL I I VH CH1 4
PlacZ ' i giti
rbs g CK rbs S CH1 t %
^ € o l E 1 ori ^ AMP' M13 interg^nic regi-o^
Phagemid vector pCES1
Figure 1.31 Phagemid vector pCESl.

Vector (left) contains E. coli and M13 origins of replication, antibiotic selection
gene, phage gene III and a multiple cloning site for insertion of antibody genes.
Addition of helper phage allows packaging of the phagemid into phage particles
with Fab fragments displayed as a g3p fusion (right).
(Figure from Hoogenboom et al., 1998a)
Early library construction experiments used phage vectors which carried all of the genes
required for viral assembly, but these were soon replaced by phagemid vectors which
were better suited for generating large repertoires. Phagemids are small plasmid vectors
which utilize some phage genes and contain both phage and bacterial origins of
replication (Figure 1.31). This allows them to replicate in E. coli and also be packaged
as phage particles. They contain restriction sites to allow insertion of foreign DNA
adjacent to a phage coat protein gene (usually g3p although g9p has been used
successfully for library construction; Gao et al., 2002). When the phagemid is packaged
into phage particles, the foreign DNA is expressed along with the coat protein and is
displayed on the surface of the phage. A recombinant phage particle contains both the
DNA sequence and the protein product of that DNA and it is this link between genotype
and phenotype that is the key to the technique.
The use of phagemid vectors which do not contain all the genes required for viral
assembly (rather than phage vectors) necessitates the use of helper phage for production
of phage particles. The helper phage (such as M13K07: Vierra and Messing, 1987)
provides all of the missing phage proteins, including g3p, and allows phagemid DNA to
be packaged into viral particles displaying the g3p fusion protein. This process of
conversion from phagemid to phage is referred to as 'phage rescue' and is a key
component in the biopanning process. The helper phage itself has a defective origin of
replication and therefore competes poorly with the phagemid for packaging into viral
particles. Theoretically it is possible for a phage particle to carry between zero and five
antibody molecules on its surface. This is a disadvantage as multivalent phage will
appear as very strong binders due to the effect of avidity, but once the antibody is
expressed as a monomer, its affinity may turn out to be much poorer. Use of helper
phage reduces this problem as helper g3p competes with g3p-antibody fusion for
incorporation into the particles and results in a population with an average of less than
one antibody per phage particle, referred to as 'monovalent phage' (Lowman et al.,
1991).
The impact of Smith's technique of generating 'fusion phage' was not recognized
initially as he put it forward as a 'simple way of cloning a gene when an antibody
against the product of that gene is available'. The real power of the technique was
revealed five years later when he went on to generate a library displaying 4x10^ unique
hexapeptide epitopes and screened via 'tight binding' to an antibody of interest (Scott
and Smith, 1990). Smith wasn't the only one to see this potential as another two groups
published similar studies in the same year (Devlin et al., 1990; Cwirla et al., 1990) with
Devlin et al. generating a 15-mer peptide library containing 2x10^ clones which was
used to isolate peptides binding to streptavidin. At this time there was much skepticism
about whether larger molecules could be displayed on the surface of phage (Rader and
Barbas, 1997) but the doubters were soon silenced.
Figure 1.32 Colony lift screening of antibody library clones.
Library clones are grown on antibiotic selection plates and overlaid with either
DNA or protein binding membranes. Membranes are then probed for positive
clones either by using a labeled oligonucleotide (as in this example) or antigen
probe respectively.
Filter a: Positive anti-lysozyme clones after one round of affinity purification
(biopanning) out of a total of 900 colonies on the plate. Filter b; Positive clones
after a second round of biopanning out of a total of 372 colonies on the plate.
(Figure from McCafferty et al., 1990)
The first antibodies from a combinatorial library were isolated by Huse et al. (1989)
from the Scripps Institute in the USA, who used a bacteriophage lambda vector system
to construct a library of 2.5 x 10 clones and isolate murine Fab fragments against the
hapten, p-nitrophenyl phosphonamidate. Clones were screened using a 'colony lift'
assay where plated colonies were overlaid with protein-binding membrane and the
membranes probed for antigen binding. Using this method 10^ to 10^ clones could be
screened per day but the screening technique was cumbersome and not suitable for
larger repertoires.
Soon afterwards, McCafferty et al. (1991) from Cambridge in the UK, effectively
combined antibody cloning with Smith's work on peptide display and thereby invented
phage display technology. They proved the principle of the technology by expressing
functional alkaline phosphatase on the surface of phage (McCafferty et al, 1991) and
also produced a single chain antibody against lysozyme (McCafferty et al., 1990). The
50
proteins were displayed as a g3p fusion using an fd-CATl phage vector (based on fd-tet
filamentous phage cloning vector produced by Zacher et al, 1980) and positive phage
were enriched by affinity chromatography (in the latter case using a lysozyme sepharose
column) then analysed by phage ELISA and colony screening (Figure 1.32).
Enrichment of the library for the phage of interest (using Clackson et al., 1991 as an
example; from undetectable levels before biopanning, to 13 % then 93 % of the total
phage after two rounds) meant that only a small proportion of clones needed to be
screened in order to find the positive binders (Figure 1.34).
In the same year, Bass et al. (1990) applied Smith's peptide display method to the
production of 'hormone phage'. Their aim was to display human growth hormone
(hGH) on the surface of phage and use biopanning to isolate new receptor binding
mutants. However, they had noted from the literature that in some cases it had proved
difficult to isolate high affinity peptides from libraries, or to separate moderate from
high affinity binders (Cwirla et al., 1990) and that this had been attributed to the
multivalency of phage particles. They solved this 'chelate effect' problem by
assembling particles with their g3p fusion protein under tight transcriptional control
using a novel phagemid vector along with a controlled amount of normal g3p from
M13K07 helper phage. Using this technique they estimated that about 10 % of phage
particles contained one copy of the hGH-glll fusion protein and were able to distinguish
between a mixture of two variant hormones with 7.1 and 0.34 nM Kd receptor binding
affinities after one round of biopanning.
QdritlGnii
HuRTfeAfi BhCAili^ f r m natvft
or immun« ctonors
ifY iîtfO
VH (Of VMCH1) VL (or VLCL)
Gffm and
f tf/fift/fl^ OP S&spwfee fi/oflJ/ig
Immune. Naive
Of SynifHpAlc
Display Aivtibody Library
vector (rf Hurtpn sinftiborfy
J lragm«nftB
/nffocÛiEip EcwTr
fffiSCili
Figure 1.33 Construction of a phage display antibody library.

Antibody variable region genes are PCR amplifled from B cells and cloned into a
phagemid vector in frame with the g3p gene. The phagemid library is transformed
into E. coli and phage particles rescued by infection with helper phage. This
results in a library of antibodies displayed on phage ready for the biopanning
process.
(Figure from Hoogenboom et al., 1998a)
The following year both the Scripps and Cambridge groups developed phagemid
vectors and used them to isolate phage antibodies, although their approaches differed.
Phagemid vectors were thought to be better than phage vectors for this purpose as the
former have much higher transformation efficiencies (by 2-3 orders of magnitude)
allowing construction of larger repertoires (Hoogenboom et al., 1991). The Scripps
group produced Fab antibody fusions with the major coat protein, g8p, resulting in
phage with multiple copies of antibody along the length of the particle (Kang et al.,
1991). It is interesting to note that they put this multivalency forward as an advantage
of the technique as the resulting strength of antibody binding due to avidity effects
allowed 'the capture of a wider range of affinities'. Multivalent phage are now viewed
as disadvantageous since the aim of most library construction is the isolation of high
affinity antibodies, and the group soon transferred to a g3p fusion methodology
'complementing their g8p approach' (Barbas et al., 1991).
Phsge antibody Iftrary
Prepare pha<g& partldes
Seiection Amplify bacteria
Jncyi3>a(te with Antigen

Wash
Cycle
/
Roinf&ci sclcctcd
phages
Etule
0& Anáiy^ moricpclofi^l ^fe

ai ajilitxidifrB
Throw away non binders
Figure 1.34 The phage display biopanning cycle.
Library phage are exposed to antigen, phage carrying a specific antibody bind and
non-specific phage are washed away. Specific phage are eluted, reinfected into E,
coli and grown on large antibiotic selection plates (re-amplification). Phage are
rescued from the plated colonies, re-exposed to antigen and the panning cycle is
repeated. Typically 3 or 4 rounds of biopanning are carried out and colonies from
the latter rounds are screened for positive clones by phage ELISA.
(Figure from Hoogenboom et al., 1998b)
The Cambridge group continued their g3p fusions but with single chain Fv display
(Marks et al., 1991), as well as making Fab libraries (Hoogenboom et al, 1991). They
generated a library of human single chain Fv antibodies by PGR amplifying genes from
unimmunised donor lymphocytes (referred to as a naive library) and cloning them into a
phagemid vector as linked heavy and light chain variable regions. Specific antibodies
were isolated against lysozyme, bovine serum albumin and a hapten; 2-phenyloxazol-5-
one. The Fab library was constructed in a similar way, however the heavy and light
chains were not linked, but expressed separately. This was believed to generate more
diversity as the heavy and light chains could recombine randomly into Fab fragments in
the bacterial periplasm. The other difference in approach was in the screening
technique. Unlike the Scripps researchers, Marks et al. moved on from 'colony lift'
screening and developed a phage ELISA which is still widely used. In this method,
single colonies are picked from an agar plate and grown up as individual cultures in a
96-well plate. Phage are rescued from these cultures and assayed for antigen binding
activity by ELISA (Figure 1.35).
Substrate (ABTS)
Colour development
Figure 1.35 Phage ELISA.
Antigen is immobilised on to a solid phase
(in this example, Fy^ extracellular
< — Anti-M13 Conjugate domain). Phage particles are incubated
with antigen and those carrying specific
Fab are retained after extensive washing.
Bound phage are detected using an
enzyme conjugate which binds to phage
< — Antigen specific Fab coat protein and a chromogenic substrate.
1.9 Biopanning Strategies
3 Immobilized Ag f Cells displaying k In vivo selection

> antigerVinternalizing Ag
JT
b Biotinylated Ag 9 Alternating selections I Trypsin digestion

on Ag+/- cells
I
C Recombinant Ag h Subtractive cell sorting m Mild reduction

w (release phage only)
•sSS
d Bacteria displaying Ag Tissues with Ag n Mild reduction

(release phage +Ag)
^ AA
0-Si
G Subcellular fractions J Proxirnity to other Ag 0 Competitive elution
I a
Figure 1.36 The wide range of approaches to antibody selection from libraries.
Antigen (Ag) is represented as red triangles. Biotin is represented as orange
circles and streptavidin coated beads as a purple cross. Blue circular and oval
shapes represent liposomes and immunoadhesins respectively. Figure h represents
flow cytometry results with the population of selected cells gated inside the red
circle. Figure i represents a tissue section prepared on a slide. Other antigen in
Figure j is represented in pink. Figure k illustrates direct injection of a library
into a living animal. Figures 1 to o illustrate phage elution conditions with the
antibody shown in orange and the linked phage in purple. SS represents
disulphide bonds. See text for further explanation and citations.
(Figure from Hoogenboom, 2005)
There are many different approaches to phage display biopanning (reviewed by
Bradbury and Marks, 2004a; Hoogenboom, 2005), the most common being the use of
soluble antigen which is immobilised to a solid support (Figure 1.36, a). A possible
drawback of this approach is that the antigen may be denatured upon immobilisation
and not present the correct epitopes for antibody selection. However, this is not very
common and can be overcome by indirect coating using an antigen specific ligand.
Alternatively the antigen can be labelled, typically by biotinylation, and biopanning
carried out on a solid phase (Figure 1.36, b) or in solution. After phage binding in
solution, the antigen is recovered by binding to streptavidin coated beads. Antibodies
may also be isolated to streptavidin but this can be avoided by alternating the recovery
step using avidin, which has a different structure although its function is the same. The
concentration of antigen is more easily controlled in solution phase biopanning and this
can be important as higher affinity antibodies can be selected for by the use of low
antigen concentrations.
If soluble antigen is not available, as in the case of many membrane proteins that lose
their native conformation upon extraction, then whole cells carrying the antigen can be
used for biopanning (Figure 1.36, d-f). This is always a more difficult technique as
cells will invariably carry many other epitopes along with the ones of interest. In
addition, the wash steps are more labour intensive as cells are centrifuged and
resuspended each time. Isolation of specific phage from cell based biopanning is
usually achieved by a subtraction biopanning step (Figure 1.36, g). In the subtraction
method, library phage are pre-incubated with cells that are as similar as possible to the
antigen carrying cells but are antigen negative. In this way, phage are depleted that
would bind specifically to the carrier cells and be eluted along with the antigen specific
phage making it more likely for the antibodies of interest to be isolated. Figure 1.36, f,
shows antigen intemalisation which is useful for isolating antibodies to be used in
targeted therapeutics such as tumour specific immunotoxins. Phage bind to cell surface
receptors, are internalised and then recovered from inside the cells for use in the next
round of biopanning.
In some methods of cell based biopanning, the cell/phage complex is fiuorescently

labelled and positive clones are isolated by FACS (fluorescence activated cell sorting;
Figure 1.36, h). Other biopanning methods involve the use of tissue sections as a source
of antigen or even in vivo tissue selection v^here phage are injected into a mouse and
then tissues recovered to look for specifically bound phage (Figure 1.36, i and k). The
standard method for recovering bound phage after exposure to antigen and washing to
remove non-specific phage is to use high or low pH to disrupt the Ab/Ag interaction.
Figure 1.36,1 to o, illustrates other methods of phage recovery which include
proteolytic digestion of the polypeptide chain between Ab and phage to release the
phage particles. Other methods involve the use of disulphide bonds which can be
reduced to release either the phage or the whole phage/Ab/Ag complex, or the phage/Ab
complex can be competed off the antigen by introduction of an antigen specific
antibody.
Figure 1.37 Ludwik Hirszfeld Institute of

Immunology and Experimental Therapy logo.
Ludwik Hirszfeld was a renowned Polish
microbiologist and serologist.
The best chance of recovering specific antibodies from

phage display biopanning is to use soluble antigen
(Bradbury and Marks, 2004a). Unfortunately, soluble
antigen is rarely available when biopanning against
RBC surface antigens. As mentioned previously, many
of them have multiple transmembrane domains making it impossible to maintain the
protein conformation when isolated from the membrane. Among the antigens of
interest in this project, the Duffy glycoprotein has 7 transmembrane domains, Kell has a
single domain, Kidd has 10 and the Rhesus glycoproteins have 12. As previously
mentioned (Section 1.2), RBC antigens are carried on the extracellular domain of the
RBC protein. Since many of these proteins have been sequenced, it is possible to use
gene technology to clone and express just the extracellular domain of the protein. This
was achieved for the Duffy protein by Wasniowska et al. (2000) from the Ludwik
Hirszfeld Institute in Wroclaw, Poland (Figure 1.37), who expressed the amino terminal
extracellular domain of the Duffy antigen from amino acids 3 to 60. Several samples of
this polypeptide used in this project were a gift from Wasniowska et al.
Soluble antigen was not available for any of the other antigens of interest in this project
and cell based biopanning was the only alternative. As mentioned, RBCs have a
complex surface structure and carry many antigens. Biopanning against cells carrying
just one RBC antigen may be advantageous in isolating specific antibodies.
Yazdanbakhsh et al. (2000) generated cell lines transfected with RBC antigens
including Duffy, Kell and Kidd antigens. They used these cell lines for immunisation to
generate antibodies and also in identifying complex mixtures of irregular antibodies in
human serum samples. Several human embryonic cell lines transfected with RBC
surface antigens used in this project were a gift from Marion Reid at the New York
Blood Centre. These samples were characterised by flow cytometry and used for
panning. Whole RBCs from a typed panel (CSL Phenocell Panel) were also used for
panning.
As well as biopanning the immune library generated in this project, a large naive library
was also obtained for biopanning experiments against the same purified and cell based
antigens. This library was obtained from Cambridge Antibody Technology in the UK
and consisted of 1.3 x lO'^ individual single chain Fv clones (Vaughan et al., 1996).
Antibody variable genes for library construction were sourced from 43 non-immunised
human donors. The repertoire was constructed in the phagemid vector pCantab 6 (a
derivation of pUCl 19) and was shown to be highly diverse by clone fingerprinting
techniques. Expression of soluble scFv antibody from the library gives rise to fusion
protein products consisting of antibody plus hexahistidine and myc tags and Vaughan et
al. (1996) isolated sub-nanomolar affinity antibodies from the library.
1.10 Phage Displayed Antibodies to Blood Group Antigens
The first phage displayed human antibody fragments against blood group antigens were
isolated by Marks et al. in 1993. The library was constructed from peripheral blood
lymphocytes of two unimmunised donors and scFv antibodies were obtained to ABO
(B), Rh (D and E) and Kell (Kp^) antigens. However antibody affinities were relatively
low and the antibodies did not recognise weak B or D variant RBCs (Hughes-Jones et
al., 1994). Subsequently several groups pursued the isolation and characterisation of
anti-D antibodies by phage display (Dziegiel et al., 1995; Tonye Libyh., 1997; Siegel et
al., 1997; Miescher et al., 1998; Furata et al., 1998; Hughes-Jones et al., 1999). The
high level of interest in the production of anti-D antibodies arose from the difficulties in
producing murine antibodies of this specificity and also the demand for antibody
generated by anti-D prophylaxis for HDN. One of the groups also isolated high affinity
Fab antibodies to M and N antigens from a murine library (Czerwinski et al., 1999) and
more recently, anti-A and anti-B Fab and Fv antibodies have been isolated from human
phage libraries (Chang and Siegel, 2001; Santos-Esteban and Curiel-Quesada, 2001).
Recombinant antibodies have the potential to replace polyclonal human antibodies in

RBC phenotyping assays. Harvesting of polyclonal human antibodies is complicated by
ethical and practical issues since immunising individuals against RBC antigens can have
adverse effects on health and is a costly process with inherent variability. Recombinant
antibody technology offers a consistent, unlimited supply of cost effective reagents.
Hence the aim of this study is to isolate recombinant antibodies to clinically significant
RBC surface antigens which could be used in routine blood grouping and phenotyping.
The desired specificities include antibodies to antigens in the Rhesus, Duffy, Kell and
Kidd blood groups systems. To date, no high affinity antibodies to Duffy, Kell or Kidd
antigens have been isolated by recombinant methods.
2 Materials and Methods
2.1 Biological Reagents
2.1.1 Library Primers
Oligonucleotide primers for library construction were synthesised by Gibco Life

Technologies. Sequences for primers were according to de Haard et al. (1999) except
for IgG specific primer which was from Marks et al. (1993; see Appendices 3 and 4 for
primer sequences).
2.1.2 PGR Screening Primers

Primers for PGR screening of clones were designed in house (P. Sturgess, personal
communication, UNSW) and synthesised by Invitrogen (Life Technologies; see
Appendix 1 for primer sequences).
2.1.3 E. coli XL 1 Blue Bacterial Stock

E. coli XL 1 Blue (Bullock et al., 1987) bacterial stock was obtained from stocks held in
the School of Biotechnology and Biomolecular Sciences (SBBS), University of New
South Wales (UNSW).
Phenotype:
recAl endAl gyrA96 thi-1 hsdR17(rk[+], mk[+]) supE44 relAl lac [F'proAB
lacPZAMlS TnlO (Tef)]
2.1.4 E. coli TGI Bacterial Stock

E. coli TGI bacterial stock was obtained from stocks held in SBBS, NSW.
Phenotype:
F' traD36 lacPA (lacZ)M15proA^B^/supE A (hsdM-mcrB)5 (rk'rrik'McrB') thi A (lac-
proAB)
2.1.5 Peripheral Blood Mononuclear Cells
Peripheral Blood Mononuclear Cells (PBMC) expressing the antibodies of interest and
their concomitant serum samples were obtained from stocks held in the Department of
Cell Biology at the ARCBS-NSW (Table 2.1).
Table 2.1 Peripheral blood mononuclear cells expressing antibodies of interest

shown by ARCBS typing assays to be present donor and patient serum samples.
The blood group systems of interest to which the antigens belong are shown in
brackets (*see Section 3.3.2 for an explanation of Saline lAT).
PBMC Sample Antibodies found in corresponding serum

sample (other than ABO System).
Donor 1 D, C, E, Fy'(by Saline lAT*)
Donor 2 D, C, Fy'(by Saline lAT*), PI
Donor 3 D, C,K
Donor 4 D, C, E, Fy^
Donor 5 K (by Saline lAT*), M
Patient 1** E, Jk", K.
Patient 2 F/
Patient 3 Jk^
Patient 4 C
** Cell line could not be maintained, no RNA obtained.
2.1.6 Helper Phage

M13K07 Helper phage was obtained from stocks held in SBBS, UNSW.
2.1.7 Recombinant Fy^ Extracellular Domain Fusion Polypeptide

Recombinant Fy^ extracellular domain expressed as a fusion with Glycophorin A
supplied as a freeze dried powder was a gift from the Ludwik Hirszfeld Institute of
Immunology and Experimental Therapy in Wroclaw, Poland.
2.1.8 Naïve Library
A large naïve library (1.3 x lO'^ individual recombinants) of single chain Fv fragments
displayed on phage was obtained from Cambridge Antibody Technology, UK. The
library was supplied as caesium chloride gradient purified phage particles at a
concentration of 2 x phage/mL, ready for biopanning.
2.1.9 pCESl Phagemid Vector

pCESl vector was obtained from stocks held in SBBS,IJNSW. The vector was a gift
from Prof. H. Hoogenboom, Department of Pathology, Maastricht University, The
Netherlands (see Appendix 2 for full sequence).
2.1.10 Transfected Human Embryonic Kidney 293 Cell Lines

Human embryonic kidney (HEK) 293 cell lines transfected with human red blood cell
(RBC) surface proteins (Table 2.2) were a gift from Marion Reid at the New York
Blood Centre, USA.
Table 2.2 Human embryonic kidney 293 cell lines transfected with human RBC
surface proteins.
Cell Line Antigen Expressed
293T.Vector5 Control cell line transformed with
'empty' vector (no antigens present).
293T.Fya.4 F/
293T.Fyb.9wt Fy"
293T.Kellwt.82 Wild type Kell allele expressing k, Kp^
and Js^ antigens.
293T.K1.41 K1
293T.Kpa.69 Kp^
293T.Jsa.lO Js^
2.1.11 Epstein Barr Virus

Epstein Barr Virus was obtained from stocks held in the Department of Cell Biology at
the ARCBS-NSW.
2.2 Bacterial Culture Media
All autoclaving was carried out at 15 Ib/sq.in for 20 min on general purpose, fluids
cycle.
Milli-Q water is purified water from the Millipore Milli-Q system and RO water is
purified water from the Millipore reverse osmosis system.
LB (Luria-Bertani) Media
10 g tryptone (Oxoid), 5 g yeast extract (Oxoid) and 10 g sodium chloride (Univar -
analytical grade) were dissolved in 1 L Milli-Q water and autoclaved.
LB Amp Media
Ampicillin stock (Section 2.3) was added to autoclaved, cooled (-60 °C), LB Media at a
ratio of 1:1000 to give a final concentration of 100 jig/mL.
LB Amp Agar Plates

Before autoclaving, agar (Agar Technical, No.3, Oxoid) was added to LB media at 15
g/L. Ampicillin stock (Section 2.3) was added to autoclaved, cooled (-60 °C), LB agar
at a ratio of 1:1000 to give a final concentration of 100 jig/mL. Solution was poured
into 90 mm Petri dishes (Sarstedt), allowed to cool completely and dried in a laminar
flow hood.
LBTet Agar Plates

LB media was made as described. Before autoclaving, agar (Agar Technical, No.3,
Oxoid) was added to LB media at 15 g/L. Tetracycline stock (Section 2.3) was added to
autoclaved, cooled (-60 °C), LB agar at a ratio of 3:1000 to give a final concentration of
30 jig/mL. Solution was poured into 90 mm Petri dishes (Sarstedt), allowed to cool
completely and dried in a laminar flow hood.
Minimal Agar Plates

M9 minimal salts (Sigma) were dissolved in Milli-Q water at 56.4 g/L and autoclaved to
give 5X stock. Thiamine hydrochloride (Calbiochem) was dissolved in Milli-Q water at
10 mg/mL and sterilised by filtration through a 0.22 ^m syringe driven filter unit
(Millipore, Millex GP). 15 g agar (Agar Technical, No.3, Oxoid) was added to 750 mL
Milli-Q water, autoclaved and cooled to -60 °C. To the cooled agar was added; 200 mL
5X M9 salts, 10 mL 40 % w/v glucose, 1 mL 1 M magnesium chloride (Section 2.3)
and 0.5 mL thiamine hydrochloride 10 mg/mL stock and the solution was made up to 1
L with sterile Milli-Q water. Solution was poured into 90 mm Petri dishes (Sarstedt),
allowed to cool completely and dried in a laminar flow hood.
SB Media
30 g tryptone (Oxoid), 20 g yeast extract (Oxoid) and 10 g MOPS (3-[N-
Morpholino]propane-sulphonic acid >99.5 % by titration, Sigma) were dissolved in 1 L
Milli-Q water and autoclaved.
SBTet Media
Tetracycline stock (Section 2.3) was added to autoclaved, cooled (-60 °C), SB media at
a ratio of 3:1000 to give a final concentration of 30 jj,g/mL.
SOB Media
analytical grade) were dissolved in 1 L Milli-Q water and 2.5 mL/L 1 M potassium
chloride (Section 2.3) was added before autoclaving. After autoclaving, 10 mL/L 1 M
magnesium chloride (Section 2.3) was added.
SOBAG Agar Plates

SOB media was made as described. Before autoclaving, agar (Agar Technical, No.3,
Oxoid) was added to LB media at 15 g/L. After autoclaving, 10 mL/L 1 M magnesium
chloride and 100 mL/L 40 % glucose were added (Section 2.3). Ampicillin stock
(Section 2.3) was added to autoclaved, cooled (-60 °C), SOB agar at a ratio of 1:1000
to give a final concentration of 100 |ig/mL. Solution was poured into 90 mm or 150
mm Petri dishes (Sarstedt), allowed to cool completely and dried in a laminar flow hood.
SOC Media
40 % glucose (Section 2.3) was added to SOB media at 10 mL/L to make SOC media.
2TY Media
analytical grade) were dissolved in 1 L Milli-Q water and autoclaved.
2TYAG Media
40 % glucose (Section 2.3) at 50 mL/L (final concentration 2 %) and ampicillin stock
(Section 2.3) at 1:1000 dilution (final concentration of 100 |ig/mL) were added to 2TY
media to make 2TYAG media.
2TYAG Agar Plates

2TY media was made as described. Before autoclaving, agar (Agar Technical, No.3,
Oxoid) was added to 2TY media at 15 g/L. 40 % glucose (Section 2.3) at 50 mL/L
(final concentration 2 %) and ampicillin stock (Section 2.3) at 1:1000 dilution (final
concentration 100 jig/mL) were added to autoclaved, cooled (-60 °C), 2TY agar.
Solution was poured into 90 mm Petri dishes (Sarstedt), allowed to cool completely and
dried in a laminar flow hood.
2TYAK Media
Ampicillin and kanamycin stocks (Section 2.3) at 1:1000 dilution (final concentration of
100 |xg/mL and 50 |ig/mL respectively) were added to 2TY media to make 2TYAK
media.
2TYKG Agar Plates

2TY media was made as described. Before autoclaving, agar (Agar Technical, No.3,
Oxoid) was added to 2TY media at 15 g/L. 40 % glucose (Section 2.3) at 50 mL/L
(final concentration 2 %) and kanamycin stock (Section 2.3) at 1:1000 dilution (final
concentration 50 |xg/mL) were added to autoclaved, cooled (-60 °C), 2TY agar.
Solution was poured into 90 mm Petri dishes (Sarstedt), allowed to cool completely and
dried in a laminar flow hood.
2.3 Buffers and Solutions
ABTS Substrate Solution

ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, Sigma) was dissolved at
1.1 mg/mL in 50 mM citric acid pH 4.0 and hydrogen peroxide (30 % w/w, Sigma) at 1
% v/v was added immediately before use.
Alkaline Lysis Solutions:

Stock solutions:
0.5 M glucose: 9 g D-glucose (anhydrous, Univar) was dissolved in 100 mL Milli-Q
water and sterilised by filtration through a 0.22 [im syringe driven filter unit (Millipore,
Millex GP).
1 M tris pH 8.0: 60.6 g tris (BDH biochemical grade) was dissolved in 450 mL Milli-Q
water, adjusted to pH 8.0 with conc. HCl and made up to a final volume of 500 mL.
Solution was sterilised by autoclaving.
0.5 M EDTA pH 8.0: See individual entry in this section.
10 M sodium hydroxide: See individual entry in this section.
10 % SDS: 10 g SDS (sodium dodecyl sulphate -Univar) was dissolved in sterile Milli-
Q water to give a final volume of 100 mL.
7.5 M ammonium acetate: 57.8 g ammonium acetate (Univar) was dissolved in Milli-Q
water to give a final volume of 100 mL and sterilised by filtration through a 0.22 jim
syringe driven filter unit (Millipore, Millex GP).
RNase A: 200 mg RNase (bovine pancreatic ribonuclease A, USB) was dissolved in 9
mL sterile Milli-Q water plus 1 mL 1 M tris pH 8.0 to give a final concentration of 20
mg/mL in 100 mM tris. Stored as aliquots at -20 °C.
Lysozyme: 100 mg lysozyme (Sigma) was dissolved in 9.9 mL sterile Milli-Q water
plus 0.1 mL 1 M tris pH 8.0 to give a final concentration of 10 mg/mL in 10 mM tris;
stored as aliquots at -20
Working solutions:
Soln I: Resuspension
2.5 mL 0.5 M glucose , 625 [iL 1 M tris , 0.5 mL 0.5 M EDTA , 2.5 mL lysozyme (10
mg/mL) and 50 [iL RNase A (20 mg/mL) were made up to 25 mL with sterile Milli-Q
water and chilled on ice before use.
Soln II: Lysis
5 mL 10 % SDS and 1 mL 10 M sodium hydroxide were made up to 50 mL with sterile
Milli-Q water.
Soln III: Neutralisation
40 mL 7.5 M ammonium acetate chilled on ice before use.
Ampicillin Stock
Ampicillin (Sigma) was dissolved in Milli-Q water at 100 mg/mL and sterilised by
filtration through a 0.22 jim syringe driven filter unit (Millipore, Millex GP). Solution
was stored as aliquots at -20 °C.
Carbonate Binding Buffer
Di-sodium carbonate (Univar analytical grade) at 1.59 g/L and sodium hydrogen
carbonate (Univar analytical grade) at 2.93 g/L were dissolved in Milli-Q water to give
a 50 mM carbonate solution at pH 9.5.
50 mM Citric Acid pH 4.0

Citric acid (Sigma reagent grade) at 96.06 g/L was dissolved in Milli-Q water and the
pH adjusted to 4.0 with 10 M sodium hydroxide solution.
6X DNA Gel Loading Dve

25 mg bromophenol blue (Sigma analytical grade) and 25 mg xylene cyanol (BDH
analytical grade) were dissolved in 7 mL sterile Milli-Q water, 3 mL glycerol (Univar
analytical grade) added and the solution thoroughly mixed by inversion.
0.5 M EDTA pH 8.0
186.1 g EDTA (ethylenediaminetetraacetate, Univar) and 15 g sodium hydroxide pellets
(Univar) were dissolved in 800 mL Milli-Q water and the pH adjusted to 8.0 with 10 M
sodium hydroxide solution. Solution was sterilised by autoclaving.
70 % Ethanol
Ethanol (absolute, Univar analytical grade) was mixed with Milli-Q water at 70 % v/v.
lOX FA Gel Buffer
20.93 g MOPS (3-[N-Morpholino]propane-sulphonic acid, Sigma), 2.05 g sodium

acetate (anhydrous, BDH AnalaR) and 1.86 g EDTA (ethylenediaminetetraacetic acid
di-sodium salt- BDH AnalaR) were dissolved in a final volume of 500 mL Baxter water
(water for irrigation, Baxter Healthcare). The buffer pH was adjusted to pH 7.0 using
10 M sodium hydroxide solution.
IX FA Gel Running Buffer

100 mL lOX FA gel buffer and 20 mL 37 % formaldehyde (Sigma) were made up to a
final volume of 1 L with Baxter water (water for irrigation, Baxter Healthcare) and
mixed.
40 % Glucose
Glucose (Univar analytical grade) was dissolved in Milli-Q water at 400 g/L by heating
in a 60 °C water bath and sterilised by filtration through a 0.22 |im syringe driven filter
unit (Millipore, Millex GP).
10 % Glycerol
Glycerol (Univar analytical grade) was mixed with Milli-Q water at 10 % v/v and then
autoclaved.
50 % Glycerol
Glycerol (Univar analytical grade) was mixed with Milli-Q water at 50 % v/v and then
autoclaved.
Kanamycin Stock
Kanamycin (Sigma) was dissolved in Milli-Q water at 50 mg/mL and sterilised by
filtration through a 0.22 |im syringe driven filter unit (Millipore, Millex GP). Solution
was stored as aliquots at -20 °C.
1 M Magnesium Chloride
Magnesium chloride hexahydrate (Univar analytical grade) was dissolved in Milli-Q
water at 203.3 g/L and sterilised by filtration through a 0.22 jim syringe driven filter
unit (Millipore, Millex GP).
5X PBS (Phosphate Buffered Saline)

Di-sodium hydrogen phosphate (BDH) at 27.3 g/L, sodium di-hydrogen phosphate
(BDH) at 8.0 g/L and sodium chloride at 45 g/L (BDH) were dissolved in Milli-Q water.
IX PBS (Phosphate Buffered Saline)

5X PBS stock was diluted 1 in 5 into Milli-Q water to give a pH (without adjustment) of
7.1 to 7.2.
PBS/BSA
BSA (bovine serum albumin, fraction V, Sigma molecular biology grade) was dissolved
in IX PBS at 1% w/v.
PBS/EDTA
EDTA (ethylenediaminetetraacetate- Univar) at 0.02 % w/v was dissolved in PBS and
sterilised by filtration through a 0.22 [im syringe driven filter unit (Millipore, Millex GP)
in a laminar flow cabinet.
PBS/Tween (PBST)
Tween 20 (BDH AnalaR) was added to IX PBS at 0.1 % v/v and mixed thoroughly.
PEG/NaCl
Polyethylene glycol (PEG), average molecular weight 8000 (Sigma) at 20 % w/v and
sodium chloride (Univar analytical grade) at 146.1 g/L were dissolved in Milli-Q water
and autoclaved.
70
Phage Decontamination Solution
SDS (Sigma) at 1 % w/v and sodium hydroxide (Univar) at 0.4 % w/v were dissolved in
RO water. Solution was added to autoclaved, rinsed flasks and incubated overnight
before normal glassware washing.
PNPP Substrate Solution

One substrate tablet (SIGMAivi.ST^'^ p-Nitrophenyl phosphate tablets, Sigma) and one
buffer tablet were dissolved in Milli-Q water according to manufacturer's instructions
and the resulting solution was used immediately.
1 M Potassium Chloride
Potassium chloride (BDH AnalaR) was dissolved in Milli-Q water at 74.55 g/L and
sterilised by filtration through a 0.22 jxm syringe driven filter unit (Millipore, Millex
GP).
5X RNA Loading Buffer

25 mg bromophenol blue (Sigma), 80 ^L 0.5 M EDTA pH 8.0, 720 |iL 37 %
formaldehyde (Sigma), 2 mL glycerol (Univar analytical grade), 3084 |iL formamide
(BDH AnalaR) and 4 mL lOX FA gel buffer were placed in a centrifuge tube. Volume
was made up to 10 mL with Baxter water (water for irrigation, Baxter Healthcare) and
the solution mixed thoroughly by inversion.
3 M Sodium Acetate pH 5.0

Sodium acetate (anhydrous, BDH AnalaR) was dissolved in Milli-Q water at 24.6 g/100
mL, the pH adjusted to 5.0 with glacial acetic acid (Univar analytical grade) and
sterilised by filtration through a 0.22 |im syringe driven filter unit (Millipore, Millex
GP).
1OM Sodium Hydroxide

40 g sodium hydroxide (Univar) was dissolved in sterile Milli-Q water to give a final
volume of 100 mL.
Staining Wash (Tris/EDTA) for Flow Cytometry
1.21 g tris (Sigma), 8,77 g sodium chloride (Ajax), 8.09 g EDTA (potassium salt, BDH),
1 g sodium azide (Sigma) and 1 g BSA (bovine serum albumin, fraction V, Sigma
molecular biology grade) were dissolved in Milli-Q water to give a final volume of 1 L.
The pH of the buffer was adjusted to 7.4 with 10 M sodium hydroxide solution.
50X TAE
Tris base (Sigma), 242 g, 57.1 mL glacial acetic acid (Univar analytical grade) and 100
mL 0.5 M EDTA pH 8.0 were made up to 1 L with Milli-Q water and stirred until
dissolved.
IX TAE
50X TAE was diluted to working concentration by the addition of 20 mL stock to 980
mL Milli-Q water and mixing by inversion.
lOX TBE
Tris base (Sigma), 108 g, 55 g boric acid (Univar analytical grade) and 40 mL 0.5 M
EDTA pH 8.0 were made up to 1 L with Milli-Q water and stirred until dissolved.
IX TBE
lOX TBE was diluted to working concentration by the addition of 100 mL stock to 900
mL Milli-Q water and mixing by inversion.
IM Tris HCl pH 7.4

60.6 g tris (BDH biochemical grade) was dissolved in 450 mL Milli-Q water, adjusted
to pH 7.4 with conc. HCl and made up to a final volume of 500 mL. Solution was
sterilised by autoclaving.
100 mM Triethylamine
Triethylamine (Sigma, Min. 99 %) 138.6 jiL, was added to 10 mL Milli-Q water and
mixed by inversion to give a 100 mM solution. Solution was prepared immediately
before use.
Tetracycline Stock
Tetracycline (Sigma) was dissolved in ethanol (absolute, Univar analytical grade) at 10
mg/mL, aliquoted and stored at -20 °C protected from light.
2.4 Equipment
Bench centriñige: Sigma, 3-10.

CO2 Incubator: Forma Scientific, 3164.
Electroporator: BioRad, Gene Pulser.
Fluorescent Microplate Reader: Molecular Devices, fmax and SOFTmax Pro Software,
Gel Documentation System: Syngene, GeneGenius and GeneTools Software.
Horizontal gel electrophoresis unit: IBI, QSH (Quick Screening Horizontal) or MPH
(Multi-Purpose Horizontal).
Immuftige: Dade, 569E.
Incubator: Thermoline, I60FD.
Inverted Microscope: Olympus, CK2.
Laminar Flow Cabinet: Gelman, CF43S.
Microcentriftige: Eppendorf, 5415D.
Microscope: Olympus, BH.
PCR Machine : Hybaid, PCR Express.
: Hybaid, Omni E.
pH Meter: Radiometer, PHM 92.
Pipettes: Gilson P2, PIO, P200, PIOOO, P5000.
Plate Reader: Molecular Devices, Versamax and SOFTmax Pro Software.
Power Supply: BioRad, Power Pac 300.
Refrigerated Bench Centriftige: Beckman, Allegra 6R
Shaking Incubator: Paton Scientific, 01 2116.
Spectrophotometer - Hitachi, U-1100.
- NanoDrop Technologies, NanoDrop ND-1000.
Thermal Sequencing Block: Corbett Research, FTS-320.
Vertical Mini Gel Electrophoresis Unit: Novex Xcell II Mini-Cell
Water bath: Thermoline, NBIT.
2.5 RBC Phenotyping and Cell Culture Methods
2.5.1 Indirect Antiglobulin Technique (Saline lAT)
Human serum samples were tested for the presence of antibodies of interest by
agglutination assay using reagent RBCs from a Phenocell panel (11 Cell panel for use in
identification of atypical antibodies- CSL). Depending on sample availability, 2-4
drops of serum were added to each of 11 appropriately labelled 1 0 x 7 5 mm glass test
tubes. The 3 % v/v washed red cell preparations were resuspended by gentle inversion
and one drop of each cell type was added to the appropriate tube. Monoclonal anti-D
IgGl (produced at the ARCBS from BTSN6 cell line) was used as a positive control
and human AB serum (Batch 1/99 from ARCBS-NSW) was used as a negative control.
The samples were mixed by shaking the test tube rack, the rack covered with aluminium
foil and incubated at 37 °C in a water bath for 30-60 min.
Samples were centrifuged at low speed for 10 sec in an Immufuge and analysed for
macroscopic agglutination or hemolysis by gentle resuspension under a magnifying
glass mounted over a light box. The level of macroscopic agglutination was graded
using a numerical scale (Figure 3.5). Results were recorded on the Phenocell Antigen
Composition Sheet. Cells were washed by resuspending gently, filling tubes with PBS
and then centrifuging at high speed for 30 sec. The supernatant was aspirated and the
washing process repeated a total of four times. After the last aspiration, two drops of
anti-human globulin were added (Epiclone anti-IgG, -C3d; polyspecific rabbit and
murine monoclonal, CSL) and the samples mixed by shaking the test tube rack.
Samples were centrifuged at low speed for 15 sec in an Immufuge and then analysed for
macroscopic agglutination or hemolysis as before.
2.5.2 Thawing Cells from Liquid Nitrogen
Two water baths were required for this procedure, one set at 37 °C and the other at 42
°C. Media was prepared (25 mL per vial to be thawed) by adding foetal calf serum
(PCS; CSL) to Iscove's modified Dulbecco's media (IMDM; ICN) to a final
concentration of 20 % v/v. This media was placed in the 37 °C water bath to pre-warm.
The vial or vials to be thawed were removed from storage in liquid nitrogen and quickly
transferred to dry ice. Vials were thawed singly by gentle agitation in the 42 °C water
bath until only a small frozen crystal remained. Samples were not allowed to thaw
completely. The tube was swabbed with 70 % ethanol and transferred to a laminar flow
cabinet.
The contents of the tube were transferred to a 15 mL centrifuge tube using a transfer
pipette and 0.5 mL PCS was added in a drop-wise fashion (-one drop per second) with
mixing. Using a sterile pipette, 10 mL of pre-warmed media was added drop-wise
whilst the tube was agitated gently. A further 5 mL of media was added more quickly.
The cells were then centrifuged at 1900 rpm (500 g) in a bench centrifuge for 5 min.
The supernatant was discarded and the cells resuspended in 10 mL pre-warmed media.
A small aliquot was taken for viability testing (Section 2.5.5).
2.5.3 EBV Transformation of PBMC
A fresh or thawed PBMC suspension was centrifuged at 2100 rpm (600 g) in a bench
top centrifuge for 10 min. A vial of EBV viral supernatant (Section 2.1.11) was thawed,
swabbed with 70 % ethanol and transferred to a laminar flow cabinet. A 1.2 mL aliquot
of viral supernatant was transferred to a 5 mL tube and an equal volume of IMDM/20 %
PCS added. The PBMC supernatant was discarded and the cells resuspended in the
diluted viral supernatant and mixed well. A small aliquot was taken for viability testing
(Section 2.5.5). The cells were incubated at 37 °C in a 5 % carbon dioxide incubator for
90 min, with gentle mixing every 20 min. The cells were then centrifuged as before for
8 min and the supernatant discarded. Cells were resuspended in IMDM media
containing 20 % PCS and 1 % PHA (phytohaemagglutinin, Murex Biotech reagent
grade) such that viable cells were at a fmal concentration of 0.5 x lO^cells/mL. The
cell suspension was transferred to either a 12-well cell culture plate (1 mL per well) or
25 cm^ flask (5 mL per flask) depending on the volume of suspension obtained
(Coming Costar) and placed in a 37 °C in a 5 % carbon dioxide incubator.
2.5.4 Maintenance of Lymphoblastoid Cell Lines
When growth of the lymphoblastoid cell lines (LCLs) was extensive, as indicated by the
appearance of clumps of cells (observed by light microscope) and a media pH indicator
colour change from red to orange/yellow, the cells were passaged. This was typically
carried out every 3-4 days using IMDM media containing 10 % v/v PCS, 50 lU/mL
penicillin (Trace Biosciences), 50 |ig/mL streptomycin (Trace Biosciences), 50 |ig/mL
gentamycin (Sigma) and 2 mM L-glutamine (Trace Biosciences). In a laminar flow
cabinet, cells were transferred from plates or flasks to a suitably sized centrifuge tube.
The cell suspensions were centrifiiged at 1800 rpm (450 g) in a bench top centrifuge for
5 min and the supernatant retained for saline lAT testing if required (Section 2.5.2).
The cell clumps were broken up and the pellet resuspended by flicking the tube. Cells
were resuspended in 5-10 mL media depending on the size of cell pellet obtained, and a
small aliquot was taken for viability testing (Section 2.5.5). The volume of media
required to give a cell density of 0.4 to 0.5 x 10^ cells/mL was calculated and added to
the cells. The cell suspension was transferred to suitably sized cell culture plate or flask
and placed in at 37 °C in a 5 % carbon dioxide incubator.
2.5.5 Cell Culture Viability Test (Trypan Blue Exclusion)
Eight drops of cell suspension were transferred to a glass test tube, one drop of 0.5 %
trypan blue solution (Trace Biosciences) added and the sample mixed well. The sample
was loaded into the loading groove of a haemocytometer with a cover slip on top. Cells
in the centre large square of the grid were counted with viable (unstained) and dead
(blue stained) cells counted separately. The volume of the centre square of the
haemocytometer was 0.1 mm^ (or 10""^ mL) so to obtain the number of cells per mL, the
count was multiplied by 10"^. The percent viability of the cell suspension was calculated
by dividing the number of viable cells by the total number of cells (viable and dead) and
multiplying by 100.
2.5.6 Freezing Cells in Liquid Nitrogen
A viability test (Section 2.5.5) was performed on the cell sample to be frozen and the
number of vials to be used was calculated based on the freezing of 5 x 10^ cells per vial.
Cryovials (Nunc) were labelled and placed in a solid plastic test tube rack that had been
stored in the freezer (-20 °C) for several hours before use, and the rack returned to the
freezer. IMDM plus 20 % v/v DMSO (BDH) was prepared and placed in an ice bath
(ice and water mixture) to pre-chill. IMDM plus 40 % v/v FCS was prepared and
placed in a 37 °C water bath to pre-warm. Cell suspensions were centrifuged at 1900
rpm (500 g) in a bench top centrifuge and, in the case of LCLs, the supernatant retained
for testing by lAT if required (Section 2.5.1). The cells were resuspended in IMDM/40
% FCS, in a total volume corresponding to 0.5 mL multiplied by the number of vials to
be frozen. The cell suspension was then placed in the ice bath to cool. Cryovials were
transferred from the freezer to a laminar flow cabinet and the lids removed aseptically.
An equal volume of IMDM/20 % DMSO was added to the cell suspension in a drop-
wise fashion with gentle mixing. Cells were immediately transferred to the vials as 1
mL aliquots and then placed in the ice bath. Vials were transferred to -70 °C storage
and then to liquid nitrogen storage the following day.
2.5.7 Maintenance of Human Embryonic Kidney 293 Cell Lines
To initiate cultures from frozen stocks, cells were thawed (Section 2.5.2) checked for
viability (Section 2.5.5) and resuspended in IMDM media containing 20 % FCS such
that viable cells were at a final concentration of 0.5 x 10^ cells/mL. The cell suspension
was transferred to a 25 cm^ flask (5 mL per flask; Coming Costar) and placed in a 37 °C
in a 5 % carbon dioxide incubator. When growth of the cell suspensions was extensive,
as indicated by the appearance of a confluent cell layer on the base of the flask
(observed by light microscope) and a media pH indicator colour change from red to
orange/yellow, the cells were passaged. This was typically carried out every 3-4 days
using IMDM media containing 10 % v/v FCS, 50 lU/mL penicillin (Trace Biosciences),
50 |xg/mL streptomycin (Trace Biosciences), 50 [ig/mL gentamycin (Sigma) and 2 mM
L-glutamine (Trace Biosciences). In a laminar flow cabinet, cell supematants were
transferred from plates or flasks to a suitably sized centrifuge tube. A 2 mL volume of
0.02 % PBS/EDTA was added to each flask to cover the adherent cell layer and was
78
incubated at RT for 5-10 min. The flasks were agitated gently to dislodge the cells and
the cell suspension was transferred to the same tube as the cell supernatant.
The cell suspensions were centrifuged at 1900 rpm (500 g) in a bench top centrifuge for
5 min and the supernatant discarded. The cell clumps were disrupted and the pellet
resuspended by flicking the tube. Cells were resuspended in 5-10 mL media depending
on the size of cell pellet obtained and a small aliquot was taken for viability testing
(Section 2.5.5). The volume of media required to give a cell density of 1 to 2 x 10^
cells/mL was calculated and this was added to the cells. The cell suspension was
transferred to suitably sized cell culture plates or flasks and placed in a 37 °C in a 5 %
carbon dioxide incubator.
2.5.8 Preparation of Samples for Flow Cytometry
Human embryonic kidney 293 Cells were harvested and tested for viability as described
(Section 2.5.5). Cells were resuspended in cold staining wash at a density of 2 x 10^
cells/mL. Microcentrifuge tubes were labelled before the addition of 0.5 mL cell
suspension. Cells were centrifuged for 4 sec at 10,000g in a microcentrifuge and then
the supernatant removed by aspiration. Primary antibody (antigen-specific polyclonal
human serum) was added at 50 jaL per tube and gently mixed by vortex to ensure that
the cell pellet was resuspended. Samples were incubated in an ice bath (ice plus water)
for 30 min with gentle mixing every 10 min.
Staining wash was added, 1 mL per tube, the sample mixed by vortex and then
centrifuged as before. The wash solution was aspirated and the washing process
repeated a total of three times. Secondary antibody (anti-human Ig-FITC labelled,
Silenus) was diluted 1 in 40 with staining wash and added to washed cells at 25 |iL per
tube. Samples were mixed by vortex and incubated at 4 °C for 30 min before being
washed as described. Cellfix (lOX concentrate, Becton Dickinson) was diluted 1 in 10
with Milli-Q water and then added to samples at 1 mL per tube. Samples were mixed
by vortex and then stored at 4 °C, protected from light, for no more than 24 h before
analysis by the ARCBS-NSW flow cytometrist.
2.6 Library Construction Methods
2.6.1 RNA Extraction
RNA was extracted from between 10^ and 10^ cells using RNeasy Mini Kit (Qiagen)
according to manufacturer's instructions. The process was carried out in a laminar flow
cabinet. After addition of disruption buffer (Buffer RLT) the cell preparation was
homogenised by five passages through a 20-gauge needle attached to a sterile 1 mL
syringe. RNA was eluted from the spin columns with two 50 jiL washes with RNase
free water which were collected into the same tube. Two aliquots were removed
immediately, 2 jiL for spectrophotometric analysis (Section 2.6.7) and 10 |iL for
analysis by FA gel electrophoresis (Section 2.6.2) and all samples were stored at -70
2.6.2 Formaldehvde Agarose (FA) Gel Electrophoresis (for RNA)
Method from Qiagen RNeasy Mini Handbook.
1.2 g agarose (molecular biology grade, Progen Biosciences) and 10 mL lOX FA buffer
were made up to a total volume of 100 mL with Baxter water (water for irrigation,
Baxter Healthcare) and heated in a microwave oven on ftill power for 30 sec bursts,
with mixing, until agarose was dissolved. This solution was allowed to cool to ~60 °C
before the addition of 1.8 mL 37 % formaldehyde 9Sigma) and 5 ^L ethidium bromide,
10 mg/mL (stock prepared by SBBS staff). Molten agarose was poured into a casting
tray with a 1 mm comb and allowed to cool completely. The solidified gel was
transferred to a horizontal gel electrophoresis unit. The gel tank was filled with IX FA
gel running buffer until the gel was just covered and left for 45 min before use to allow
for RNase inactivation. Samples were prepared by adding 5 |xL RNA loading buffer to
10 [iL RNA samples, heating at 65 °C for 5 min and then storing at 4 °C prior to use.
Samples were loaded onto the gel and electrophoresed at 70 V until the bromophenol
blue had travelled approximately two thirds of the total gel length. The gel was then
photographed on a UV light box.
2.6.3 Reverse Transcription
RNA samples were converted to cDNA using reverse transcriptase (First Strand cDNA
Synthesis Kit, Life Technologies). Preparation of reactions was carried out in the
laminar flow cabinet using plugged pipette tips (ART, Molecular BioProducts). An
aliquot from either a single patient or donor RNA sample or from pooled donor or
patient RNA, containing 4 to 5 jig was added to 1 jiL random hexamer primers (50
ng/^L) and made up to a total volume of 10 jiL with DEPC-treated water from the kit.
This sample was denatured by heating to 65 °C for 5 min and then placed on ice. In a
separate tube the following reagents were combined (typically more than one sample
was transcribed and reagents combined as a master mix):
Reagent Volume (jiL)

cDNA Synthesis Buffer, 5X 4
DTT, 0.1 M 1
RNaseOUT, 40 U/^L 1
DEPC-treated Water 1
dNTP Mix, 10 mM 2
Thermoscript RT, 15 U/|iL 1
A 10 jiL volume of master mix was added to the denatured RNA sample and incubated
at 25 °C for 10 min (primer extension) then 50 °C for 50 min (synthesis) before
terminating reaction by heating to 85 °C for 5 min. The RNA template was removed by
the addition of 1 jiL E. coli RNase H (2 U/|iL) and incubation at 37 °C for 40 min. The
resulting cDNA samples were stored at -20 °C prior to use in PCR experiments.
2.6.4 Primary and Secondarv Polymerase Chain Reactions (PCR)
Preparation of PCR Reagents:
Each primer was resuspended in sterile Milli-Q water to give a final concentration of
250 jxM. These primary stocks were diluted 1 in 10 in sterile Milli-Q water to give 25
|iM working solutions. All samples were stored at -20 °C.
Nucleotide mix (dNTP mix) was prepared by adding 50 |iL of each nucleotide (100 mM
nucleotide set, dATP, dTTP, dCTP, dGTP, MBI Fermentas) to 1800 ^L sterile Milli-Q
water to give a total of 10 mM or 2.5 mM of each nucleotide. This working solution
was aliquoted and stored at -20 °C.
Initial PGR experiments were carried out according to de Haard et al. (1999) as follows:
Reagent Volume (|iL)
PGR Buffer, lOX 5
dNTP Mix, lOmM 5
Magnesium Ghloride, 50 mM 2
Sterile Milli-Q water** 33.5
Taq DNA Polymerase * 0.5
cDNA Template for primary PGR 2
Forward Primer Total of 1 |j,L
X PGR had 2 forward primers- (XFOR 0.5 ^L each)
Secondary H PGR had 4 forward primers- (JHFOR 0.25 ^L each)
Reverse Primer 1
(*Recombinant, Life Technologies)
For secondary PGR conditions (marked ** in Table above) 5 ng of purified primary

PGR product was used as template and the required amount of sterile Milli-Q water
added to give a 35.5 [iL final volume.
Gycling Gonditions:
Stage Temperature (°G) Time (Min) Number of cycles
Initial Denaturation 94 3 1
Denaturation 94 1
Annealing 54 1 30
Extension 72 2
Final Extension 72 10 1
Not all reactions were successful under these conditions therefore the following matrix
was used for PGR optimisation:
MgGl Template* Forward Reverse
(mM) (^L) Primer (jiM) Primer (jiM)
1 1 1 0.5 0.5
2 2 1 0.5 0.5
3 3 1 0.5 0.5
4 4 1 0.5 0.5
5 5 1 0.5 0.5
6 2 5 0.5 0.5
7 2 3 0.5 0.5
8 2 2 0.5 0.5
9 2 0.5 0.5 0.5
10 2 0.25 0.5 0.5
11 2 0.1 0.5 0.5
12 2 1 0.4 0.4
13 2 1 0.3 0.3
14 2 1 0.2 0.2
15 2 1 0.1 0.1
Template concentration optimisation (marked * in Table above) was not carried out for
secondary PGR; all optimisation reactions utilised 5 ng of purified primary PGR product
as template. The best conditions were selected based mainly on product yield but also
specificity and subsequently reactions were optimised with respect to annealing
temperature, testing in the range of 51 to 61 °G for primary PGR and up to 68 °G for
secondary PGR.
After this optimisation, some reactions were still not working satisfactorily, so the
following DNA Polymerases were tested:
Platinum Taq DNA Polymerase (Invitrogen).
Platinum Taq DNA Polymerase High Fidelity (Invitrogen).
HotStarTaq (Qiagen).
2.6.5 Agarose Gel Electrophoresis (DNA)
All gels were 1 % agarose unless stated otherwise.

The required amount of agarose (typically 1 g; Progen Biosciences molecular biology
grade) was added to 100 mL IX TAE buffer and heated in a microwave oven on iull
power for 30 sec bursts, with mixing, until agarose was dissolved. This solution was
allowed to cool to ~60 and then poured on to a glass plate in the casting tray with
either an analytical (10 tooth) 1 mm comb or preparative (1 sample, 1 marker) 1 mm
comb and allowed to cool completely. Solidified gel was transferred to horizontal gel
electrophoresis unit (Model QSH: Quick Screening Horizontal or MPH: Multi-Purpose
Horizontal, from IBI). The unit was filled with IX TAE until the buffer just covered
the gels completely. This buffer was reused several times.
DNA samples were prepared by addition of DNA gel loading dye at a ratio of 5:1,
sample to dye and loaded on to the gel along with lambda Hindlll or kb ladder DNA
markers (MBI Fermentas). Gels were electrophoresed at between 80 and 120 V
depending on time constraints until the bromophenol blue and xylene cyanol had
migrated approximately one third and two thirds of the way down the gel respectively.
Gels were stained for approximately 15 min in 1 )ag/mL ethidium bromide in Milli-Q
water (prepared by SBBS staff), destained briefly in Milli-Q and then photographed on
a UV light box either digitally (SynGene, GeneGenius) or with film (Polaroid, Polapan
667).
2.6.6 Agarose Gel Purification of DNA
DNA samples were purified with exposure to UV light by illuminating agarose gel on a
UV light box and excising the requisite band (as quickly as possible to minimise UV
exposure) using a single-edged industrial blade. DNA samples were purified without
exposure to UV light (see Figure 4.12) as follows:
Samples were electrophoresed using a preparative (1 sample, 1 marker) 1 mm comb and
stained as described (Section 2.6.5), with 5 |LIL of the sample loaded into the small
'marker' lane and the remainder in the large 'sample' lane. The gel was placed on the
UV light box without illumination, and the marker lane and a 2-3 mm strip of the
sample lane on the opposite edge of the gel were cut off using a single-edged industrial
blade. The centre part of the gel was removed from the light box, the gel illuminated
and the requisite band cut out of the two remaining strips. The light box was switched
off, the centre part of the gel replaced, and all of the gel pieces re-assembled to their
original position. This enabled the alignment and excision of the section of the gel
containing the DNA band without UV exposure. After the band had been removed, the
remaining gel was illuminated to check that the correct area of gel had been removed
for extraction. DNA was recovered from the gel slice using a commercial kit (Wizard®
SV Gel and PGR Glean-Up System, Promega).
2.6.7 Nucleic Acid Quantitation
Spectrophotometry:
The concentration of DNA or RNA solutions was estimated using a UV

spectrophotometer. The spectrophotometer was blanked against Milli-Q water using a
quartz cuvette (Stama) at 260 nm. The sample was diluted in Milli-Q water to give a
reading in the linear range of the spectrophotometer (0.1 to 1.0) and measured using the
same cuvette. All RNA samples were diluted 1 in 500 (2 [iL RNA + 998 jaL Baxter
water (water for irrigation, Baxter healthcare) using 2 jiL aliquots stored at -70 °G
immediately after extraction. DNA or RNA concentration was calculated based on the
following formulae:
1 Absorbance Unit (AióOnm) = 50 ^g/mL dsDNA
1 Absorbance Unit (AióOnm) = 40 [ig/mL RNA
Spectrophotometry was also carried out using a NanoDrop spectrophotometer blanking

against Milli-Q water. This system only required 1 îL of sample and had a very broad
dynamic range thus obviating the need for sample dilution. DNA concentration was
automatically calculated by the associated software.
Picogreen :
DNA concentration was measured using PicoGreen dsDNA Quantitation Reagent and
Kit (Molecular Probes). A standard curve was prepared by diluting lambda DNA
standard (100 iig/mL stock) to give 100, 50, 20, 10, 5, 2, 1 and 0.5 ng/mL in TE buffer.
Samples were diluted appropriately into TE buffer (typically 1 in 250) and transferred
along with the standards to a microtitre plate (Sarstedt) at 100 [iL per well, in duplicate.
The PicoGreen reagent was prepared according to manufacturers instructions and added
to the DNA samples at 100 |iL per well. Samples were mixed by gently shaking the
plate and protected from light as much as possible before reading in a fluorescence
microplate reader. Results were analysed using SoftMax Pro software (Molecular
Devices).
GeneTools (Gel Estimation):

DNA samples to be analysed were electrophoresed on an agarose gel along with a
known quantity of lambda DNA/Hind III marker (MBI-Fermentas). A digital image of
the gel was obtained using GeneSnap software on the GeneGenius Biolmaging System
(Syngene). DNA estimation was carried out by comparison of sample and standard
band density using GeneTools software.
2.6.8 Plasmid Extraction
pCESl was extracted from E. coli either by the alkaline lysis method (Sambrook et al.,
1989) or using a commercial kit, according to manufacturer's instructions (PureYield^^
Plasmid Midiprep System, Promega).
Preparation of bacterial cultures for extraction:

Bacteria transformed with the required plasmid, stored as a glycerol stock, were
streaked onto an LB Amp agar plate and incubated at 37 °C overnight. A single, well
defined colony was picked from this plate into 10 mL LB Amp media in a 100 mL
baffled flask and placed in a shaking incubator at 37 °C; 200 rpm for 8 h. This culture
was then used to inoculate a larger volume of LB Amp media (50 to 250 mL) at a ratio
of 1:500, which was placed in a shaking incubator at 37 °C; 200 rpm overnight.
Typically, the absorbance readings at 600 nm of the resulting cultures (blanked against
LB Amp media) were between 1.0 and 3.0 absorbance units. Bacteria were harvested by
centrifugation at 4-5 krpm for 15-30 min, the supernatant discarded and pellets drained
on paper towel. Pellets were subjected to extraction by the alkaline lysis method or
using a commercial kit, according to manufacturer's instructions (PureYield™ Plasmid
Midiprep System, Promega).
Alkaline Lysis Method:
The pellet was resuspended by vortex, 10 mL of ice-cold resuspension buffer (Soln I)
was added and the cell suspension mixed ensuring that no cell clumps remained. The
cell suspension was incubated at RT for 10 min, then 20 mL lysis buffer (Soln II) at RT
was added and the sample was mixed gently by inversion five or six times before
incubation on ice for 10 min. After incubation, 15 mL of ice-cold neutralisation buffer
(Solnlll) was added to the sample, mixed by inversion as before and incubated for 10
min on ice. The extract was centrifuged at 10,000 rpm for 10 min to pellet the
precipitated material and the supernatant decanted through gauze into a fresh tube to
remove floating cell debris. Extracted DNA was precipitated by the addition of an
equal volume of isopropanol (AR grade, Univar) followed by incubation at -20 °C for 1 -
2 h or overnight. DNA was recovered by centrifugation at 10,000 rpm for 10 min, the
supernatant discarded and the DNA pellet air-dried. The DNA pellet was resuspended
in an appropriate volume (0.1 to 1 mL) of sterile Milli-Q water and incubated with a
final concentration of 10 |Lig/mL RNase A at 37 for 30 min. This sample was phenol
chloroform extracted (Section 2.6.16) and ethanol precipitated (Section 2.6.17).
2.6.9 Polvacrylamide Gel Electrophoresis of DNA (DNA PAGE)
Pre-cast TBE gels, 1 mm x 15 well (Novex, Invitrogen) were used for DNA PAGE.
Gradient gels, 4-12 %, were used for analysis of PGR insert samples (-500 bp) and
homogenous 20 % gels were used for analysis of low molecular weight fragments from
BstOI digestion of PGR screening products. DNA samples were prepared as for
agarose gel electrophoresis and 10-15 jiL volumes loaded per lane. Gels were
electrophoresed using a Novex Xcell II Mini-Cell containing IX TBE buffer. Gels
were electrophoresed at 150-200 V until, in the case of the 20 % gels, the bromophenol
blue tracking dye reached the bottom of the gel; in the case of the 4-12 % gels,
electrophoresis was continued until the xylene cyanol tracking dye reached the bottom
of the gel in order maximise resolution. Acrylamide gels were stained with ethidium
bromide and visualised as for agarose gels (Section 2.6.5).
2.6.10 DNA Extraction from Acrvlamide Gels
DNA bands were excised from PAGE gels with and without exposure to UV light as for
agarose gels (Section 2.6.6). Pestle and mortars were wiped with 70 % ethanol and pre-
chilled on dry ice. Gel slices were placed in the mortar (on dry ice) and crushed to a
fme powder. The powdered gel was recovered into a microfuge tube and 200 |iL IX
TAE buffer added. The sample was mixed thoroughly and subjected to phenol
chloroform extraction (Section 2.6.16) followed by ethanol precipitation (Section 2.6.17)
to recover the DNA sample.
2.6.11 SvbrGreen Staining of DNA Gels

SybrGreen staining solution (Molecular Probes) was prepared according to
manufacturers instructions and DNA gels were stained for approximately 15 min,
destained briefly in Milli-Q water and then visualised on a UV light box.
2.6.12 Restriction Digestion of pCESl Phagemid Vector for Ligation

pCESl vector or pCESl containing heavy chain antibody repertoire was digested for
light chain ( V L C L ) insertion by first digesting with either ApaLI (Apa) or AscI (Asc),
(New England Biolabs) as follows:
Asc Digest Apa Digest
Reagent
Volume (|iL) Volume (|xL)
pCESl (50 |ig/mL) 125 123.5
Buffer NE4, lOX 15 15
BSA, lOOX 0 1.5
Enzyme 10 10
Digests were incubated at 37 for 16-20 h in a water bath and the Hnearised vector
was dephosphorylated by the addition of 2 ^L calf intestinal alkaline phosphatase (CIP),
10,000 U/mL (New England Biolabs) and incubated in a 37 °C water bath for 30-90
min. Undigested vector was removed from the preparation by gel purification (Section
2.6.6). DNA was eluted from the purification columns with 2 x 50 |iL nuclease free
water and second digests with the opposite enzyme were set up as follows:
Apa Digest Asc Digest
Reagent
Volume (jiL) Volume (|iL)
pCESl Single Digest 90 90
Buffer NE4, lOX 15 15
BSA, lOOX 1.5 0
Nuclease-free Water 33.5 35
Enzyme 10 10
Digests were incubated at 37 °C for 16-20 h in a water bath and the double digested
vector was dephosphorylated by the addition of 2 jiL CIP, 10,000 U/mL (New England
Biolabs) and incubated in a 37 °C water bath for 30-90 min. Samples were purified
using Wizard® SV Gel and PCR Clean-Up System (Promega) according to
manufacturer's instructions and eluted from the purification columns with 50 jxL
nuclease-free water. Third digests with Xhol (to digest any remaining single digested
vector within the unwanted CL region in order to reduce vector background) were set up
as follows:
Xhol Digest
Reagent
Volume (}iL)
pCESl Double Digest 45
Buffer NE2, lOX 15
BSA, lOOX 1.5
Nuclease-free Water 78.5
Enzyme 10
Digests were incubated at 37 °C for 16-20 h in a water bath and the triple digested
vector was dephosphorylated by the addition of 2 jiL CIP, 10,000 U/mL (New England
Biolabs) and incubated in a 37 °C water bath for 30-90 min. Inserts were removed from
the preparation by gel purification (Section 2.6.6). DNA was eluted from the
purification columns with 50 [iL nuclease free water and quantitated by Nanodrop
spectrophotometry (Section 2.6.7).
pCESl vector and pCESl containing light chain antibody repertoire was digested for
heavy chain (VH) insertion by first digesting with either Sfil (Sfi) or BstE II (Bst), (New
England Biolabs) as follows:
Sfi Digest Bst Digest
Reagent
Volume (|aL) Volume (}xL)
pCESl (50 |ig/mL) 123.5 125
Buffer, lOX 15 (NE2) 15(NE3)
BSA, lOOX 1.5 0
Enzyme 10 10
Digests were processed as for light chain preparation, except that Sfi digests were
incubated at 50 °C and Bst digests at 60 °C. Second digests with the opposite enzyme
were set up as follows:
Bst Digest Sfi Digest
Reagent
Volume (|iL) Volume (}xL)
pCESl Single Digest 90 90
Buffer, lOX 15(NE3) 15 (NE2)
BSA, lOOX 0 1.5
Nuclease-free Water 35 33.5
Enzyme 10 10
Digests were processed as for light chain preparation except that Sfi digests were
incubated at 50 °C and Bst digests at 60 °C. Third digests with Ncol (to digest any
remaining single digested vector within the unwanted 'stuffer' region in order to reduce
vector background) were set up as follows:
Ncol Digest
Reagent
Volume (jiL)
pCESl Double Digest 45
Buffer NE2, lOX 15
BSA, lOOX 1.5
Enzyme 10
Digests were processed and quantitated as for light chain preparation.
2.6.13 Purification and Restriction Digestion of PCR Amplified Antibody Repertoire
Kappa, lambda and heavy chain PCR products were pooled separately and purified by
phenol chloroform extraction (Section 2.6.16) followed by ethanol precipitation
(Section 2.6.17) and gel purification (Section 2.6.6), eluting from the purification
columns with 50 jiL nuclease-free water. Purified products were digested as follows:
Lambda or Kappa Heavy chain Sfi
Reagents (|iL)
Apa Digest Digest
PCR Insert 45 45
Buffer, lOX 10(NE4) 10(NE2)
BSA, lOOX 1 1
Nuclease-free
34 34
Water
Enzyme 10 10
Digests were incubated at 37 °C for Apa and 50 °C for Sfi for 16-20 h in a water bath.
Samples were purified using Wizard® SV Gel and PGR Glean-Up System (Promega)
according to manufacturer's instructions and eluted from the purification columns with
50 |iL nuclease-free water. Second digests were set up as follows:
Lambda or Kappa Heavy chain Bst
Reagents (|xL)
Asc Digest Digest
PGR Insert Single
45 45
Digest
Buffer, lOX 10 (NE4) 10(NE3)
Nuclease-free
35 35
Water
Enzyme 10 10
Digests were incubated at 37 °G for Asc and 60 °C for Bst for 16-20 h in a water bath.
according to manufacturers instructions, eluted from the purification columns with 50
|iL nuclease-free water and quantitated by Nanodrop spectrophotometry (Section 2.6.7).
2.6.14 Purification of Antibody Repertoires from pGESl Single Ghain Libraries.
The heavy chain repertoire was digested from the pGES 1 heavy chain library for
insertion into the pGESl light chain library, and vice versa, as follows:
Lambda or Kappa Heavy chain
Reagents (jxL)
Repertoire Repertoire
pGESl Single
170 170
Ghain Library
Buffer, lOX 20 (NE4) 20 (NE3)
Enzyme 10 (Asc) 10 (Bst)
Digests were incubated at 37 °C for Asc and 60 for Bst for 16-20 h in a water bath.
according to manufacturer's instructions and eluted from the purification columns with
2 X 60 fiL nuclease-free water. Second digests were set up as follows:
Lambda or Kappa Heavy chain
Reagents (|xL)
Repertoire Repertoire
pGESl Single Ghain
100 100
Library
Buffer, lOX 15 (NE4) 15 (NE2)
BSA, lOOX 1.5 1.5
Nuclease-free Water 23.5 23.5
Enzyme 10 (Apa) lO(Sfi)
Digests were incubated at 37 °G for Apa and 50 °G for Sfi for 16-20 h in a water bath.
Antibody repertoires were separated from the vector by gel purification (Section 2.6.6).
DNA was eluted from the purification columns with 50 jiL nuclease free water and
quantitated by Nanodrop spectrophotometry (Section 2.6.7).
2.6.15 Preparation of Samples for Ligation
Vector (pGESl) to insert molar ratios of 3:1, 1:1 and 1:3 were tested and the 1:3 ratio
was used for all subsequent ligations. All ligation reactions were set up on ice using T4
DNA ligase (New England Biolabs) as follows:
L Ghain H Ghain L Ghain Insert Heavy Ghain Gontrol:
Insert + Insert + + H Ghain Insert + L pGESl +/-
pGESl pGESl Library Ghain Library Ligase
Vector 100 ng 100 ng 100 ng 100 ng 100 ng
Insert 42 ng 21 ng 39 ng 21 ng Ong
lOX Buffer 1.5 îL 1.5 îL 1.5 îL 1.5 ^L 1.5 laL
Ligase 1 |iL 1 ^L 1 jiL 1 ^L 1 ^L/0 [iL
Water To 15 ^L To 15 îL To 15 ^L To 15 îL To\5[iL

In addition, control reactions were set up containing digested vector only (no insert)
plus and minus ligase. To ligate larger amounts of DNA, multiples of the above
composition were made as a master mix, divided into 15 [iL aliquots for incubation and
pooled for purification. All samples were incubated for 12-16 h at 15 °C in a thermal
sequencing block. Samples were purified by adding sterile Milli-Q water to a final
volume of 250 |xL and purifying by phenol/chloroform extraction (Section 2.6.16). A
200 jiL aliquot was recovered and the remaining mixture re-extracted after the addition
of 200 |iL sterile Milli-Q water. A 200 \iL sample was recovered, pooled with the first
extraction and purified by ethanol precipitation (Section 2.6.17) with the addition of
glycogen as a co-precipitant. Samples were resuspended immediately before use in 5 or
10 jiL nuclease-free water.
2.6.16 Phenol Chloroform Extraction
Protein was extracted from samples by the addition of an equal volume of phenol:
chloroform: isoamyl alcohol, 25:24:1, saturated with 10 mM tris pH 8.0, 1 mM EDTA
(Sigma Chemical Company). Samples were mixed thoroughly (~30 sec) by vortex and
then centrifuged at 13,000 rpm for 15 min in a microfiige. An aliquot of approximately
80 % of the original sample volume was removed from the upper phase, taking care not
to disturb the interphase, and transferred to a fresh 1.5 mL tube. The same volume of
sterile Milli-Q was added to the original extraction and the process repeated to increase
yields. The resulting sample was ethanol precipitated (Section 2.6.17).
2.6.17 Ethanol Precipitation
DNA was recovered and desalted by the addition of 1/10 volume of 3 M sodium acetate
pH 5.0 and two volumes of absolute ethanol (Univar analytical grade,), mixing between
additions. The sample was incubated at -20 °C for 1-2 h or overnight before
centrifuging at 13,000 rpm for 15 min in a microfuge to pellet DNA. The supernatant
was removed, mL cold 70 % ethanol added and the tube mixed by inversion. The
sample was centrifuged as before for 5 min and the 70 % ethanol wash repeated. As
much as possible of the final wash solution was removed and the pellet was dried by
placing in a desiccator under vacuum (~lk Pa) for 1-2 h or until dry. When small
amounts of DNA were precipitated (e.g. ligation reactions), IjiiL of 20 mg/mL Glycogen
(MBI Fermentas, molecular biology grade) was added to the sample before precipitation
to improve yield and allow the pellet to be visualised.
2.7 Phage Display and Biopanning Methods
All glassware used in panning procedures was decontaminated before re-use (see phage
decontamination solution, Section 2.3).
2.7.1 Preparation of Electrocompetent Cells
The E. coli strain, XL 1 Blue, stored as a glycerol stock, was streaked onto an LBTet agar
plate and incubated at 37 °C overnight. A single, well defined colony was picked from
this plate into 10 mL SBTet media in a 100 mL baffled flask and placed in a shaking
incubator at 37 °C; 200 rpm overnight. This culture was used to inoculate 2 x 250 mL
SB media (with 2.5 mL of 40 % glucose and 2.5 mL 1 M MgCh per flask) in 1 L
baffled flasks at a ratio of 1:200. Cultures were placed in a shaking incubator at 37 °C;
200 rpm until absorbance at 600 nm was 0.35 to 0.40 (blanking against SB media).
Flasks were immediately put on ice and cultures were transferred to pre-chilled 250 mL
centrifuge bottles.
Cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C. The supernatant
was discarded and cells resuspended in an equal volume of ice-cold sterile Milli-Q
water. Cells were centrifuged as before and the process repeated once more with ice-
cold Milli-Q water and twice more with ice-cold 10 % glycerol (i.e., a total of 1 L of
Milli-Q water and 1 L 10 % glycerol). Each pellet was resuspended in - 6 0 mL 10 %
glycerol and both cell suspensions were pooled into one centrifuge bottle. Cells were
centrifuged as before, the supernatant discarded and the pellet drained. The pellet was
resuspended in the residual buffer, transferred to a 1.5 mL tube on ice and the final
volume brought up to 1.0 to 1.5 mL with ice-cold 10 % glycerol. Cells were incubated
on ice for 30 min before use.
2.7.2 Electroporation
Several hours before electroporation, cuvettes (Gene Pulser Cuvette, 1 mm, BioRad)
were placed on ice. A 50 \iL aliquot of electrocompetent E. coli was added to each
resuspended ligation sample, mixed thoroughly and incubated on ice for 30 min. Each
sample was transferred to a fresh cuvette and electroporated using a Gene Pulser
(BioRad) attached to a pulse controller (made at UNSW) using the following
parameters:
Gene Pulser: 1.25 kV
Pulse Controller: 200 Q
Cells were rescued by the addition of 1 ml SOC media, transferred to a 15 mL culture
tube (Sarstedt) and incubated at 37 °C for 1 h exactly. Appropriate dilutions of each
sample were plated on to 90 mm SOB AG agar plates and remaining undiluted sample
(excluding controls) was plated on to five 150 mm SOBAG agar plates, -200 jxL per
plate. All plates were incubated in a 37 °C incubator overnight before colony counting
of dilution plates. Successful libraries were stored as glycerol stocks (Section 2.7.3),
and dilution plates were stored at 4 °C awaiting fiirther analysis.
2.7.3 Glycerol Stocking of Bacteria
Screw topped 1.5 mLvials (Sarstedt) were labelled and pre-chilled on dry ice. Cultures
were collected by adding 2-4 mL of LB media to each plate and dislodging cells gently
with a plate spreader. Cells were transferred to a suitable centrifrige tube and
centrifuged at 3500 g for 30 min in a bench top centrifuge. The supematants were
discarded, the pellets resuspended in residual media and an equal volume of 30 %
glycerol in LB media was added. After thorough mixing, ~1 mLaliquots of the cell
suspensions were transferred to screw topped vials, placed on dry ice and transferred to
-70 °C for storage.
2.7.4 PCR Screen of Transformants
Transformants were screened for the presence of insert using a 'colony pick' PCR
method where a cell sample from an agar plate was added directly to a 50 ^L aliquot of
PGR master mix using a sterile tip or toothpick. The master mix was made as muhiples
of the following volumes:
Reagent Volume (|aL)
lOX Reaction Buffer 5
Nucleotide Mix 5
Taq DNA Polymerase* 0.5
Primer scFv FOR 1
Primer scFv BAGK 1
*Recombinant, New England Biolabs
Gycling Gonditions:
Stage Temperature (°G) Time (Min) Number of cycles
Initial Denaturation 94 8:00 1
Denaturation 94 0:25
Annealing 47 0:30 40
Extension 72 1
Final Extension 72 10 1
PGR samples were analysed by agarose gel electrophoresis (Section 2.6.5) along with a
DNA marker (GeneRuler Ikb DNA Ladder, MBI-Fermentas) to allow calculation of the
PGR product size.
2.7.5 Library Diversity Analysis
Samples which showed a full-length insert according to the size of the product
generated in the PGR screening were digested with BstOI (Promega). Digestion
reactions were set up as follows:
Reagent Volume
PGR Product 20
lOX Buffer G 3
lOOX BSA 0.3
Sterile Water 5.7
BstOI 1
Samples were incubated in a 60 °C water bath for 2 h before analysis by polyacrylamide
gel electrophoresis (Section 2.6.9) or storage at-20 °C pending later analysis.
Electrophoresis was carried out using 20 % TBE gels (Section 2.6.9). Samples were
mixed with DNA loading dye (10 [iL sample plus 2 ^L dye), the entire sample loaded
on to the gel and the gel electrophoresed at 150 V until the bromophenol blue marker
dye reached the end of the gel.
2.7.6 Preparation of TGI for Phage Rescue
E. coli TGI from glycerol stocks held at UNSW were streaked on to minimal agar and
incubated at 30 for 3 days to allow colony formation. A single, well defined colony
was picked from the minimal plate into 10 mL 2TY media and incubated at 37 with
shaking overnight. Overnight TGI culture was diluted 1 in 50 into 2TY media and
incubated at 37 200 rpm until an absorbance of 0.5 at 600 nm was obtained,
indicating that the cells had reached exponential phase. Cells were used immediately.
2.7.7 Phage Rescue of Library Glycerol Stocks for Biopanning
Library glycerol stocks were diluted into 2TYAG media to give an absorbance at 600
nm of no more than 0.05. An aliquot of this culture was plated on to 2TYAG agar to
check the size of the inoculum. The remaining culture was incubated at 37 °C with
shaking until an absorbance of 0.5 at 600 nm was obtained, indicating that the cells had
reached exponential phase. Helper phage was added to the exponential culture to give a
final concentration of 5 x 10^ pfu/mL and incubated at 37 °C for 30 min stationary and
then 30 min shaking at 200 rpm to allow infection. Aliquots of this culture were plated
on to 2TYAG and 2TYKG agar plates to check the level of helper infection as indicated
by the acquisition of kanamycin resistance by the library clones in addition to ampicillin
resistance conferred by pCESl vector transformation. The culture was centrifuged at
4500 rpm for 15 min and the supernatant discarded. The pellet was resuspended in the
same volume of 2TYAK media (no glucose) and incubated overnight at 25 °C with
rapid shaking (300 rpm).
Selection plates inoculated with aliquots of the infected library were colony counted to
ensure efficient helper infection. This was indicated by the number of colonies on the
kanamycin selection plates being greater than 25 % of the number of colonies on the
ampicillin selection plates. Phage preparations not meeting this criterion were discarded.
The overnight culture was centrifuged at 8000 rpm for 15 min using pre-cooled
centrifuge bottles, centrifuge and rotor. The centrifugation was repeated in fresh
centrifuge bottles to ensure all cell debris was removed. Supernatant was collected into
a pre-chilled sterile bottle and 3/10 volume of pre-chilled PEG/NaCl added. The
sample was mixed thoroughly by inversion and incubated on ice for 60-90 min to allow
phage precipitation. Precipitated phage were pelleted by centrifugation at 8000 rpm for
15 min at 4 °C. Supernatant was removed and the bottles centrifuged again briefly to
allow removal of all remaining supernatant with a Pasteur pipette. The phage pellet was
thoroughly resuspended in 1-2 mL of PBS and then centrifuged in smaller tubes to
remove remaining cell debris. After centrifugation, resuspended phage were transferred
to a fresh tube, taking care not to disturb the bacterial pellet, and stored at 4 °C.
2.7.8 Phage Titration
Phage were serially diluted in 2TY media to give 50 |iL each of a suitable range of
dilutions. An exponential TGI culture was prepared as described (Section 2.7.6) and 50
jiL aliquots added to phage dilutions. The remainder of the TGI culture was retained.
Samples were incubated at 37 °C for 30 min stationary and then 30 min shaking at 200
rpm to allow infection. Infected samples were plated on to 2TYAG agar, 50 fiL per
plate, and incubated at 37 °C overnight. Non-infected TGI from the exponential culture
were also plated on to 2TYAG agar to check that the culture was not already
contaminated with phage. Colonies were counted to give the number of phage present
in the original samples.
2.7.9 Biopanning of Phage Library Against Solid Phase Antigen
For biopanning against recombinant Fy^ antigen, an Immunotube (Nunc) was coated
with antigen by the addition of 0.5 mL recombinant Fy^ at 5 |ig/mL in carbonate
binding buffer and incubated at 4 overnight. The following day, a culture of TGI
was set up as described (Section 2.7.6) to provide cells at exponential phase to coincide
with the final biopanning step (phage elution). The coated Immunotube was washed
three times with PBS and then blocked by filling to the brim with 3 % w/v skim milk
powder in PBS and incubating at RT for 1-2 h. Library phage were blocked by
combining 1.5 mL phage with an equal volume of 6 % w/v skim milk powder in 2X
PBS (prepared from 5X PBS) and incubating at RT for 30-60 min. The blocked
Immunotube was washed three times with PBS and 0.5 mL of blocked phage added.
The remainder of the phage were retained to determine the titre, referred to as 'input'.
The phage were incubated in the Immunotube for 1.5 h at 37 °C. After incubation the
phage were discarded and the tube washed 20 times with PBST and 20 times with PBS.
A sample of the final wash was retained and a 10 p,L aliquot mixed with 1 mL
exponential TGI culture to determine the phage titre in the final wash. Bound phage
were eluted by the addition of 0.5 mL freshly prepared 100 mM triethylamine and
incubating at RT for 15 min. Eluted phage (referred to as 'output') were transferred to a
microfuge tube containing 250 |j,L 1 M tris HCl pH 7.4 for neutralisation and stored on
ice until the TGI culture reached exponential phase (absorbance reading at 600 nm of
0.5 to 0.8 absorbance units).
Input and output phage titres were determined as described (Section 2.7.8). Half of the
output phage were mixed with 5 mL exponential TGI culture and incubated at 37 °C for
30 min stationary and then 30 min shaking at 200 rpm to allow infection. The
remainder of the output phage were stored at 4 °C in case the experiment needed to be
repeated. Infected cells were spun down at 3500 rpm for 10 min, resuspended in 500
jiL 2TY media and plated on to four 150 mm 2TYAG plates. All plates were incubated
at 37 °C overnight. Colonies on the titration plates were counted to give the input and
output titres. Colonies on the 150mm plates were stored as glycerol stocks according to
Section 2.7.3.
2.7.10 Biopanning of Phage Librarv Against Transfected HEK293 Cells
Cells were harvested from flasks, counted and used at 10^ to 10^ cells per biopanning
tube. HEK293T.Vector 5 cell line (transfected with empty vector, no RBC antigen
present) was used for subtraction biopanning and 293T.Fya.4, 293T.Fyb.9wt and
293T.K1.41 cell lines were used separately for antigen selection.
A TGI culture was set up as for solid phase biopanning (Section 2.7.9). The required
number of Immunotubes (Nunc) were pre-blocked using 3 % skim milk powder in PBS
for ~1 h at RT. Prior to use, HEK293 transfected cells were washed three times in 0.02
% PBS/EDTA by centrifugation at 1500 rpm for 3 min in a bench top centrifuge,
removal of supernatant and resuspension in 10 mL PBS/EDTA.
After washing, cells were resuspended in 3 % skim milk powder in PBS/EDTA,

transferred to a pre-blocked Immunotube and incubated for >1 h at RT to block.
Library phage were blocked as in solid phase biopanning (Section 2.7.9). Blocked cells
were centrifuged in a Dade Immufuge at low speed (Cycle 1) for 1 min and the
supernatant discarded. Cells were resuspended in 1 mL blocked phage and incubated
for 90 min at RT in one round of subtraction biopanning. The cell suspension was
centrifuged as before, subtracted phage were transferred to Immunotubes containing
antigen positive cells and incubated for 90 min at RT. Cells were centrifuged as before,
the supernatant discarded and resuspended in 2-3 mL of 3 % skim milk powder in
PBS/EDTA to remove loosely bound phage. This washing procedure was repeated 12
times with the final wash in PBS/EDTA only. Cells were transferred to a fresh tube and
bound phage were eluted by the addition of 0.5 mL freshly prepared 100 mM
triethylamine and incubating at RT for 15 min. Eluted phage were neutralised
immediately by the addition of 250 |iL 1 M tris HCl pH 7.4 and stored on ice until the
TGI culture reached exponential phase (Absorbance reading at 600 nm of 0.5 to 0.8
absorbance units). Subsequent steps were carried out as for solid phase biopanning
(Section 2.7.9).
2.7.11 Biopanning of Phage Library Against Whole RBC
RBCs (3 % v/v suspension of washed cells in PBS) for use in biopanning were selected
from a Phenocell panel (CSL). A cell suspension positive for a particular antigen was
chosen for biopanning and two other cell suspensions were chosen from the panel that
carried all the antigens present on the positive cells except for the antigen of interest
(Table 2.3). Positive antigens used in phage selection were, Fy^ Fy^, K, Jk®, D, C and E.
101
Panning using RBCs was carried out essentially as described for HEK293 cells (Section
2.7.10) with the following modifications. A 200 [iL aliquot of the 3 % Phenocell
washed RBC suspension was used for selection and 100 |iL each of the cells selected
for subtraction biopanning were mixed to give the same number of cells. All blocking
and wash buffers were the same except that EDTA was omitted.
Table 2.3. Phenocell RBC suspensions used in subtraction biopanning and

selection (see Figure 5.4 Antigen Composition Sheet for Phenocell Red Blood Cell
Reagent Panel).
Antigen of Interest Phenocell panel number Phenocell panel number
(and RBC system) used for positive selection used for subtraction
Fy' (Duffy) 10 6+9
Fy" (Duffy) 9 4+10
K (Kell) 8 7+11
Jk^ (Kidd) 9 3 + 11
D (Rhesus) 5 7+10
C (iüiesus) 6 3+5
E (Rhesus) 7 2+5
2.7.12 Preparation of Glycerol Stocks from Biopanning Output
The biopanning output cultured on the four large 2TYAG plates was recovered and
stored as glycerol stocks as follows. A 3 mL aliquot of 2TY media was added to each
plate and the cells dislodged gently using a sterile plate spreader. The resulting cell
suspension was transferred to a sterile tube and an equal volume of 50 % glycerol added.
The sample was mixed thoroughly and stored at -70 in ~1 mL aliquots in screw-
topped vials (Sarstedt).
2.7.13 Phage Rescue from Biopanning Output Glycerol Stocks
To perform second and subsequent rounds of biopanning, glycerol stocks were cultured
and re-infected with helper phage (Section 2.1.6) to produce phage as follows. A 25
mL aliquot of 2TYAG media was inoculated with 100 jiL of biopanning output stock
and incubated at 37 °C until an absorbance of 0.5 to 0.8 units at 600 nm was reached.
Helper phage were added to the exponential culture to give a final concentration of 5 x
10^ pfu/mL and incubated at 37 °C for 30 min stationary and then 30 min shaking at
200 rpm to allow infection. Aliquots of this culture were plated on to 2TYAG and
2TYKG agar plates to check the level of helper infection as indicated by the acquisition
of kanamycin resistance by the library clones in addition to ampicillin resistance
conferred by pCESl vector transformation. The culture was transferred to a 50 mL
sterile tube and centrifuged at 3500 rpm for 10 min and the supernatant discarded. The
pellet was resuspended in the same volume of 2TYAK media (no glucose), transferred
to a fresh flask and incubated overnight at 25 °C with rapid shaking (300 rpm).
Selection plates spread with aliquots of the infected library were counted to ensure
efficient helper infection. This was indicated by the number of colonies on the
ampicillin selection plates. Phage preparations not meeting this criterion were discarded.
The overnight culture was centrifuged at 3500 rpm for 10 min and the resulting
supernatant used for the next round of biopanning.
2.7.14 Rescue of Phagemid Clones in 96-Well Plates for Phage ELISA
Individual colonies from biopanning output titration plates were picked using a sterile
tip or toothpick into individual wells of a microtitre plate containing 100 jiL 2TYAG
media per well. The plate was sealed and incubated with moderate shaking at 37 °C
overnight to give the master plate. A microtitre plate containing 100 |xL 2TYAG media
per well was prepared and inoculated from the master plate using a 96-well replicating
tool (Bel-Art Scienceware) to create a replica plate. For storage, 50 \LL of 50 %
glycerol was added to each well of the master plate, the plate sealed and placed at -70
The replica plate was incubated with moderate shaking at 37 °C for 5-6 h. Helper
phage at 5x10^^ pfu/mL in 2TYAG media was added to the replica plate at 10 jiL per
well and incubated at 37 °C for 1 h to allow infection. The replica plate was centrifuged
at 2000 rpm for 10 min in a bench top centrifuge and the supernatant removed from the
wells. Cell pellets were resuspended in 100 ^L 2TYAK media (no glucose) and
incubated overnight with moderate shaking at 30 °C to produce phage.
2.7.15 Fv^ Phage ELISA
A Nunc Maxisorp 96-well plate was coated with recombinant Fy^ antigen at 5 jig/mL in
carbonate binding buffer, 50 [iL per well and incubated at 4 °C overnight. A second
plate was set up similarly with buffer only (no antigen control plate). The coated plates
were washed three times with PBS and then blocked with 200 [LL per well 3 % w/v
skim milk powder in PBS for 1 h at RT. The overnight culture replica plate (Section
2.7.14) was centrifuged at 2000 rpm for 10 min and each 100 jiL supernatant was
transferred to corresponding wells of another plate containing 50 |xL per well of 9 %
w/v skim milk powder in 3X PBS (prepared from 5X PBS) per well. Phage and
blocking solution were mixed by pipetting and incubated for -30 min at RT. Blocked
plates were washed three times in PBS and 50 ^L blocked phage transferred to each of
the antigen and control plate wells. Plates were incubated at RT for ~ 1 h. After
incubation, plates were washed three times in PBST and three times in PBS with a 2
min incubation of wash solution each time. Anti-M13 HRP conjugate (Amersham) was
diluted 1 in 5000 in 3 % w/v skim milk powder in PBS and added to both plates at 50
|xL per well. Plates were incubated and washed as in the previous step. Freshly
prepared ABTS substrate was added to both plates at 100 \iL per well and incubated at
RT until colour developed. Developed plates were analysed at 415 nm in a microplate
reader and analysed by subtracting control plate readings from antigen plate readings.
2.7.16 Screening Assays for RBC Antigen Specific Clones
A: Immobilised RBC Assay
Nunc Maxisorp wells were coated with wheatgerm agglutinin (Sigma) at 10 |ig/mL in
carbonate binding buffer, 50 jiL per well and incubated at 4 °C overnight. Wells were
washed with PBS/BSA and 50 [iL Fy^ positive RBC, 3 % suspension (CSL- Phenocell
panel RBC suspension number 10) added to each test well. Fy^ positive RBC were used
as a negative control (a mixture of RBC suspension numbers 6 and 9) and all wells
incubated at RT for h. After gentle washing (3 times with PBS/BSA) the plate was
read at 450 nm to check that RBC were bound (haemoglobin present in RBC absorbs at
this wavelength). The plate was blocked with PBS/BSA by adding lOOjiL per well and
incubating at RT for ~1 h. Positive and negative Fy^ antibody phage were rescued as
described (Section 5.3.6) and added to positive and negative RBC wells. The plate was
incubated for ~1 h at RT and washed gently as before. Anti-M13 monoclonal antibody
(Amersham Biosciences) at 1 |ag/mL in PBS/BSA was added at 50 \iL per well, and the
plate was incubated and washed as before. Plate was read again at 450 nm to check that
RBC had remained attached to the plate. Goat anti-mouse alkaline phosphatase
conjugate (Sigma) was diluted 1/5000 in PBS/BSA and the plate was processed as in
the primary antibody step. The plate was developed by the addition of PNPP substrate
and read on a microplate reader at 405 nm.
B -RBC Agglutination Assav
V-bottomed microtitre wells (Sarstedt) were blocked by the addition of 1 % BSA

(bovine serum albumin, fraction V, Sigma molecular biology grade) in carbonate
binding buffer at 200 |iL per well for ~1 h at RT. Rescued phage (Section 5.3.6; 50 jiL
per well) were added to 10 fiL Fy^ positive or Fy^ positive RBC (as per A- Immobilised
RBC Assay) in wells of the blocked plate and incubated for 60 to 90 min at RT. The
plate was centrifuged at 1000 rpm for 2 min in a bench centrifuge to sediment the RBC
and the supernatant removed. Cells were resuspended in PBS/BSA, 100|iL per well,
and the plate centrifuged. This washing step was repeated a total of three times with
cells resuspended in a fmal volume of 50 |LIL. Washed, resuspended cells were
transferred to fresh blocked v-bottomed microtitre wells and an equal volume of anti-
Mi 3 monoclonal antibody (Amersham Biosciences) at 2 \xg/mL in PBS/BSA was added.
Wells were mixed and incubated at RT for 60 min before being centrifuged at 1000 rpm
for 1 min. The plate was propped up at an angle, almost vertical, and incubated at RT
for 5 min. A positive result was indicated by a button of cells remaining in the bottom
of the well whereas a negative result was indicated by a smooth trail of cells down the
slope of the v-bottomed well (Figure 2.1). This assay was also carried out with the anti-
Mi 3 antibody replaced with anti-human globulin, supplied pre-diluted (CSL). In
addition, this assay was carried out as a competition where anti-M13 antibody was
replaced with human Fy^ positive serum (from ARCBS-NSW). In this case, a positive
phage result was indicated by the inhibition of RBC agglutination.
Figure 2.1 Representation of patterns obtained from microplate RBC
agglutination assays.
In wells where no agglutination has taken plaee (negative result), when the plate is
tilted, the RBC run in a line down the slope of the v-bottomed well. In
agglutinated sample wells (positive result) the RBC remain in a clump (which can
vary in shape) at the bottom of the well.
C -Solution Phase RBC Assay
V-bottomed microtitre wells (Sarstedt) were blocked by the addition of 1 % BSA

(bovine serum albumin, fraction V, Sigma molecular biology grade) in carbonate
binding buffer at 200 |AL per well for ~1 h at RT. Rescued phage (Section 5.3.6; 50 |LIL
per well) were added to 10 )LIL Fy^ positive or Fy^ positive RBC (as per A- Immobilised
RBC Assay) in wells of the blocked plate and incubated for 60 to 90 min at RT. The
plate was centrifuged at 1000 rpm for 2 min in a bench centrifuge to sediment the RBC
and the supernatant removed. Cells were resuspended in PBS/BSA, 100 |LIL per well,
and the plate centrifuged as before. This washing step was repeated a total of three
times with cells resuspended in a fmal volume of 50 |iL. Washed, resuspended cells
were transferred to fresh blocked v-bottomed microtitre wells and an equal volume of
anti-M13 monoclonal antibody (Amersham Biosciences) at 2 |Lig/mL in PBS/BSA was
added. Wells were mixed and incubated at RT for 60 min before being washed as
before. Rabbit anti-mouse alkaline phosphatase conjugate (Sigma) was diluted 1/5000
in PBS/BSA and the plate was processed as in the primary antibody step. Washed
resuspended cells were transferred to a flat-bottomed microtitre plate the assay was
developed by the addition of PNPP substrate at 100 |iL per well and read on a
microplate reader at 405 nm.
D -RBC Capture Assay
Nunc Maxisorp wells were coated with anti-M13 monoclonal antibody (Amersham
Biosciences) at 5 jig/mL in carbonate binding buffer, 50 }iL per well and incubated at 4
°C overnight. The wells were washed and blocked with PBS/BSA, 200 jiL per well at
RT for ~1 h. V-bottomed microtitre wells (Sarstedt) were blocked by the addition of 1
% BSA in carbonate binding buffer at 200 i^L per well and incubated at RT for ~1 h.
Rescued phage (Section 5.3.6; 50 [iL per well) were added to 10 îL Fy^ positive or Fy^
positive RBC (as per A- Immobilised RBC Assay) in wells of the blocked v-bottomed
plate and incubated for 60 to 90 min at RT. The plate was centrifuged at 1000 rpm for 2
min in a bench centrifuge to sediment the RBCs and the supernatant removed. Cells
were resuspended in PBS/BSA, 100 îL per well, and the plate centrifuged as before.
This washing step was repeated a total of three times with cells resuspended in a final
volume of 50 jiL. Washed, resuspended cells were transferred to the anti-M13 antibody
coated plate and incubated for 60 to 90 min at RT. After gentle washing (3 times with
PBS/BSA), ABTS substrate was added at 100 |iL per well to detect the presence of
bound RBC by the release of endogenous peroxidase. The plate was analysed in a
microplate reader at 450 nm.
3 Antibody Genes from Human Blood Samples
3.1 Aim
The aim of this section of the research project can be divided into three sections:
- Identify patient and donor samples (lymphocytes) suitable for using as a source of
RNA for immunoglobulin gene library construction.
- EBV transform cells. As these samples are a limited resource, EBV transformation
was required to ensure adequate supply of cells for RNA extraction.
- Extract RNA from human lymphocytes which produce clinically significant antibodies
to red blood cell (RBC) antigens.
Blood samples were collected, fractionated and stored by researchers in the Cell
Biology Department at the ARCBS-NSW. All other work described was performed as
part of this research project.
3.2 Introduction
Blood Cells
\f
Erythrocytes Leukocytes Platelets

(Red Blood Cells) (white Blood Cells)
Granulocytes
/ \ Lymphoid Cells
Eosinophils
Lymphocytes
O ( B & T Cells)
Basophils
Monocytes
Neutrophils ^ "" >

if
Figure 3.1 Cells found in human blood.

(Images from American Society for Clinical Pathology, 2002)
To construct an antibody phage display library containing antibodies to blood groups of
interest, it was first necessary to obtain the genetic information encoded in the
immunoglobulin gene repertoire for these antibodies. This genetic information was
obtained in the form of RNA from antibody-producing human white blood cells (B cells,
Figure 3.1). Samples from donors, who were shown during typing assays to have
atypical antibodies present in their blood (Table 2.1) were collected by ARCBS-NSW
personnel. The ARCBS-NSW also maintains connections with various hospitals in
order to obtain blood samples from patients who have suffered a transfusion reaction
and are likely to have generated antibodies in response to incorrectly matched RBCs.
These samples were initially collected by the Cell Biology department at the ARCBS-
NSW to generate human/mouse heterohybridomas producing monoclonal IgM for use
in for RBC phenotyping. Typically, several samples were taken from the same
individual over time and it was shown by phenotyping assays that the later samples
contained IgG (Table 3.4). IgG subclass antibody genes are ideal for library
construction as this subclass of antibodies are largely produced in the secondary,
affinity matured, immune response and as such are likely to have higher affinities than
IgM isotypes of the same specificity (Figure 1.25). Both coagulated and non-
coagulated samples were collected from donors and patients. Non-coagulated blood
samples were fractionated immediately after collection to isolate the peripheral blood
mononuclear cells (PBMC) which contain the antibody producing B cells necessary for
use in this project. PBMC were stored in liquid nitrogen and serum samples from
coagulated blood samples were stored at -20 °C.
Although antibodies to Rhesus group antigens are represented in the donor and patient
samples used in this project, they were not the main focus for recombinant antibody
production. Of particular interest were antibodies to the less widely known, although
still clinically significant, blood groups represented in the samples; Duffy, Kell and
Kidd. The PBMC samples detailed above were transformed using Epstein Barr Virus
(EBV) into lymphoblastoid cell lines allowing them to be expanded. EBV, a member of
the herpes virus family, is responsible for causing infectious mononucleosis (glandular
fever) and has been implicated in the generation of various tumours (Burkitt and
Hodgkin lymphomas; post-transplant lymphomas). Within the virus, the genome exists
in a linear form, but after infection of B cells the virus DNA forms a circle and exists as
an episome in cell nuclei (Küppers, 2003). The EBV genome contains more than 85
genes but only nine are expressed by LCLs, which include nuclear antigens important in
transcription regulation of viral and cellular proteins, and another protein which
maintains the viral episome during cell division (Bishop and Busch, 2002).
These cell lines were cultured in the presence of phytohaemagglutinin (PHA) which is
used to stimulate mitotic division of lymphocytes (Nowell, 1960). The discovery of
PHA as a mitogen was made serendipitously due to its use as an agglutinator to remove
erythrocytes from samples prior to leukemic cell culture. A chance circumstance
resulted in the culture of non-leukemic cells which unexpectedly continued to divide, an
effect eventually attributed to the use of PHA in sample preparation (Nowell, 1977).
Other mitogenic plant lectins have also been used, such as concanavalin A, and
treatment of lymphocytes with these agents results in activation similar to antigenic
stimulation (i.e. cell growth and proliferation). The cultured cells tend to remain
attached to each other after they divide, and form clumps visible to the naked eye which
are broken up during passaging (Figure 3.2). LCL cultures were maintained for several
weeks in order to obtain sufficient cell numbers to preserve cell stocks in liquid nitrogen
and extract RNA for use in library construction.
Figure 3.2 An example of EBV transformed B cells.
Ill
3.3 Methods
3.3.1 Blood Sample Collection and Storage
Donor and patient samples were collected by the ARCBS-NSW in two forms; one was
coagulated to yield a serum sample and the second was collected in the presence of
EDTA as an anticoagulant to allow the extraction of PBMCs. The serum sample was
tested at the time of collection by the ARCBS-NSW for the presence of atypical
antibodies using a reagent RBC panel (Phenocell Panel, CSL) by direct or indirect
antiglobulin technique (DAT or lAT respectively). The reagent red cells in the panel
are selected to include antigens associated with the most clinically significant antibodies.
Each cell type in the panel is obtained from a different individual donor, selected to give
distinctive patterns of positives and negatives for the antigens of interest. Phenotypes
are distributed in the panel such that common antibodies can be identified and most
others tentatively excluded.
Sample
{ ,
^ j >L/mphopifep i
Mononuclear^
Cells (pbmc's) , Mm
Erythrocytes
Figure 3.3 Example of separation phases after centrifugation of human blood.

Blood is diluted 1:1 with cell culture media and layered above an equal volume of
Lymphoprep (Nycomed) density gradient media. Samples are centrifuged at 800 g
for 30 min at RT to separate blood cell types. Heparin was added to the media to
prevent platelet aggregation.
The non-coagulated sample was purified by density gradient centrifligation by ARCBS-
NSW personnel using Lymphoprep (Nycomed) to remove RBCs and granulocytes
(B0yum, 1968), and using sucrose in a subsequent step to remove platelets (Chanarat
and Chiewsilp, 1975). The separation media contains a polymer compound
(polysaccharide) which causes erythrocytes to aggregate and therefore sediment under
centrifugation. Also present is a high density compound which separates the lower
density lymphocytes from the higher density granulocytes and aggregated erythrocytes.
The blood sample is either layered above or below the separation media and, after
centrifugation, the mononuclear cells form a distinct band at the sample/medium
interface while other cells are pelleted at the bottom of the tube (Figure 3.3). The
resulting PBMC sample contains mainly lymphocytes with a smaller number of
monocytes (20-35 % and 2-6 % respectively of the original blood sample). These
PBMC samples were stored in liquid nitrogen for several years before being accessed
for use in this project (Table 3.3).
3.3.2 Indirect Antiglobulin Technique (Saline lAT)
This technique was used to identify samples containing clinically significant antibodies
to RBC antigens suitable for use in library construction. Although information
regarding the presence of atypical antibodies in donor blood samples was available from
ARCBS records held on the NSW Blood Transfusion Services (NSWBTS) database, all
of the available donor serum samples held as frozen stocks in the Cell Biology
Department which also had a PBMC sample available, were tested by saline lAT. If
samples of serum and PBMC from the donor were not available from exactly the same
collection date, then serum samples collected close to the PBMC collection date were
tested.
In this method, a panel of fully typed Group O RBCs (Phenocell, CSL) are reacted with
serum and the level of agglutination measured to indicate the presence of any IgM
antibodies (Figure 3.5). The red cells were then washed and anti-human globulin added
which binds to any serum antibodies already bound to the RBCs, and causes
agglutination. Agglutination observed in the second step indicates the presence of IgG
antibodies. IgG antibodies alone do not bring about agglutination of RBC as they are
monomeric immunoglobulins and a higher order multimer is required for direct
agglutination such as IgM which is pentameric (Figure 3.4).
Direct Agglutination Test ^^^^^^^^ Antiglobulin Test

(Coombs Test)
Pentameric IgM Monomerie IgG
NO AGGLUTINATION
Anti-Human
Globulin
AGGLUTINATION AGGLUTINATION
Figure 3.4 Illustration of direct and indirect agglutination (Coombs test; Coombs
et al., 1946).
Presence of IgM in a sample causes agglutination due to the presence of multiple
binding sites on each molecule. The two binding sites present on an IgG molecule
are insufficient to cause agglutination and further cross-linking has to be brought
about by the addition of an anti-human immunoglobulin antibody.
Seventeen donor serum samples were thawed and tested by Saline lAT for the presence
of either IgM or IgG type atypical antibodies according to Section 2.5.1. Each assay
contained an AB serum sample (obtained from the ARCBS-NSW) as a negative control.
Only 5 out of the 11 RBC suspensions in the panel were tested with the negative control
as the sample was in short supply. Based on analysis of these results, and results from
the NSWBTS database, samples were selected for EBV transformation. Serum samples
were not available for the four patient samples used in EBV transformation and
therefore could not be tested by lAT. Instead, information supplied at the time of
sample collection was relied on for the antibody specificities of interest likely to be
present in the sample.
REACTION GRADE
4 3 2 1 Neg
Figure 3.5 Grading of RBC agglutination reactions.

Grade 5: Cell button remains in one clump or dislodges into a few large clumps,
macroscopically visible.
Grade 4: Cell button dislodges into numerous clumps, macroscopically visible.
Grade 3: Cell button dislodges into many small clumps, macroscopically visible.
Grade 2: Cell button dislodges into finely granular but definite small clumps,
macroscopically visible.
Grade 1: Cell button dislodges into fine granules, microscopically visible.
Grade 0 (Neg): Negative result: cells flow in a line when tube is tilted and no
clumps are visible.
(Figure from Watson et al., 2004)
3.3.3 Interpretation of Phenocell Panel Results
Each Phenocell panel is supplied with an antigen composition sheet which shows
antigens present on the RBCs supplied. The blood systems typically represented in the
eleven RBC suspensions include; Rhesus, Kell, Duffy, Kidd, MNSs, P Group, Lewis,
Lutheran and Colton. These antigens are selected because their concomitant antibodies
are clinically significant in blood transfusion. The antigen composition sheet also
indicates the Rhesus type of each cell type included in the Phenocell panel,
abbreviations for which are explained in Table 3.1.
Table 3.1 Rhesus nomenclature

D is referred to as the Rhesus antigen (see Section 1.1 for explanation). 'R'
represents a Rhesus positive RBC (has D antigen expressed on surface) and 'r'
represents a Rhesus negative RBC (no D antigen present). The Rhesus group of
antigens also includes the antithetical antigens C and c, and £ and e. Suffixes to
the 'R' or 'r' code indicate which of C, c, E, and e antigens are also present on the
RBC type. There are two copies of these gene complexes present in each person
which may be different, thus each cell type in the Phenocell panel is represented by
two Rhesus Type codes, e.g. Ro r (representing different alleles) or Ri Ri
(representing the same alleles).
Rhesus Type Antigens represented
Ro D, c,e
Ri D, C,e
R2 D, c,E
r d*, c, e
r' d*, C, e
r" d*, c, E
Rz D, C,E
fy d*, C, E
*Antithetical antigen'd' is postulated but has never been found.
Analysis of results from an agglutination test is a relatively complex process. The panel
identifies antibodies via a process of elimination and will show conclusively which
antibodies are n ^ present in the sample. However, antibodies not excluded in the
elimination panel may or may not be present in the sample and further testing with an
extended cell panel would be needed for confirmation.
In addition, any one of the RBCs in the panel can be either homozygous or
heterozygous for a given antigen. Using the example of the E antigen (E and e alleles)
from the Rhesus system, if a REC is homozygous for E (E+e- therefore expressing EE)
then a strong agglutination reaction will result from any anti-E present in the serum. If
a serum sample gave a negative result against these RBCs then it could be concluded
with certainty that there was no anti-E present in the sample. If however, the RBC is
heterozygous for E (E+e+ therefore expressing Ee and having a lower level of E antigen
on the RBC surface) it would only give a weak agglutination reaction if any anti-E is
present and the signal may be below the limit of detection. Therefore if a negative
signal is recorded for a heterozygous antigen, it cannot be concluded with certainty that
there is no concomitant antibody in the sample and this antibody is tentatively excluded
(circled on the composition sheet) rather than being discounted entirely. Many RBC
antigens are allelic (Table 3.2) and need to be considered in this way when analysing
agglutination reactions.
Table 3.2 Allelic antigens present in various blood group systems.

Blood Group System Alleles
Rhesus D d*
E e
C c
Cw Cx MAR
Kell K k
Kp^ Kp^ Kp'
Duffy F/ Fy"
Kidd Jk"" ¡k"
MNS M N
*The Rhesus antigen D is a special case. Its antithetical antigen'd' is postu ated but
has never been found so D must always be considered as heterozygous and therefore
can only ever be tentatively excluded during analysis.
3.3.4 Epstein Barr Viral Transformation
To enrich for antibody-producing B cells, obtain sufficient RNA for library construction
and generate a renewable resource, the PBMC samples were transformed to give
actively proliferating B lymphoblastoid cell lines (LCLs). This was carried out using
EBV, in the presence of PHA which stimulates growth of the B cells. The other
lymphocyte cells present in the PBMC preparation (e.g. T cells) are not infected by
EBV and die during the early stages of cell culture.
Due to the amount of cell culture involved, only two or three transformations were
performed at a time (see Table 3.3 for details of PBMC samples). PBMC samples from
donor 1 and donor 2 were removed from liquid nitrogen storage and thawed as
described in Section 2.5.2. The samples were EBV transformed as described in Section
2.5.3 and then counted and analysed for viability (Section 2.5.5). After cell culture of
these samples was completed (Section 2.5.4), donor 3 and patient 1 PBMC were
transformed and cultured as for donors 1 and 2. At this time a further donor PBMC
sample (donor 4) was EBV transformed in the Cell Biology department of the ARCBS-
NSW for production of a heterohybridoma. Some of the cells from the lymphocyte cell
line generated were made available for use in this project. Finally, donor 5 PBMC were
transformed along with a repeat transformation using two vials of donor 3 PBMC due to
poor RNA yields from the first experiment. Later in the project, further stocks of useful
PBMC samples were identified which had been collected from patients having
undergone a transfusion reaction. These samples were referred to as patient 2, patient 3
and patient 4 and were transformed and cultured in the same way as the donor samples.
Table 3.3 Collection dates of PBMC samples used in EBV transformation.
PBMC Sample Collection Date
Donor 1 3/6/97
Donor 2 5/7/96
Donor 3 19/4/00
Donor 5 5/5/99
Patient 1 11/8/99
Patient 2 21/11/89
Patient 3 25/11/89
Patient 4 21/7/92
3.3.5 Maintenance and Storage of Lvmphoblastoid Cell Lines
After EBV transformation, LCL cultures were maintained for several weeks (Section
2.5.4) during which time sufficient cell numbers were obtained to put several vials of
cells into liquid nitrogen storage (Section 2.5.6) and provide enough material for RNA
extraction. Storage and RNA extraction were performed as early as possible during the
cell culture process as soon as sufficient cells were available in order to maintain B cell,
and therefore antibody, diversity. The longer the cell population is cultured, the greater
the possibility of certain clones predominating along with the loss of less vigorous B
cell types.
In the case of patient 4 LCL cell counts and viability dropped steadily during culturing.
Since no stocks of this cell line had been made due to lack of growth, mouse
macrophage feeder cells from mouse peritoneal cavity (isolated by ARCBS-NSW staff)
were added to wells containing the transformed cells in an attempt to save the sample.
Macrophages were added to give a final concentration of 10"^ cells per mL of LCL
culture. During the cell culture process, samples of culture supernatant were retained
and tested for the presence of antibody by Saline lAT (Section 2.5.1).
3.3.6 Eukarvotic RNA Extraction
Total RNA was extracted from the antibody producing LCLs (Section 2.6.1) to supply
antibody gene templates for PGR and library construction. During extraction it was
important to avoid exposure of samples to endogenous RNases to ensure that RNA
extracted from LCLs was of high quality and not degraded (Figure 3.6). Non-degraded
eukaryotic RNA appears as two predominant, sharp bands on a denaturing gel referred
to as 28S and 18S ribosomal RNA. The upper band (28S) is stronger than the lower one
(18S) with other minor mRNA bands evident. Poor quality, degraded RNA appears as
smear of many small bands down the gel with little or no distinct 28S and 18S species.
Poor quality RNA would seriously compromise the library construction process as full
length antibody-coding messenger RNA may not be available for amplification. RNA
samples were analysed by denaturing agarose electrophoresis (Section 2.6.2) and
quantitated by spectrophotometry (Section 2.6.7). The RNA was analysed under
denaturing conditions as the presence of secondary structures within native RNA
molecules (formed via intra-molecular base pairing) prevents molecules from migrating
according to their molecular weight.
/
/ /
w 4
IBS
H I8S
Figure 3.6 Denaturing gel electrophoresis showing an example of degraded and

intact RNA.
3.4 Results
3.4.1 Saline Indirect Agglutination Tests
A total of seventeen donor serum samples were tested using a panel of RBCs (Phenocell,
CSL). Results were recorded on 'Antigen Composition Sheets' which were supplied
with the Phenocell kit (Figures 3.7 to 3.11). Agglutination results were recorded on a 0
to 4+ scale (1+ grade is simply recorded as +) where zero represents no agglutination of
RBCs and 4+ represents complete agglutination of all RBCs in sample or RBC
haemolysis which also indicates a strong antibody reaction. Only results sheets for the
donors subsequently selected for transformation are shown. Antibodies not excluded
during interpretation of agglutination results for all donor samples are listed in Table 3.4.
Direct agglutination of RBCs by serum indicated the presence of IgM subclass
antibodies (see Column 3, Table 3.4). Indirect agglutination of RBCs (via addition of
anti-human globulin), if no direct agglutination was observed, indicated the presence of
IgG antibodies (see Column 4, Table 3.4). Also listed for comparison are results
obtained in ARCBS blood typing laboratories (from NSWBTS database) for serum
samples from the same donors although not necessarily from the same sampling date
(see Column 5, Table 3.4). Based on these results, five donor samples were selected for
EBV transformation.
3.4.2 Maintenance of Lvmphoblastoid Cell Lines, Storage and RNA Extraction
A total of five donor and four patient PBMC samples were EBV transformed. Cell
count and viability data for donor cell lines 1, 2, 3 and 5 are shown in Table 3.5 and
data from cell lines 1 and 2 are represented graphically in Figure 3.12. Data from
patient cell lines 2, 3 and 4 are shown in Table 3.6 and Figure 3.13. Cells were
examined every 3 to 4 days for signs of growth; appearance of cell clumps and a
lowering of media pH as indicated by a media colour change from red towards yellow.
Data are shown in the table only when the cells were passaged (collected from culture,
pelleted by centrifugation, resuspended and counted). During periods of greater than 3
or 4 days between cell counts, the cultures were diluted with fresh media but not
passaged.
Cell count data is not available for donor 4. This sample was EBV transformed by
ARCBS-NSW personnel for use in the production of a heterohybridoma cell line. To
conserve resources, the sample was not separately transformed for this project but
instead, the cell line was expanded to give sufficient cell counts for both experiments.
Approximately 2x10^ cells from this cell line were obtained for RNA extraction. Cell
count data is not presented for patient 1 as cell counts could not be maintained at or
above the starting cell count of 3 x 10^. Viability declined sharply and the cell line was
lost after two passages.
3.4.3 Saline lAT of Cell Culture Supematants
Cell culture supematants from several passages of donor LCLs 1, 2 and 5 were tested by
saline lAT (Section 2.5.1). Out of the several possible antibody specificities that could
be present, only anti-D antibody could be detected in all of the samples tested (results
not shown).
3.4.4 Total RNA Extraction
A total of nine RNA extractions were performed (donor 3 transformation, culture and
RNA extraction was repeated) and samples analysed by electrophoresis and
spectrophotometry. Figures 3.14 and 3.15 show denaturing agarose gel electrophoresis
of donor and patient RNA samples. Although varying in amount of RNA, all samples
show the same band pattern of two major bands with the upper band being more intense
than the lower. In addition, no lower molecular weight RNA species were observed.
Spectrophotometric results for RNA samples are shovm in Table 3.7. Samples varied in
concentration from -0.2 to 1.6 mg/mL giving total yields for donor samples 1 to 5 of
110, 32, 18+20, 308 and 116 jig respectively. RNA yields for patient samples 2 to 4
were 98, 76 and 160 jiig respectively.
Table 3.4 Saline lAT and NSWBTS database results for donor samples.
oo3 Ü ^ IgM Ab (direct IgG Ab (tested Antibody Information from ARCBS

O ^ 1 aggluf) by lAT) Testing
(From NSWBTS Database)
1 3/6/97 None C, D, E, Cw, 14/2/96 C, D, E, Fy' (PEG I AT)

Lu^ 2/11/99 C, D, E, Fy' (Saline lAT)
21/3/00 C, D, E, Fy' (PEG lAT)
2 10/3/00 r r C, D, E, Cw, K, 16/7/98 D, C, Fy' (Saline lAT), PI
Fy^ Lu^ co'^
3 19/4/00 K, Co' K, Co' 17/11/99 K (Saline lAT), M
4 10/2/99 None D, Cw 12/8/98C, D, E 6/10/99 C , D , E , F y '
5 5/5/99 None C, D, Cw, Lu' 14/1/98 C, D, K (PEG I AT), E(w)
24/5/99 C, D, K
6 7/7/97 None C^^ ^^W i — 5 23/4/97 D, Jk' (PEG lAT)
Lu^ Co'' 7/7/97 D, Jk' (PEG lAT)
7 \/7/91 None C, D, Cw, Lu' 15/12/88 C, D, E 30/4/99 C, D, E
7 22/6/99 c C, D, Cw, Lu'
8 15/6/99 C, D, E, Cw, Lu^ C, D, E, Cw, 31/5/99 C , D , E 29/6/99 D
Lu^
8 19/4/99 D, E, C^v C, D, E, Cw,
Lu^
9 22/12/9 None C, D, E, Cw, 22/8/96 C, D, E 15/4/99 C, D, E
5 Lu^
9 28/5/97 None C, D, E, Cw, 13/7/88 C , D , E 23/7/99 C , D , E
Lu^
9 8/2/99 C, C^,Kp^ M, Lu' C, D, E, Cw, K,
Kp', M, Lu'
10 28/2/96 None D, Cw 9/1/89 C, D,E
2/7/97 D, E
11 18/6/97 C, D, E, Cyv C, D, E, Cw, 14/8/98 C, D, E
Lu^
12 30/8/95 None D, Cw 7/6/88C, D, E 17/1/97 C , D , E
13 21/5/92 None D, E, Cw 11/12/97 C, D, E
14 20/9/95 None C, D, E, Cw, 26/7/88 C, D, E 26/3/96 C, D, E
Lu^
15 20/9/95 E C, D, E, Cw, 10/11/97 C , D , E
Lu^
15 3/6/97 C, D, E, Cw, Lu' C, D, E, Cw?
Lu^
16 4/6/97 D, Cw D, Cw 20/5/97 D 2/7/97 D
17 20/9/95 None D, Cw 16/6/87 C, D, E 12/2/99 C, D, E
Table 3.5 Cell growth and viability data for donor LCLs 1, 2, 3 and 5.
Time points where cells were harvested for producing cell stocks and RNA are
shown.
Date Viable Cell Viability Freeze/RNA
count (total) Extraction
Donor 1
Transformation 4/4/00 4.56 X 10^ 90%
Passage 1 10/4/00 8.3 X 10' 75%
Passage 2 14/4/00 12.5 X 10' 70% 1 vial 4 X 10'
Passage 3 17/4/00 8.2 X 10' 61 %

Passage 4 19/4/00 6.3 X 10' 63 % RNA extr.
Donor 2
Transformation 4/4/00 11.5 X 10' 100%
Passage 1 10/4/00 2.4 X 10' 95 % 3 vials 5 X 10'
Passage 2 14/4/00 5.0 X 10' 68%

Passage 3 17/4/00 6.9 X 10' 68%
Passage 4 19/4/00 3.3 X 10' 50% RNA extr.
Donor 3
Transformation 16/5/00 13 X 10' 100%
Passage 2 29/5/00 28.4 X 10' 60% 2 vials 5 X 10'

RNA extr.
Donor 5
Passage 2 29/5/00 lOx 10' 50% 1 vial 5 X 10'

RNA extr.
Reagent Red Blood Cells N.irro !: •a:^; Ol Qitth
Phenocell
I.D. N u . Waid Bien 10/02/99
Blorri G r o u p , Rh P h e r r i l y p ? ü i i ö c l Anliülübu ii'i
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CELL REF. Rh Rh esus Keil 1 Duffvl Kidd WiNSs Lewis ILU I CO Additional Rcsul s
©
P CELL
NO. NO. TYPE C D E 6 e c"' K k © ) • ¥y ¥y Wt' ik^ N Q s fee te Lu Information NO. Test -ve +ve
I 000880 R.R, + + 0 0 + 0 + + 0 0 + 0 + + 0 + 0 0 + + 0 1 3+ 0 +
2 118116 RrR, + + 0 0 + + 0 + 0 + 0 + + + + 0 + 0 + 0 0 Bg(b+') 2 3+ 0 +
3 332666 R2R2 0 + + + 0 0 0 + 0 + + + 0 + + + + + 0 0 0 3 3+ 0 +
4 633338 R2R2 0 + + + 0 0 + + 0 0 + + 0 + 0 + 0 + 0 0 4 3+ 0 +
5 180619 Ror 0 + 0 + + 0 0 + 0 + 0 0 + + 0 + + 0 0 + 0 5 3+ 0 +
6 303090 r'r + 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 6 2+ 0
7 141422 r"r 0 0 + + + 0 0 + 0 + 0 0 + + 0 + 0 0 + 0 0 7 2+ 0
8 337463 rr 0 0 0 + + 0 0 + + 0 + 0 + + + + + 0 + 0 0 8 0 0
Y 008000 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + 0 + 0 0 + 0 + •Ch (a-) 9 0 0
10 128198 rr 0 0 0 + + 0 0 + 0 0 + + 0 0 + + + + 0 0 0 10 0 0
11 252142 rr 0 0 0 + 0 + 0 0 + + + + + + + 0 0 + 0 0 11 0 0
Auto Auto
Ab 1 Ab 1
Ab 2 Ab 2
Ab 3 Ab 3
I ^
c D E c c C" K k Kp" F y Fy Jk" Jk^ M N s s Pi Le" Le Lu" Co^ Cord
Noie: A l l c.;lls arc positive for Vel. Lu^. Kp^ and Co" unless stated in "Additifmal Intbrmation" coliimn Al
*Ch(a-) : High incidence antigen (Ref: Issitt P. D. "Applied Blood Group Serology" 3rd Edition Pgs 4 2 0 - 2 2 ) A2
Red cells provided by voluntary donors, selected, collected and tested by the Australian Red Cross Blood Service.
06640299G Prepared and packaged by CSL Limited, 45 Poplar Road, Parkville, 3052, Victoria, Australia. October, 1998
Figure 3.7 Saline I AT Results Sheet for donor 1.

Recorded results shown in blue text. Antibody specificities definitely excluded are crossed out while those tentatively excluded are
circled. Blue figures 0 to 4+ represent grades from negative to complete agglutination respectively.
Roagont R e d Blood Cells Nan-p _ Donor 2 D a : . i al Birth
Phenocell
I.D. N u Wtitd O a - c Bier: in/nr^/nn
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Antibody Wentificaton Panel T-=s:er b v SALINE lAT D a l t í TöülOÜ 29/03/0Q..
00
CELL REF Rh Rhesus Kell 1 Duffy 1 Kidd INSs P Lewis Ilu ICo Additional CELL Resul ts
NO. NO TYPE C D E e e c" K k Q f Fy^ Fy'- Jk'
© N Le" tTe** Lu" Co^ Information NO Test -ve +ve
1 0(XJ880 R,R, + + 0 0 + 0 + + 0 0 + 0 + + 0 + 0 0 + + 0 1 3+ 0 3+
2 118116 R^Ri + + 0 0 + + 0 + 0 + 0 + + + + 0 + 0 + 0 0 Bg(b+') 2 3+ 0 3+
3 332666 R2R2 0 + + + 0 0 0 + 0 + + + 0 + + + + + 0 0 0 3 3+ 0 3+
4 633338 R2R2 0 + + + 0 0 + + 0 0 + + + 0 + 0 + 0 + 0 0 4 3+ 0 3+
5 180619 Ror 0 + 0 + + 0 0 + 0 + 0 0 + + 0 + + 0 0 + 0 5 3+ 0 3+
6 303090 r'r + 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 6 2+ 0
7 141422 r"r 0 0 + + + 0 0 + 0 + 0 0 + + 0 + 0 0 + 0 0 7 2+ 0
8 337463 rr 0 0 0 + + 0 0 + + 0 + 0 + + + + + 0 + 0 0 8 0 0
9 008000 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + 0 + 0 0 + 0 + *Ch ia-) 9 2+ 0

10 128198 rr 0 0 0 + + 0 0 + 0 0 + + 0 0 + + + + 0 0 0 10 0 0
11 252142 rr 0 0 0 + + 0 + 0 0 + + + + + + + + 0 0 + 0 0 11 2+ 0
Auto Auto
Rífercíc N.i
Ab 1 Ab 1
Ab 2 Ab 2
Ab 3 Ab 3
C" Kp^ Fy Fy h Jk"
Jk^ Pl Le Le Lu" Co"^
T- Í1 r a I h
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N o t e : A l l c cU s a r c p o s i t i v e l o r V e l , l,u'', K p ' ' a n d C o " u n l e s s s t a l e d i n " A d d i t i o n a l I n f o r m a t i o n " c o l u m n
A,
*Ch(a-) : High incidence antigen (Ref; Issitt P. D. "Applied B l o o d Group Serology" 3rd Edition Pgs 4 2 0 - 2 2 ) A2
Figure 3.8 Saline lAT Results Sheet for donor 2. Note: Figure 3.7 describes recording and interpretation of agglutination results.
Reagent R e d B l o o d C e l l s I N.'irrp _ Donor 3 Da:»i ol Qitth
Phenocell
I.D. N o . Waid OaK- Bier: 19/04/00
B l o r rI G r o u p . Rh Pherr:1ypo ÜIIÜC1 A n i i g l o b u 111
liUü'ptHiatioi' K, C o '
A n t i b o d y IdentHicaton Panel bv SALINE lAT ualtì I 16/0^/Qg-
CELL REF. Rh Rh esus Kell 1 Duffy 1 Kidd IVINSs p Lewis ILU I CO Additional CELL Resili s
NO. NO TYPE G © E e e © K k © ' Fy^' ik" M N S s Q 0 0 Co' Information NO Test -ve +VC
1 13078.1 RiRi + + 0 0 + 0 + 0 0 + + 0 + 0 + 0 + 0 + 0 + 1 2+ 0 4+
2 017604 + + 0 0 + + 0 + 0 0 + + + + + + + + 0 0 0 2 0 0 4+
3 348038 R2R2 0 + + + 0 0 0 + 0 0 + + 0 + 0 + + 0 0 0 0 3 + 0 4+
4 160886 R:R2 0 + + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 0 *Yt(bf) **Sd(a+)'' 4 0 0 4+
5 122253 R<)r 0 + 0 + + 0 0 + 0 0 + + + + + + 0 0 0 + 0 0 #Lu:14Pos Bg(b+) 5 0 0 4+
6 335448 r'r + 0 0 + + 0 0 + 0 + + + 0 + + 0 + + 0 0 0 6 0 0
7 213043 r"r 0 0 + + + 0 0 + 0 + + + + + 0 + + 0 + 0 0 0 7 0 0
8 153984 rr 0 0 0 + + 0 + + 0 0 + f 0 + 0 + 0 0 + 0 0 8 2+ 0
9 104438 rr 0 0 0 + + 0 0 + 0 0 + 0 -1- 0 + 0 + 0 + + 0 9 0 0
10 148965 rr 0 0 0 + + 0 0 + + 0 0 + + + 0 + + 0 + 0 0 10 0 0
11 118877 rr 0 0 0 + + 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 0 0 11 0 0
Auto Auto
Ab 1 Ab 1
Ab 2 Ab 2
Ab 3 Ab 3
„ IL I ') I
c D E c e K k K p " F y " Fy J k " Jk'^ M N s s Pi Le Le Lu" C o Cord
Nolo All cc lis are positive lor Vel, l.ii'', Kp'' uiul Co'' unless sliHed in "AcUlitionul Inloniiiilion " cokunn
A,
*Yt'' : A low i n c i d e n c e antigen (Ref: Issitt P. D, " A p p l i e d B l o o d G r o u p S e r o l o g y " 3rd Edition Pgs 3 8 7 - 8 8 )
A2
**Sd'' : Strong Expression (Ref: Issitt P. D. " A p p l i e d Blood G r o u p S e r o l o g y " 3rd Edition Pgs 4 2 6 - 2 7 ) B
#Lu: 14 : A low i n c i d e n c e antigen (Ref: Issitt P. D. " A p p l i e d Blood G r o u p S e r o l o g y " 3rd Edition Pg 380)
Rftagont R e d Blood Cells Nan'p _ Donor 4 •ä'tTf DI Biith
I.D. N u Waid Oaie Bier 10/02/99
Phenocell Blnr rl G r o u p .
InlO'p'ulalior n, r/
flh P h e r n l y p s Diiücl AntiQlübu ll^
Antibody Identrticaton Panel bv SALINE lAT Dalfc! T y ü l ö ü 29/03/00
CELL REF. Rh Rh esus Kell 1 Duffvl Kidd MNSs P Lewis ILU ICO Additional CELL Results
NO. NO TYPE £ D © e e c" K k ¥y ik^ M N S s Li'C LRII IRTI Go Information NO Test -ve +ve
1 000880 R.RI + + 0 0 + 0 + + 0 0 + 0 + + 0 + 0 0 + + 0 1 4+ 0 4+
2 118116 + + 0 0 + + 0 + 0 + 0 + + + + 0 + 0 + 0 0 Bg(b+') 2 4+ 0 4+
3 332666 R2R2 0 + + + 0 0 0 + 0 + + + 0 + + + + + 0 0 0 3 4+ 0 4+
4 633338 R2R2 0 + + + 0 0 + + 0 0 + + + 0 + 0 + 0 + 0 0 4 4+ 0 4+
5 180619 Ror 0 + 0 + + 0 0 + 0 + 0 0 + + 0 + + 0 0 + 0 5 4+ 0 4+
6 303090 r'r + 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 6 0 0
7 141422 r"r 0 0 + + + 0 0 + 0 + 0 0 + + 0 + 0 0 + 0 0 7 0 0
8 337463 rr 0 0 0 + + 0 0 + + 0 + 0 + + + + + 0 + 0 0 8 0 0
9 008000 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + 0 + 0 0 + 0 + *Ch (a-) 9 0 0
10 128198 rr 0 0 0 + + 0 0 + 0 0 + + 0 0 + + + + 0 0 0 10 0 0
11 252142 rr 0 0 0 + + 0 + 0 0 + + + + + + + + 0 0 + 0 0 11 0 0
Auto Auto
Rofoencc N,.
Ab 1 Ab 1
Ab 2 Ab 2
Ab 3 Ab 3
c D E c e K k Kp" Fy" Fy Jk" Jk" M N s s PI Le" Le" Lu' Co Cord
Nole: All ce Us are positive Cor Vel, l.u''. K.p'' a n d Co" unless stated in " A d d i t i o n a l I n f o r m a t i o n " e o l u m n
A,
* C h ( a - ) : H i g h i n c i d e n c e a n t i g e n (Ref: Issitt P. D. " A p p l i e d B l o o d G r o u p S e r o l o g y " 3rd Edition Pgs 4 2 0 - 2 2 ) A2
Reagent Red Blood Ceils Narro _ Donor 5 D a : ^ Ol B i n h
Phenocell
I.D. N u ... W a i d . Bier nt^/ng/QQ
B l n r ri G r o u p . Rh Pherr.lvps ü i i ü c i A n i i a l o L u ii't —-
Inlü'prHiatioi' C, D, C^, K, Lui'
Antibody WentHicaton Parel bv SALINE lAT Daití 06/04/00,
CELL REF. Rh Rhesus Keil 1 Duffvl Kidd V [NSs P Lewis ILU ICO Additional CELL Resul ts
NO. NO. TYPE c D © e e K k © * Fy" Hi" © N S s tre" Lu'' Ge*^ Information NO Test -ve +ve
1 (K)0880 RlRl + + 0 0 + 0 + + 0 0 + 0 + + 0 + 0 0 + + 0 1 4+ 0 4+
2 118116 Ri'Ri + + 0 0 + + 0 + 0 + 0 + + + + 0 + 0 + 0 0 Bg(b+') 2 4+ 0 4+
3 332666 R2R2 0 + + + 0 0 0 + 0 + + + 0 + + + + + 0 0 0 3 4+ 0 4+
4 633338 R2R2 0 + + + 0 0 + + 0 0 + + + 0 + 0 + 0 + 0 0 4 4+ 0 4+
5 180619 Ror 0 + 0 + + 0 0 + 0 + 0 0 + + 0 + + 0 0 + 0 5 4+ 0 4+
6 303090 r'r + 0 0 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 6 3+ 0
7 141422 r"r 0 0 + + + 0 0 + 0 + 0 0 + + 0 + 0 0 + 0 0 7 0 0
8 337463 rr 0 0 0 + + 0 0 + + 0 + 0 + + + + + 0 + 0 0 8 0 0
9 008000 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + 0 + 0 0 + 0 + *Ch (a-) 9 0 0

10 128198 rr 0 0 0 + + 0 0 + 0 0 + + 0 0 + + + + 0 0 0 10 0 0
11 252142 rr 0 0 0 + + 0 + 0 0 + + + + + + + + 0 0 + 0 0 11 2+ 0
Auto Auto
Ab 1 Ab 1
Ab 2 Ab 2
Ab 3 Ab 3
IR I " ^ h
c D E c e c" K k K p ' F y " Fy Jk" Jk'^ M N s s Pi Le Le Lu" C o Cord
Note; All cclls are ix>sili\e for Vcl. I.u'', Kp^' and Cn^ unless stated in "Additional Infomiation" column A,
* C h ( a - ) : High incidence antigen (Ref: Issitt P. D. " A p p l i e d B l o o d G r o u p Serology" 3rd Edition Pgs 420-22) A2
-r 1 0 0
I
j- 90
-f 80
t 70
i
^ 60
- 50
n
>
40
30
- 20
Cells harvested - 10
for RNA Extraction t t
-- 0
6 8 10 12 14 16
Days Post Transformation
Donor 1 Cell Count Donor 2 Cell Count
<>• • • Donor 1 Viability • - • Donor 2 Viability
Figure 3.12 LCL cell growth and viability curve plotted against days after EBV transformation for donor LCLs 1 and 2.
Time points where cells were harvested for producing cell stocks and RNA are shown. See Table 3.5 for data.
100
•
30 T3 90
I 80
70
h
60
I ^ ^ i Cells H a r v e s t e d
for freezing.
3
I 50
f f
I
40
30
i 20
i 10
Added Macrophages
0
0 10 20 30 40 50 60
Days Post Transformation
Patient 2 Cell Count Patient 3 Cell Count Patient 4 Cell Count

o- - - Patient 2 Viability - - - A - - Patient 3 Viability - - Patient 4 Viability
Figure 3.13 LCL cell growth and viability curve plotted against days after EBV transformation for patient LCLs 2,3 and 4.
Time points where cells are harvested for producing cell stocks and RNA are shown. See Table 3.6 for data.
Table 3.6 Cell growth and viability data for patient LCLs 2 , 3 and 4.
Time points where cells were harvested for producing cell stocks and RNA are shown.
Time point of macrophage addition to patient 4 LCL is also indicated.
Date Viable Cell Viability Freeze/RNA
Count (total) Extraction
Patient 2
Transformation 23/01/01 4.5 X lO'' 95%
29/01/01 2.7 X 10^ 73%
05/02/01 3.2 X lO'^ 50%
09/02/01 1.6 X 10^ 39%
16/02/01 16.4 X 10^ 80% 10'RNA extr.
20/02/01 25.6 X 10^ 77% 5 vials 5 X 10'
Patient 3
Transformation 23/01/01 14x 10' 82%
29/01/01 8.6 X 10' 60%
01/02/01 20 X 10' 65% 2 vial 5 X 10'
05/02/01 5.7 X 10' 38%
09/02/01 5.6 X 10' 32%
13/02/01 4.6 X 10' 32%
16/02/01 3.1 X 10' 16%
20/02/01 1.0 X 10' 7%
23/02/01 5 x 10^ 2%
Patient 4
29/01/01 9.6 X 10' 86%
01/02/01 9.5 X 10' 72%
05/02/01 6.4 X 10' 44%
09/02/01 6.6 X 10' 39%
13/02/01 8.6 X 10' 48%
16/02/01 5.8 X 10' 35%
20/02/01 3.8 X 10' 25%
23/02/01 1.4 X 10' 10%
02/03/01 1.2 X 10' 20%
09/03/01 0.8 X 10' 11 % Add m'phages
20/03/01 29.2 X 10' 95% 10'RNA extr.
4 vials 5 X 10'
.'fk
4
•
Donor Donor Donor Donor Donor

1 RNA 2 RNA 3 RNA 5 RNA 4 RNA
28Sj
Figure 3.14 Denaturing agarose gel electrophoresis of donor RNA samples extracted
from lymphoblastoid cell lines.
RNA was extracted using a commercial kit from approximately 10^ cells in a final
volume of 100 jiiL RNase free water. A 5 jiiL aliquot of this sample was loaded on to
the gel.
28S
18S
Figure 3.15 Denaturing agarose gel electrophoresis of patient RNA samples extracted
from lymphoblastoid cell lines.
RNA was extracted using a commercial kit from approximately 10^ cells in a final
volume of 100 |LIL. A 5 ^L aliquot of this sample was loaded on to the gel.
Table 3.7 Spectrophotometric analysis of purified RNA samples.
A 2 ^L sample of RNA was made up to 1 mL volume with Baxter water (water for
irrigation, Baxter Healthcare) and its absorbance measured at 260 and 280 nm to give
an estimate of concentration and purity. Calculations based on 1 OD Unit (260 nm)
being equivalent to 40 ^g/mL RNA.
Sample A260 A280 Cone (^g/mL) Ratio260/280**
Donor 1 0.055 0.034 1100 1.6
Donor 2 0.016 0.016 320 1.0
Donor 3 0.009 0.004 180 2.3
Donor 3* 0.010 0.003 200 3.3
Donor 4 0.077 0.027 1540 2.9
Donor 5 0.029 0.016 580 1.8
Patient 2 0.049 0.024 980 2.0
Patient 3 0.038 0.019 760 2.0
Patient 4 0.080 0.042 1600 1.9
•Repeat transformation to obtain more R NA.
**The A260/280 ratio is greatly affected by pH and since samples were diluted in water and are not buffered,
the ratios can vary. Lower pH results in lower ratios and best results are obtained by dilution in tris buffer pH
7.5. This was not performed in this case as RNA samples were limited due to availability of donors. In
addition, a good indication of purity can be observed from electrophoretic analysis. Samples must be diluted
in water for concentration determination as the RNA extinction coefficient was determined in this way and
would give a different value if calculated in a buffered system.
3.5 Discussion
A total of five donor and four patient PBMC samples were selected and EBV transformed
into lymphocyte cell lines (LCLs). All donor samples and three out of four of the patient
samples were successfully propagated and used to generate cell banks and RNA samples.
Purified LCL RNA samples were of excellent quality making them ideal for use in
subsequent library construction.
3.5.1 Saline lATs
Results obtained from Saline lATs were not always consistent with details from the same
individuals held on the NSW Blood Transfusion Service database. This could be due
operator variation in observation of agglutination results. Macroscopic agglutination was
observed over a light box with a relatively low powered magnifying glass and microscopic
techniques were not employed. As a result, weak positive reactions could have been
missed especially as many of the antibodies of interest occur at low titres in the serum
samples. The purpose of the donor samples used in this study (except donor 3) from an
ARCBS-NSW perspective is to generate heterohybridomas producing anti-D antibody for
use in the prevention of HDN. As a result, donor serum samples contain high titres of anti-
D antibodies which were always easily detected in lATs. The other antibodies produced as
a by product of D immunisation were at much lower titres and therefore were much more
difficult to detect. In order to detect low titre antibodies it may have been necessary to
employ a more sensitive version of lAT such as PEG lAT where polyethylene glycol is
added to promote agglutination and make weak positive results more obvious.
It is also possible that further testing of a sample with an extended panel of RBC types is
required to confirm the presence of antibodies not excluded using the first panel.
Additional cell types were not available during this testing but would be available in the
ARCBS-NSW typing laboratories, hence the more specific results. There are only eleven
types of cells in the Phenocell panel which are used to characterise antibodies of twenty
two possible specificities. Some antigens may only be represented in one RBC type within
the panel, such as C^ and Co^ in the Phenocell panel used here. These antibodies are
therefore less likely to be excluded and further testing would be needed to confirm their
presence. Using the example of C^, this antigen is only present on cell number 2 in the
Phenocell panels used in this study. This cell is also positive for C and D and therefore
most of the donor results obtained, since they are strongly D positive, also indicated the
presence of C^. A further cell type would be required which was D and C negative but C^
positive in order to elucidate the source of the positive result. In some cases, the strength of
agglutination can shed some light on this difference. When considering the two antigens in
question, if one is heterozygous and one homozygous, then the former would be expected
to give a stronger agglutination reaction than the latter. However, this is not an entirely
reliable indicator, as antibody titre will also affect agglutination. It is also possible to
consider the frequency (likelihood of occurrence) of a particular antigen when evaluating
results, which vary greatly between ethnicities.
As donor 3 serum sample did not contain any C, D or E antigens from the Rhesus group, it
was more simple to determine the antibody specificities as K and Co''. It is not likely that
Co'' antibody is present but it was not excluded for reasons previously mentioned. The
NSWBTS database results for this donor also mention the presence of anti-M antibody
which was not detected during testing. This is likely to be due to a sensitivity issue as
mentioned; the anti-K antibody was reported to be present at a titre of 512 whereas that of
anti-M was <1.
3.5.2 Generation and Maintenance of LCLs
Based on previous cell culture experience with hybridomas and human embryonic kidney
cell lines, an overall observation was made that the lymphoblastoid cell lines were difficult
to grow and maintain. Regardless of the viability of the starting PBMC population, some
cell lines did not grow as well as others. This may be explained by the fact that although B
cells are easily transformed by EBV into proliferative lymphoblastoid cell lines, they may
not necessarily be immortalized (Sugimoto et al., 1999). Some cell lines from normal
individuals are not immortal as they undergo telomere shortening and eventual apoptosis
after a certain number of population doublings. Other cell lines are truly immortalised by
developing telomerase activity and other changes determined by the genetic background of
the LCL (Sugimoto et al., 2004).
In one case the use of feeder cells was required to maintain viability of the LCL. Cellular
crisis and cell death can sometimes be avoided by the use of feeder cells (such as
macrophages) in cell culture to promote cell growth. Macrophages are phagocytic cells
involved in the mammalian immune system which also act as antigen presenting cells.
Macrophages secrete many growth factors or cytokines, some of which are targeted to B
cells. For example, B cells carry receptors for Interleukin-1 which is produced by
macrophages (and also by B cells themselves) which enhances B cell viability and cell
proliferation (Habicht et al., 1987).
Despite these difficulties, eight out of the nine samples transformed were successfully
propagated such that cell stocks could be made and RNA samples obtained. The PBMCs
derived from the patient sample that was not maintained in culture had an altered
morphology from the beginning of cell culture and experimentation. The PBMC sample
was not pure, as many RBCs and platelets were present. Since the individual from which
the sample was obtained had recently undergone a transfusion reaction, it may be expected
that the cellular components of the blood would behave atypically during fractionation. In
addition, after EBV transformation the cells appeared to be clumped together which does
not usually occur until cells begin to grow and divide. It is unclear which of these factors
was the cause of the poor viability, but cell counts could not be maintained at or above
initial levels and the PBMCs were not propagated.
The successful transformations were also subject to considerable variations in growth and
viability. Initial viability of the PBMC samples was consistently high (between 80 and 100
%) so this is unlikely to be a cause of the variation. It is possible that the efficiency of EBV
transformation is variable resulting in differences in cell growth. Reported figures of
transformation are between 1 % and 40 % efficiency (Bird et. al., 1981; Stein and Sigal,
1983). It is also worth noting that these LCLs are polyclonal and their constituent cells
may vary with respect to immunoglobulin expression as well as size and shape. There is
also discussion in the literature about the extent of immortalisation of LCLs as distinct from
transformation (Sugimoto et al., 1999 and 2004). The lymphocytes are easily transformed
by EBV but are not necessarily immortalised according to definition (i.e. infinite
proliferation). The level of infection and expression of the EBV genes required for growth
may also vary between cells.
It is a concern that the viability of LCLs was suboptimal in some cases. It is likely that
decrease in viability results in certain B cell specificities being lost. This would mean that
those antibody specificities would not be represented in the extracted RNA and therefore
not present in the final antibody library.
3.5.3 Saline lAT of Cell Culture Supernatants
Cell culture supernatants from several passages of donor LCLs 1, 2 and 5 were tested by
saline lAT. In theory, any antibodies detectable in the original serum sample should be
detectable in the culture supernatants as the same B cell antibody producing population
should be present. Out of several possible antibody specificities which could be present in
the samples, only anti-D antibody could be detected. This could simply be a sensitivity
issue as many of the antibodies of interest in donor samples were at a very low titre in the
serum. Since the distribution of antibody producing cells in the LCL is likely to be similar
to the original PBMC population, the inability to detect low titre antibodies may be
expected. Anti-D is present at high titres in all donor samples (except donor 3) so its
detection in the supernatants from the resulting LCLs is to be expected.
3.5.4 RNA Extraction
Denaturing agarose electrophoresis of RNA indicated that the samples were all of excellent
quality (Figure 3.14 and 3.15) and therefore suitable for library construction. Two strong
bands were observed for each sample on ethidium bromide staining, representing 28S and
18S ribosomal RNA species. The upper band showed approximately twice the intensity of
the lower band which is a good indication of intact RNA. No smearing or ladder of bands
was observed below the lower band in any of the samples, also indicating that the samples
had not undergone any degradation during extraction and purification.
Spectrophotometric analysis of RNA (Table 3.7) demonstrated that yields of RNA,

although low is some cases, were sufficient for reverse transcription and library
construction. The absorbance values obtained were a little lower than is optimal to ensure
significance of the readings. A larger sample size was not used for analysis as the sample
could not be recovered for further use and RNA conservation was of high importance.
Spectrophotometry was also used to determine RNA purity by calculating the ratio of
absorbance at 260 and 280 nm. Ratios should be between 1.9 and 2.1 for pure RNA but
values up to 2.3 can be obtained with some spectrophotometers. The values obtained for
the RNA samples purified from LCLs were in the range 1.0 to 2.9. This is not likely to
indicate that the RNA is impure, rather that the samples were not buffered before
measurement. The ratio should be determined in tris buffer at pH 7.5 as values are
considerably affected by pH. Since water is not buffered, its pH and therefore the ratios
obtained can vary greatly. Lower pH results in lower ratios and a decreased sensitivity to
protein contamination. However, RNA samples must be diluted in water for correct
concentration estimation as the extinction coefficient used during the calculation was
originally determined in water. The same sample was used for both concentration and
purity analyses in order to conserve sample.
3.5.5 In Summarv
The majority of PBMC samples transformed into LCLs were propagated successfully to
give sufficient cell numbers for RNA extraction and to generate frozen cell banks. All of
the cultured cell lines were used for RNA extraction (except for patient 1 LCL which could
not be maintained) and all of the RNA samples obtained were of excellent quality with
regard to purity and absence of RNA degradation products. Thus, all of the RNA samples
were suitable for use in library construction which gave the opportunity to generate a
diverse antibody library representing all of the antibody specificities present in the original
PBMC samples.
4 Phage Display Fab Library Construction
4.1 Aim
The aim of this section of work was the PCR amplification of antibody genes using
lymphocyte cell line (LCL) RNA samples as a template. Amplified DNA was inserted into
a phagemid vector before electroporation into E. coli and the resulting libraries
characterised with respect to size and diversity.
4.2 Introduction
amp prom
.arni:- marker
lacZ.a reporter
Gene
pBR322 origin
Origin of replication
Other gene
Promoter
Reporter gene
Selectable marker
Mac prom Tag
Human C H I Gene / \ L^^der

Pel B Leader Human CK Gene
Figure 4.1 Plasmid map of pCESl with cloning sites, genes and other features
marked.
Prepared using PlasMapper (Dong et al., 2004).
RNA samples obtained from the LCLs contain transcripts of many genes, not just the
antibody genes of interest. The transcripts of interest are likely to be in relatively low copy
number as antibody titres in the original samples were low for some antibodies. The first
step in library construction was to convert the RNA to cDNA by reverse transcription,
followed by amplification of the immunoglobulin sequences by PGR with antibody gene-
specific primers.
Three sets of primers were synthesised for the amplification of immunoglobulin genes; one
set each for IgG heavy chain, kappa light chain and lambda light chain (see Appendix 3 for
primer sequences). Duplicate sets of primers with restriction sites appended were also
produced to facilitate cloning of the PGR products (see Appendix 4 for primer sequences).
Primary PGR products were purified and used as template for secondary PGR to append the
restriction sites. The PGR products, along with the phagemid vector, were digested with
restriction enzymes to give fragments with incompatible ends to minimise self-ligation.
PGR products were subsequently ligated into digested pGESl phagemid vector for the
creation of single chain (light or heavy chain) immunoglobulin gene libraries. The
rationale behind the two step amplification method was that the use of short, fully-
complementary, primary PGR primers results in more efficient amplification and optimal
PGR product diversity. Once products are obtained, restriction sites are appended with the
longer, part-complementary, and therefore possibly less efficient, secondary PGR primers.
Once single chain immunoglobulin gene libraries were generated, heavy (or light) chain
insert was removed from the vector and ligated into pGESl phagemid vector harbouring a
library of immunoglobulin light (or heavy) chains to yield Fab libraries. This two step
library construction method was designed to improve ligation efficiencies as restriction
digestion of cloned genes in vectors is more efficient than digestion of PGR products.
Despite the inclusion of extra bases on the secondary PGR primers to facilitate enzyme
binding and digestion, restriction enzymes are nevertheless more efficient at digesting
vector DNA.
The sequence of primers used for immunoglobulin gene amplification were based on
antibody sequences in the Kabat database (Kabat et al., 1987). Primer sets were
synthesised for kappa, lambda and heavy chain amplification according to the method of de
Haard et al. (1999), except that primers were designed for amplification of IgG rather than
IgM class of heavy chain. The large, naïve immunoglobulin gene library constructed by de
Haard et al. targeted the IgM class of antibodies, in an attempt to obtain antibody gene PGR
products from the primary repertoire without bias due to antigens to which the B cell
donors may have been exposed. For the construction of an immune library using
lymphocytes from donors pre-sensitised to antigens of interest, primers specific for
amplification of the IgG class of antibody were chosen (Marks et al., 1991). The primers
were designed with reference to V BASE (MRC Gentre for Protein Engineering, 1997) to
ensure amplification of RNA transcripts from all antibody gene families present in the
sample.
Each antibody chain-specific set of primers (heavy, kappa or lambda) consisted of forward
primer (or primers) which annealed to the constant antibody regions (the 3' or 'G-terminal'
end of GL or GHI regions) and reverse primers which annealed to the variable antibody
regions (the 5' or 'N-terminal' end of VL or VH regions). The forward primers were few in
number as they annealed to sequences in the constant region of the antibody structure.
These regions are highly conserved and have very little variation in sequence. The reverse
primers however, annealed in the variable region of the antibody, which by definition has a
large sequence diversity (Figure 4.2, A). In order to maximise the diversity of antibody
transcripts that are amplified during PGR, as many as possible of these variations must be
included in the primer set used. The primers also include some degeneracy which further
increases the number of variable sequences recognised and amplified during PGR. For
example, within the nine heavy chain primary PGR reverse primers , there are eleven sites
of degeneracy at which either of two bases may be incorporated into the sequence. Thus 22
different primers are used in amplification rather than 9.
The primers in the secondary PGR sets were of identical sequence to primers in the primary
sets, but with an extra section of non-complementary DNA which incorporated the
restriction site to allow cloning of the PGR fragment (Figure 4.2, C). The other major
difference in primers of the secondary PGR primer set was in the forward H chain primers.
The primary PGR H chain primers amplified the whole of the Fab region of the antibody
heavy chain (VH plus GHI). However, the secondary PGR primers amplified only the VH
region, as forward primers annealed in the J (joining) region between VH and GHI rather
than in the 3' end of GHI (Figure 4.2, B. The rationale for this strategy was that the initial
amplification maintains high diversity by utilising a highly conserved forward primer site.
Once the antibody genes have been obtained by PGR, secondary amplification of the VH
region only can be performed using more degenerate primers. Vector construction of
pGESl permits the choice of cloning the Fd (VH-GH,) or light chain (VL-GL), or solely the
variable region (VH or VL), the latter fragment being subsequently fused to the constant
region upon ligation into phagemid vector harbouring a constant region gene(Figure 4.1).
For the heavy chain, cloning of the VH region was performed, as it was demonstrated that
digestion by BstEII (which yields the VH) is more efficient than that by NotI (which yields
the Fd), and would therefore result in greater cloning efficiency (D. Ghin, personal
communication, UNSW). The J chain primers (for amplification of VH) also take
advantage of a naturally occurring BstEII site within the amplified heavy chain regions and
therefore do not need the large section of non-annealing DNA containing the restriction site
that is present in the other primer sets (Figure 4.2, D). For kappa and lambda light chains,
the entire light chain (VLGL) was cloned.
Forward
A Primer
IgGFOR
V^ Region
Ch-1 Region J chain VH1B/7ABACK

VH1CBACK
VH2BBACK
VH3BBACK
Reverse
VH3CBACK
Primers
VH4BBACK
VH4CBACK
VH5BBACK
JH1/2F0R
B VH6ABACK
Forward JH3F0R
Primers JH4/5FOR
JH6F0R V^ Region
Region J chain VH1B/7ABACKSFI

VH1CBACKSFI
VH2BBACKSFI
VH3BBACKSFI
Reverse
Primers VH3CBACKSFI
VH4BBACKSFI
VH4CBACKSFI
VH5BBACKSFI
VH6ABACKSFI
C VH1B/7ABACK 5
VH1B/7ABACKSF! 5' GTCCTCGCAACTGC G G C C C i ^ G C G G G C C ATGGCC
Sfi Restriction Site Annealing s e q u e n c e

D
JH1/2F0R 5'TGAGGAGAC GGTGA(i;AGGGTGCC 3'
Naturally occurring
BstEII Restriction Site
Figure 4.2 Diagram of PGR primers used in library construction (not to scale).
Figure A and B show annealing positions of forward and reverse primers for primary
and secondary PGR respectively. Figure G is an example of reverse primers
(VH1B/7ABAGK) highlighting the appended sequence containing the restriction site.
Figure D is an example of a secondary PGR forward primer (JH1/2FOR) containing a
naturally occurring BstEII restriction site.
4.3 Methods
4.3.1 DNA Electrophoresis
All DNA electrophoresis was carried out in 1 % Agarose/TAE and stained with ethidium
bromide according Section 2.6.5 unless stated otherwise.
4.3.2 Reverse Transcription
All eight donor and patient RNA samples were transcribed separately according to Section
2.6.3.
4.3.3 Primary PGR Optimisation
Since large quantities of RNA were obtained from donor 4, this sample was used for all of
the PGR optimisation reactions. PGR was optimised using variables listed in Section 2.6.4.
4.3.4 Primary PGR Amplification and Purification
Donor and patient cDNA samples produced by reverse transcription were PGR amplified
(Section 2.6.4) as two separate pools as the donor pool was likely to contain a high
representation of anti-D antibody transcripts. These transcripts may have complicated the
subsequent biopanning process for rare antibody clones such as Fy^ or Fy^. Optimised PGR
conditions determined using donor 4 cDNA were applied to the donor and patient pooled
cDNA samples and are listed in Table 4.1. Multiple PGR reactions were carried out for
each primer pair to generate sufficient DNA for purification and subsequent re-
amplification. Primary PGR products were purified by gel extraction according to Section
2.6.6. Purified products were quantitated using Picogreen reagent according to Section
2.6.7.
4.3.5 Secondary PCR Amplification and Purification
Purified primary PCR product was used as a template for secondary PCR according to
Section 2.6.4. Optimisation, where required was carried out according to Section 2.6.4 and
optimised conditions are listed in Table 4.2.
4.3.6 Trouble Shooting of Ligation Process
After several failed attempts to ligate vector and insert (results not shown), the preparation
method for restricted vector was investigated. The possible failure modes considered were:
damage to DNA components during purification by UV exposure; inhibition of ligation due
to gel impurities or staining reagent; and inefficient digestion of PCR product for insertion.
Vector was digested with Sfil according to Section 2.6.12 to give single cut, linearised
DNA. This digested sample was gel extracted by various methods (Table 4.3) and
quantitated by GeneTools gel estimation (Section 2.6.7). A 25 ng aliquot from each sample
was ligated (according to Section 2.6.15) and transformed into E. coli to show ligation
efficiency (Section 2.7.2). A positive control sample of digested vector was purified by
Wizard® SV Gel and PCR Clean-Up System (Promega) according to manufacturer's
instructions, eluting DNA from the purification columns with 50 |iL nuclease free water.
DNA extraction from agarose gels was carried out both with (at 100 % and 70 % UV light
box intensity) and without UV exposure (Section 2.6.6). DNA extraction from acrylamide
gels was carried out according to Section 2.6.10, and SybrGreen staining was performed
according to Section 2.6.11.
Table 4.1 Primary PCR conditions optimised for PCR product yield and specificity.
All reactions contained 0.5 ^M Primer (in total), 2 ju-L cDNA Template and 0.5 ^L
Thermoscript DNA Polymerase. Cycling conditions; Denaturation: 3 min at 94 °C, 35
cycles of 1 min at 94 °C/ 1 min at annealing temp (see Table below)/1 min at 72 °C,
Final extension 10 min at 72 °C.
Primer Pair Donor cDNA Pool Patient cDNA Pool
Forward Reverse [MgCb] mM °C Annealing [MgCb] mM °C Annealing
Temperature Temperature
IgGFOR VH1B/7A 2 55 2 55
IgGFOR VHIC 2 59 2 59
IgGFOR VH2B 2 59 2 59
IgGFOR VH3C 2 55 2 55
IgGFOR VH4C 2 55 2 55
IgGFOR VH6A 2 61 2 61
CkapFOR VkaplB 2 55 2 55
CkapFOR Vkap2 2 55 55
CkapFOR Vkap3B 2 55 2 55
CkapFOR Vkap4B 2 55 2 55
CkapFOR Vkap5 2 55 2 55
CkapFOR Vkap6 2 55 2 55
ClamFOR VlamlA 2 55 2 55
ClamFOR VlamlB 2 57 2 57
ClamFOR VlamlC 2 55 2 55
ClamFOR Vlam2B 2 57 2 57
ClamFOR VlamSA 2 53 2 53
ClamFOR VlamSB 2 53 2 51
ClamFOR Vlam4 2 53 2 53
ClamFOR VlamS 2 55 2 55
ClamFOR Vlam7/8 2 55 2 53
Table 4.2 Secondary PCR conditions optimised for PCR product yield and specificity.
All reactions contained 0.5 ^M Primer (in total), 2 ^L cDNA Template and 0.5 ^L
Taq DNA Polymerase; QHS: Qiagen Hot Start, IP: Invitrogen Platinum, IR:
Invitrogen Recombinant. Cycling conditions for IR Taq same as primary PCR.
Cycling conditions for QHS and IP Taq same as for primary PCR except for initial
denaturation step which is increased to 10 min for polymerase activation.
Primer Pair Donor Primary PCR template Patient Primary PCR template
Forward Reverse [MgCb] Taq Annealing [MgCb] Taq Annealing
mM Temp. °C mM Temp. °C
IgGFOR VH1B/7A 1.5 QHS 60 1.5 QHS 60
IgGFOR VHIC 1.5 QHS 60 1.5 QHS 60
IgGFOR VH2B 1.5 QHS 60 1.5 QHS 60
IgGFOR VH3C 1.5 QHS 60 1.5 QHS 60
IgGFOR VH4C 1.5 QHS 60 1.5 QHS 60
IgGFOR VH6A 1.5 QHS 60 1.5 QHS 60
CkapFOR VkaplB 1.5 QHS 60 1.5 QHS 60
CkapFOR Vkap2 1.5 QHS 60 1.5 QHS 60
CkapFOR Vkap3B 1.5 QHS 60 1.5 QHS 60
CkapFOR Vkap4B 1.5 QHS 60 1.5 QHS 60
ClamFOR VlamlA 5 IP 60 5 IP 60
ClamFOR VlamlB 5 IP 60 5 IP 60
ClamFOR VlamlC 5 IR 55 5 IR 55
ClamFOR Vlam2B 5 IR 55 5 IR 55
ClamFOR VlamSA 5 IP 60 5 IP 60
ClamFOR Vlam3B 5 IP 60 5 IP 60
ClamFOR Vlam4 5 IP 60 5 IP 60
ClamFOR Vlam5 5 IR 55 5 IR 55
ClamFOR Vlam7/8 5 IR 55 5 IR 55
Table 4.3 Gel extraction conditions for Sfíl digested pCESl.
Sample Gel Type DNA Stain UV Source
1 Agarose Ethidium Bromide 100%UV
2 Agarose Ethidium Bromide 70 % UV
3 Agarose Ethidium Bromide None
4 Agarose SybrGreen 100 % UV
5 Agarose SybrGreen 70 % UV
6 Agarose SybrGreen None
7 Acrylamide Ethidium Bromide 70 % UV
8 Acrylamide Ethidium Bromide None
9 Acrylamide SybrGreen 70 % UV
10 Acrylamide SybrGreen None
In addition, the efficiency of restriction digestion of PCR inserts (Section 2.6.13) was tested
by molecular weight calibration of digested and undigested samples. DNA PAGE was
carried out according to Section 2.6.9 and digital images of the gels were analysed using
GeneTools software from Syngene.
Another problem encountered in library construction was a high level of transformants

obtained from re-ligation of digested pCES 1 in the absence of insert, referred to as vector
background (results not shown). This was addressed by performing a further restriction
digestion, designed to digest between the two cloning sites within the region that was to be
discarded from the vector (Figure 4.3). The rationale behind this strategy was to convert
any remaining single cut vector into double cut vector, which was much less likely to re-
anneal. The resulting vector preparations were referred to as triple digested vector. In
addition to this digestion strategy, the vector DNA was dephosphorylated after every digest
step using calf intestinal alkaline phosphatase (Section 2.6.12) to prevent vector re-ligation
in the absence of insert.
Cloning Sites
ApaLI
Light Chain Constant Region
Xhol ^ E x t r a restriction site to reduce vector background Ncol

Figure 4.3 Site of third restriction digests within pCESl sequence utilised to reduce
vector background upon ligation and transformation.
DNA regions represented as pale blue bars are removed from pCESl during cloning
and are replaced with VL-CL in the case of the light chain and V„ in the case of the
heavy chain (alongside the CHI region already present in the pCESl vector).
4.3.7 Preparation of Single Chain Libraries
pCESl vector was propagated in E. coli, purified by the alkaline lysis method (Section
2.6.8), quantitated by UV spectrophotometry (Section 2.6.7) and analysed by agarose gel
electrophoresis (Section 2.6.5). Triple digested vector was prepared using Sfil, BstEII and
NotI according to Section 2.6.12 for heavy chain insertion. Triple digested vector was
prepared using ApaLI, AscI and Xhol according to Section 2.6.12 for light chain insertion.
Secondary PCR products were purified and digested according to Section 2.6.13. Heavy,
kappa and lambda antibody fragment PCR product pools were processed separately. In
addition, donor and patient pools were processed separately resulting in a total of six insert
samples. Heavy chain PCR product was digested with Sfil and BstEII and light chain PCR
product digested with ApaLI and AscI.
Digested vector and insert were quantitated by Nanodrop spectrophotometry according to

Section 2.6.7 and ligated according to Section 2.6.15. Vector to insert molar ratios of 3:1,
1:1 and 1:3 were tested (results not shown); the 1:3 ratio was used for all subsequent
ligations as this ratio gave the highest number of transformants. Electrocompetent E. coli
XLlBlue were prepared according to Section 2.7.1 and used for electroporation of ligated
samples according to Section 2.7.2.
The six single chain libraries (donor kappa, donor lambda, donor heavy, patient kappa,
patient lambda, patient heavy) were PCR screened for the presence of insert according to
Section 2.7.4. Clones showing an insert of the correct size were screened for diversity
Library screening primers (P. Sturgess personal communication, UNSW) were designed to
flank the cloning site of the light and heavy chains into pCES 1. The forward primer
annealed to pCESl vector -150 bp upstream of the ApaLI light chain cloning site and the
reverse primer annealed -180 bp downstream from the end of the heavy chain constant
region (Table 4.5). Using unmodified pCESl as a template, PCR using these primers
would yield a product of 1146 bp. Other possible PCR products from colony screening are
shown in Table 4.5. Presence of a light chain insert would give a -1500 bp PCR product
whereas a heavy chain insert would give a slightly smaller product due to removal of the
stuffer fragment.
4.3.8 Preparation of Fab Libraries
Large scale cultures of the six single chain antibody libraries were grown and the
recombinant plasmids purified using the Pure Yield™ Plasm id Midiprep System (Promega;
Section 2.6.8). Each purified plasmid was digested in two ways (Section 2.6.14). For
example, a plasmid containing a light chain insert was sequentially digested with ApaLI
and AscI and purified to recover insert for subsequent ligation into a vector containing
heavy chain. The same starting material was also digested with Sfil and BstEII and
purified to give linearised light chain vector ready for heavy chain insertion. This strategy
resulted in the construction of a total of eight Fab libraries (Table 4.4). Methods used for
purification, quantitation, ligation, transformation and screening were as for single chain
library construction.
Fab library construction was also attempted whereby PGR insert was used rather than
vector derived insert, as in single chain library construction. Transformation efficiencies of
vector constructs with PGR derived inserts were lower than those for constructs where the
inserts were digested from vectors; thus the former approach was not pursued any further
(results not shown).
Table 4.4 Cloning strategy for Fab library production.

Each light chain insert was cloned into a heavy chain vector and vice versa to give a
total of eight Fab libraries.
Library Vector Insert
1 Kappa Donor Heavy Donor
2 Lambda Donor Heavy Donor
3 Kappa Patient Heavy Patient
4 Lambda Patient Heavy Patient
5 Heavy Donor Kappa Donor
6 Heavy Donor Lambda Donor
7 Heavy Patient Kappa Patient
8 Heavy Patient Lambda Patient
Table 4.5 Possible PCR products from library colony screening.
Diagrams are not to scale.
Diagram A shows the heavy and light chain cloning sites in pCESl along with the
PCR primer annealing sites which would result in a 1146 bp PCR product. Diagrams
B-F show possible vector constructs that may arise from the cloning process and their
respective PCR product sizes.
PCR
Vector
Product
A. Unmodified pCESl.
Asci Sfil BstEII
pm Light Chain
f
H
\ /
Heavy Chain pel 1146 bp

""feoR Constant Region 'Stuffer' c m Region teBi"
147bp 368bp 84bp 36bp 325bp 186bp
B. pCESl without Light Chain Constant Region

ApaLI AscI 778 bp
4PCF
m
' K H
'Stuffer'
Heavy Chain
CH1 Region mm
C. pCESl without Heavy Chain 'Stuffer' Region

Sfil BstEII
1110 bp
PCH Light Chain Heavy Chain PCR
FOR Constant Reqion CH1 Reaion REV
D. pCESl with Heavy Chain Insert

1450
4 bp
pm Light Chain Heavy Chain Heavy Chain PCR
-FOR Constant Reqion Variable Reqion CH1 Reaion REV
E. pCESl with Light Chain Insert

ApaLI Asci
1500
PcR ^^ Light Chain Light Chain
\ !
H Heavy Chain PCR

bp
FOR Variable Reqion Constant Reqion 'Stuffer' CH1 Reqion REV
F. pCESl with Light and Heavy Chain Inserts

ApaLI AscI Sfil BstEII
1850
PCR j Light Chain Light Chain Heavy Chain Heavy Chain
bp
IFOR- Variable Region Constant Region Variable Region CH1 Region
Results
4.3.9 Primary PGR Amplification and Purification
Primary PGR of donor 4 cDNA using published reaction conditions (55 °G annealing
temperature) was not optimal for all primer pairs (Figure 4.4; de Haard et al., 1999). Kappa
light chain PGRs resulted in high yields, with a strong band at -650 bp and few non-
specific products. Heavy chain and lambda light chain PGRs were less efficient resulting in
a low yield of specific product and greater non-specific banding thus required further
optimisation.
Before combining cDNA samples to give a donor pool and patient pool for PGR
amplification, each sample was tested separately to check that it would yield a PGR product.
This was performed by carrying out PGRs using a single primer pair already shown to be
successful for donor 4 cDNA amplification; kappa forward and kappa IB reverse primers.
All template cDNA samples resulted in significant amounts of PGR product (Figure 4.5)
and all subsequent primary PGRs were carried out on pooled donor or patient cDNA
samples.
Optimised PGRs were replicated to produce more DNA, pooled, gel extracted and
quantitated. This process was repeated until enough of each PGR product was generated
for use as a template in secondary PGR. Yields of purified DNA product ranging from - 5 0
to 1200 ng were obtained depending on the efficiency of the PGR. A total of 78 purified
samples were generated; nine heavy chain samples, six kappa and eleven lambda light
chain samples for both donor and patient cDNA pools and for donor 4. Donor 4 samples
were generated separately from the donor pool (in which donor 4 was also present) for use
in optimisation of secondary PGR. Gel analysis of these purified primary PGR products is
shown in Figure 4.6. All products were approximately 650 bp in size.
1kb—> K L Chain
750bp — >
500bp—> H Chain
250bp—>
Std 1 Std 1
X L Chain
X L Chain
Std 1 Std 10 11
Figure 4.4 Agarose gel electrophoresis of donor 4 primary PGR products before
optimisation.
A: Lanes 1-9 Heavy chain variable primers: IB, IC, 2B, 3B, 4B, 4C, 5B, 6A.
B: Lanes 1-6 Kappa chain variable primers: IB, 2,3B, 4B, 5, 6.
C: Lanes 1-11 Lambda chain variable primers: lA, IB, IC, 2,3A, 3B, 4, 5, 6, 7/8, 9.
K L Chain
Std 1
Figure 4.5 Agarose gel electrophoresis of primary kappa light chain PGR products
using KIB primer and separate donor and patient cDNA samples as template.
Lanes 1 to 7; Donor 3, donor 5, donor 2, donor 1, patient 2, patient 3, donor 4.
Lanes 8 and 9: Negative controls (no template).
1kb—» r.
750bp—>
500bp—»
250bp—>
Std 1 6Std1 9 Std 1 2 Std 3 11
11 Std Std 1 9
Std 1 6 Std 1 11 Std
Figure 4.6 Agarose gel electrophoresis of purified primary PGR products.

A: Donor pooled cDNA template; B: Patient pooled cDNA template; C: Donor 4
cDNA template. All gels: Lanes 1 to 6 , kappa L chain products; Lanes 1 to 9, H chain
products; Lanes 1 to 11, lambda L chain products. Primers as in Figure 4.4.
4.3.10 Secondary PGR Amplification and Purification
Secondary PGR of donor 4 primary PGR products using reaction conditions published in de
Haard et al. (1999; 55 °G annealing temperature) were not successful for all primer pairs
(Figure 4.7). Heavy chain PGRs yielded a strong band at -350 bp (only V H region is
amplified in secondary PGR), but also resulted in significant amounts of non-specific PGR
product. Kappa and lambda light chain PGRs were less efficient, giving a low yield of
specific product or no product in some cases and required further optimisation.
Secondary heavy chain PGR employs four forward primers which anneal in the J (joining)
region in the antibody gene structure. Before combining these primers for use in PGR, each
primer was tested separately to check amplification efficiency. This was performed by
carrying out PGR using a reverse primer already shown to be successful for donor 4
primary template amplification; IB reverse primer. Each of the four JH primers when used
with HIB reverse primers yielded significant amounts of PGR product in separate reactions
as shown in Figure 4.8. The PGR products appeared to be of a similar size and yield by
agarose gel electrophoresis analysis, whether JH primers were used separately or together.
Thus, all subsequent heavy chain secondary PGRs were carried out using pooled JH forward
primers.
Kappa and heavy chain PGRs of donor and patient samples were improved by optimisation
(Figure 4.9). Lambda secondary PGR samples proved difficult to optimise and the two
forward primers were used separately in an attempt to improve product yield (Figure 4.10).
Some PGR products were improved by this strategy but despite many rounds of
optimisation, other PGR products (such as those resulting from PGR using lambda 4
reverse primer) were still suboptimal and could not be further improved.
1kb—>
750bp—>
500bp—> K L Chain
250bp vH Chain
Std 1 -> 9 Std 1 ^ 6
L Chain A, L Chain
Std 1 7 Std 8 ^ 11
Figure 4.7 Agarose gel electrophoresis of donor 4 secondary PGR products before
optimisation.
A: Lanes 1-9 Heavy chain variable primers; IB, IC, 2B, 3B, 4B, 4C, 5B, 6A.
B: Lanes 1-6 Kappa chain variable primers; IB, 2, 3B, 4B, 5, 6.
C: Lanes 1-11 Lambda chain variable primers; l A , IB, IC, 2, 3A, 3B, 4, 5, 6, 7/8, 9.
vH Chain
Std 1 ^ 5
Figure 4.8 Agarose gel electrophoresis of donor 4 secondary PGR products from
heavy chain amplification using separate JH primers.
PGR was performed using separate (1 îL of 25 fiM stock) and combined (0.25 ^L of
each 25 îM stock to give a total volume of 1 ^L) JH forward primers with HIB reverse
primer.
Lane 1: JHl/2
Lane 2: JH3
Lane 3: JH4/5
Lane 4: JH6
Lane 5: All JH primers .
K L Chain
•>6Std
vH Chain
Std 1 ^ 9 > 9 Std
heavy and kappa light chain, patient and donor amplification.
A: Lanes 1-6 Donor kappa chain PCR. B: Lanes 1-6 Patient kappa chain PCR. C:
Lanes 1-9 Donor heavy chain PCR. D: Lanes 1-9 Patient heavy chain PCR. Primer
pairs as in Figure 4.7.
750bp—>
500bp—
250bp—>
À L Chain
Std 1 9 10S 11 ->16 Std 17 ^ 24
Pi L Chain
Std 25 31 32 Std 33 -> 39 Std 40 >44
lambda light chain, patient and donor amplification.
In table below showing lane loadings, D represents donor template and P represents
patient template.
Lane# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Template/Forward Primer D/2 D/2 D/2 D/2 D/2 D/2 D/7 D/7 D/7 D/7 D/7 D/7 P/2 P/2 P/2 P/2
Reverse Primer IC 2 5 6 7/8 9 IC 2 5 6 7/8 9 IC 2 5 6
Lane # 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Template/Forward Primer P/2 P/2 P/7 P/7 P/7 P/7 P/7 P/7 D/2 D/2 D/2 D/2 D/2 D/7 D/7
Reverse Primer 7/8 9 IC 2 5 6 7/8 9 lA IB 3A 3B 4 lA IB
Lane # 32 33 34 35 36 37 38 39 40 41 42 43 44
Template/Forward Primer D/7 D/7 D/7 P/2 P/2 P/2 P/2 P/2 P/7 P/7 P/7 P/7 P/7
Reverse Primer 3A 3B 4 lA IB 3A 38 4 lA IB 3A 3B 4
4.3.11 Purification of pCESl Vector
In-house stocks of XLlBlue strain of E. coli harbouring pCESl were used for purification
by the alkaline lysis method (Section 2.6.8) and Figure 4.11 shows purified pCESl vector.
The pCESl vector is 5.3 kb in size and appears as two bands at approximately 9 kb and 3.5
kb representing open circle and supercoiled forms of the plasmid respectively.
10kb<- Open circle plasmid

4kb<- "U Supercoiled plasmid
3.5kb<- ilii
3kb<- n
S t d 5 2 1}iL
Figure 4.11 Agarose gel electrophoresis of purified pCESl at 5,2 and 1 îL loading
volumes.
4.3.12 Trouble Shooting of Ligation Process
Figure 4.12 shows the resulting gel when a DNA band is excised using a method that
avoids UV exposure. This demonstrates that the correct section of gel was removed for
extraction. The method used to purify restricted pCESl vector had a significant impact on
its subsequent ligation and transformation efficiency (Figure 4.13). Purifying the restricted
vector without exposing it to UV light gave results similar to the positive control sample
which had not been gel extracted. No significant improvement in efficiency resulted from
the use of acrylamide gel rather than agarose nor from the use of SybrGreen stain rather
than ethidium bromide.
PAGE analysis of single and double digested kappa light chain insert showed a difference
in size between digested and undigested species (gel and molecular weight calibration
results shown in Figure 4.14). The molecular weight estimation of undigested light chain
gave an average size of - 6 9 0 bp (sample was analysed in triplicate). The size of single
digested light chain was -670 bp and double digested light chain was -650 bp. ApaLl and
AscI digestion both result in the removal of 13 bases of DNA from the PGR product
causing its molecular weight to decrease by this amount upon each digest. This analysis
method was also used to test digestion efficiency of heavy chain insert (results not shown).
Although small differences in molecular weight were apparent from the acrylamide gel
image, the analysis software was unable to discern these differences on the digital image.
step 1. Both edg es of gel are cut and left in place (UV light is off).
A
' I I 'H » \T
Í
Step 3 . - ^ i n s t e p 3. UV light is switched on, linearised

plasmid band is cut on both gel edges and
left in place
Step 5. UV light is off, linearised plasmid

band excised using cut edges as a guide.
Gel illuminated to check that the correct
section of gel was removed.
Step 2. Centre section of Step 4. Centre section of

gel is removed from the gel is replaced and
light box. realigned.
Figure 4.12 Excision of linearised vector from agarose gel avoiding UV exposure.
Table 4.6 PAGE molecular weight calibration results for undigested, single and
double digested kappa light chain secondary PGR product.
Lane # Sample Size (kb) Average Size (kb)
1 Undigested K L chain. 692 692
4 Undigested K L chain. 703
7 Undigested K L chain. 680
2 Single digest (ApaLI) 670 670
5 Single digest (AscI) 670
3 Double digest (ApaLI-ÂscI) 648 648
6 Double digest (Ascl-ÂpaLI) 648
+ with ligase
100000000
- without ligase ie. Vector background
10000000
1000000
100000
10000
1000
100
10
1 >—
+
>+ >+ >+ >+ >+ >+ >+ >+
>
D
o
o
>
3
o
O
ZD
O
g1 >
D
O
2
3
O
O
>
D
O
o
D
O
>
D
o
D
o
z
>
D
O ZD
O
>
D
D
o
>
3O
O
>
o
D
Z
o
>
=)
o
z
^
c
o
CO
N
^
Agarose Ethidium Agarose SybrGreen PAGE Ethidium PAGE SybrGreen NO

GEL
Figure 4.13 Effect of purification method on singly digested pCESl vector re-ligation and transformation.
DNA was purified either on agarose or acrylamide gel stained with either ethidium bromide or SybrGreen. For excision of the
DNA band, the gel was either exposed to full power UV light box (100 Vo), 70 % intensity UV or not exposed at all.
K L Chain
250bp—>
.*! 670
oaid
• 660
Undigested Apa Digest Apa-Asc Digest Undigested Asc Digest Asc-Apa Digest Undigested
Figure 4.14 PAGE molecular weight calibration results for undigested, single and
double digested kappa light chain secondary PGR product
The 4-12 % gel was electrophoresed at 100 V until the xylene cyanol marker
reached the bottom of the gel in order to obtain maximum resolution. Molecular
weight determination was carried out using GeneTools software from Syngene.
Samples in lanes 1 to 7 on the gel correspond to bars on the graph reading from
left to right. See Table 4.6 for data.
4.3.13 Restriction Digestion and Purification of PGR Insert and pCESl Vector
Pooled and purified PGR products (separate donor and patient sample pools) were
double digested in sequential steps and purified (Figure 4.15). Digests yielded PGR
products of size -650 bp and -300 bp for lambda and kappa, and heavy chains
respectively.
Purified pGESl vector was triple digested in three sequential steps (Figure 4.16).
Agarose gel analysis of primary digests showed that all digests gave a single band of
linearised plasmid at kb. Second and third digests all showed the plasmid band at a
similar molecular weight. ApaLI and AscI double and triple digests resulted in DNA
fragments of -350 bp in size as expected for excision of the GL region of the vector.
Sfil and BstEII double and triple digests were shown to contain a lower molecular
weight band when electrophoresis of the whole sample for gel extraction was performed
(image not recorded) but this band was not evident in the small sample loaded on to
these gels.
X L Chain
«—KL Chain
<—H Chain
Std 1 Std 5 6
Figure 4.15 Agarose gel electrophoresis of digested PGR products.
Light chains were digested with ApaLI and Asci, and heavy chains with BstEII
and Sfil.
Lane 1: Donor heavy chain; Lane 2: Patient heavy chain; Lane 3: Donor kappa
light chain; Lane 4: Patient kappa light chain; Lane 5: Donor lambda light chain;
Lane 6: Patient lambda light chain.
8kb<
6kb«
5kb«
Std 5
std 9—^12
Figure 4.16 Agarose gel electrophoresis of purified pCESl single, double and
triple restriction digests.
LaneliApaLI; Lane 2: Asci; Lane3:SfiI; Lane 4: BstEII. Lane 5:
ApaLI->AscI ; Lane 6: AscI^ ApaLI ; Lane 7: SfiI-> BstEII; Lane 8: BstEII->
Sfil. Lane 9: ApaLI->AscI^XhoI ; Lane 10: AscI^ ApaLI^XhoI ; Lane 11:
SfiI-> BstEII^NcoI; Lane 12: BstEII^ Sfil^NcoI.
4.3.14 Production and Characterisation of Single Chain Libraries
Purified vector and insert samples were ligated and transformed into E. coli XLl Blue
electrocompetent cells. Unmodified pCESl (1 ^g) and vector preparations ligated in
the absence of insert were included as positive and negative controls respectively.
Table 4.7 shows the total number of transformants obtained. The resulting samples
were analysed by agarose gel electrophoresis to determine PCR product size (Figures
4.17 and 4.18). A total of twenty transformants were screened by PCR from each single
chain library to determine whether they contained an antibody chain insert. The
presence of a full length insert resulted in a PCR product of -1500 bp in size.
Frequency of full length insert ranged from 55 to 90 % for the six libraries (Table 4.8).
Table 4.7 Total number of transformants obtained for the six single chain
libraries and control samples.
An aliquot of each transformed sample was subjected to a serial dilution and
grown on selection media to show the number of successfully transformed cells.
Sample Total number of transformants
Kappa Donor 2.0 X 10'
Kappa Patient 1.0 X 10'
Lambda Donor 0.7 X 10'

Lambda Patient 2.1 X 10'
Heavy Donor 1.4 X 10'

Heavy Patient 1.0 X 10'
pCESl Positive Control (1 \ig) 1.0 X 10'
Vector background (Heavy chain) 3.0 X 10'

Vector background (Light chain) 8.0x10'
«
h i.
r f
2kb- <—2kb
1.5kb- <—1.5kb
1kb-
750bp-
Kappa Donor PGR Screen
i> » '
<—2kb
<—1.5kb
<—1kb
2kb—> <— 750bp
1.5kb—>
1kb—>
750bp—>
Kappa Patient PGR Screen
2kb—>
1.5kb—> •2kb
1kb—>
750bp—> -1.5kb
1kb
750bp
Heavy Donor PGR Screen
Figure 4.17. Agarose gel electrophoresis of PCR screen samples from single chain
libraries; kappa donor, kappa patient, and heavy donor.
A total of twenty colonies from library selection plates were picked into PCR
mixture to show the presence of insert within single clones. A full length insert is
indicated by a PCR product of -1500 bp in size.
<—2kb
^^—1.5kb
-1kb
•750bp
... -
Heavy Patient PGR Screen
2kb—> <—2kb
1.5kb—> <—1.5kb
1kb—> <—1kb
750bp—> i ».f <—750bp
? H
Lambda Donor PGR Screen
2kb—>
1.5kb—>
1kb
750bp
Lambda Patient PGR Screen

Figure 4.18 Agarose gel electrophoresis of PCR screen samples from single chain
libraries; heavy patient, lambda donor, and lambda patient.
A total of twenty colonies from library selection plates were picked into PCR
mixture to show the presence of insert within single clones. A full length insert is
indicated by a PCR product of -1500 bp in size.
Table 4.8 Proportion of single chain library clones containing a full length insert.
Results show the number of clones giving the correct size insert upon gel analysis
of PCR screening products as a percentage of the number of clones tested.
Single Chain Library Clones containing full length insert
Kappa Donor 85%
Kappa Patient 55%
Lambda Donor 75%
Lambda Patient 60%
Heavy Donor 90%
Heavy Patient 75%
4.3.15 Restriction Digestion and Purification of Insert and Vector from Single Chain
Libraries
Single chain library glycerol stocks were cultured and plasmids (containing single
chains) purified. Analysis by agarose gel electrophoresis showed that the purified
vectors appeared as a major band at kb and two minor bands at and above 10 kb
(Figure 4.19). Purified vectors were restriction enzyme digested to recover single chain
inserts (Figure 4.20). The light chain inserts can be observed in the double digested
samples at -650 bp and the heavy chain inserts at -300 bp. Vectors were also digested
to give linearised single chain vector ready for ligation of insert. All samples were
purified by gel extraction (Figure 4.21). Linearised single chain vectors appeared as a
major band at ~5 kb. Insert sizes were the same as in pre-purification samples. Purified
samples were quantitated using the Nanodrop prior to ligation.
10kb«
8kb<
5kb<
4kb<
Std 1 6
Figure 4.19 Agarose gel electrophoresis of purified pCESl from single chain
library stocks.
Lane 1: Heavy donor; Lane 2: Heavy patient; Lane 3: Kappa donor;
Lane 4: Kappa patient; Lane 5: Lambda donor; Lane 6: Lambda patient.
X'i'.'vv.-;
250bp
Std 1 7 12 Std
Figure 4.20 Agarose gel electrophoresis of single and double restriction digests of
pCESl from single chain libraries.
Double digestion of heavy chain libraries results in linearised vector (~5 kb) plus
an insert of ~300 bp, and light chain libraries show a -650 bp insert.
Lane 1: Heavy donor library digested with SfìI (single digest).
Lane 2: Heavy donor library digested with Sfil followed by BstEH.
Lane 3: Heavy patient library digested with Sfil (single digest).
Lane 4: Heavy patient library digested with SfìI followed by BstEH.
Lane 5: Kappa donor library digested with ApaLI (single digest).
Lane 6: Kappa donor library digested with ApaLI followed by Asci.
Lane 7: Kappa patient library digested with ApaLI (single digest).
Lane 8: Kappa patient library digested with ApaLI followed by Asci.
Lane 9: Lambda donor library digested with ApaLI (single digest).
Lane 10: Lambda donor library digested with ApaLI followed by Asci.
Lane 11: Lambda patient library digested with ApaLI (single digest).
Lane 12: Lambda patient library digested with ApaLI followed by Asci
6kb<
5kb<
4kb<
750bp—>1
500bp—>
250bp—>J
Std 1 8 12 Std
Figure 4.21 Agarose gel electrophoresis of purified, digested vector and insert
from single chain libraries.
Lanes 1 to 6 show linearised vector (-6 kb), lanes 7 to 10 show light chain insert
(-650 bp) and lanes 11 and 12 show heavy chain insert (-300 bp).
Lane 1: Heavy donor library digested with ApaLI followed by Asci.
Lane 2: Heavy patient library digested with ApaLI followed by Asci.
Lane 3: Kappa donor library digested with Sfil followed by BstEII.
Lane 4: Kappa patient library digested with Sfil followed by BstEII.
Lane 5: Lambda donor library digested with Sfil followed by BstEII.
Lane 6: Lambda patient library digested with Sfil followed by BstEII.
Lane 7: Kappa donor library insert.
Lane 8: Kappa patient library insert.
Lane 9: Lambda donor library insert.
Lane 10: Lambda patient library insert.
Lane 11: Heavy donor library insert.
Lane 12: Heavy patient library insert.
4.3.16 Production and Characterisation of Fab Libraries
Purified single chain vector and vector insert samples were ligated and transformed into
E. coli XLl Blue electrocompetent cells. Unmodified pCESl (1 jig) and vector
preparations ligated in the absence of insert were included as positive and negative
controls respectively. The number of transformants obtained is shown in Table 4.9.
Table 4.9 Total number of transformants obtained for the eight Fab libraries, six
vector only negative controls and unmodified pCESl positive control.
Vector Insert Total number of transformants
Kappa Donor Heavy Donor 3 x 10^
Lambda Donor Heavy Donor 3 x 10^
Kappa Patient Heavy Patient 4 x 10^
Lambda Patient Heavy Patient 5 x 10^
Heavy Donor Kappa Donor 2 x 10^
Heavy Donor Lambda Donor 3 x 10^
Heavy Patient Kappa Patient 3 x 10^
Heavy Patient Lambda Patient 2 x 10^
Kappa Donor None 1x10'
Lambda Donor None 2 x 10'
Kappa Patient None 1x10"
Lambda Patient None 8 x 10'
Heavy Donor None 2 x 10'
Heavy Patient None 8 x 10'
pGESl 1 x 10'
A total of twenty transformants were screened by PGR from each Fab library to
determine whether they contained both heavy and light antibody chain inserts. The
resulting samples were analysed by agarose gel electrophoresis to determine PGR
product size (Figure 4.22). The presence of a full length insert gave a PGR product of
-1850 bp. Frequency of full length insert ranged from 25 to 90 % for the eight libraries
(Table 4.10). As a result of the cloning strategy, two libraries of each type were
generated. For example, heavy chain donor vector with lambda light chain insert
effectively resulted in the same library as lambda light chain donor vector with heavy
chain insert. In this case the former construct had a much higher proportion of full
length inserts than the latter (90 % compared to 25 %). The four libraries with optimal
full length insert ratios were chosen for biopanning (Table 4.10).
Table 4.10 Proportion of Fab library clones containing a full length insert
indicating the presence of both a light and heavy chain.
Results show the number clones giving the correct size insert upon gel analysis of
PCR screening products as a percentage of the number of clones tested. The four
best libraries (marked *) were used for biopanning.
Vector Insert Clones containing full length insert
Kappa Donor Heavy Donor 85 %*
Lambda Donor Heavy Donor 25%
Kappa Patient Heavy Patient 45%
Lambda Patient Heavy Patient 35%
Heavy Donor Kappa Donor 60%
Heavy Donor Lambda Donor 90 %*
Heavy Patient Kappa Patient 65 %*
Heavy Patient Lambda Patient 85 %*
PCR samples showing the presence of a full length insert by gel analysis were analysed
for diversity. Samples were digested with a restriction enzyme having a frequently
occurring recognition site (BstOI) and the resulting fragments analysed by PAGE
(Figure 4.23). All clones appear to be different with the exception of two clones in the
kappa donor/heavy Insert library (lanes 10 and 11, bottom right hand gel in Figure 4.23).
2.0kb—>
2.0kb
1.5kb
Heavy Donor vector with Kappa Insert Heavy Donor vector with Lambda Insert
Heavy Patient vector with Kappa Insert Heavy Patient vector with Lambda Insert
2.0kb-
1.5kb-
Kappa Donor vector with Heavy Insert Kappa Patient vector with Heavy Insert
2.0kb
Lambda Donor vector with Heavy Insert Lambda Patient vector with Heavy Insert
Figure 4.22 Agarose gel electrophoresis of PCR screen samples from Fab libraries.
Twenty clones per library, from library selection plates were PCR screened and
analysed by agarose electrophoresis to show the presence of insert. A full length
insert is indicated by a PCR product of-1850 bp in size.
fm Mi
BSaSS giSiNgs
•i'. Ji 4 l»,.^
Heavy Donor vector with Lambda Insert Heavy Patient vector with Kappa Insert
!1 I1
ti* mê
ÎSS
M
w "^"t ^ S 1
00
Heavy Patient vector with Lambda Insert Kappa Donor vector with Heavy Insert
Figure 4.23 PAGE analysis of BstOI digested PGR products.

Frequent cutting of the DNA into small fragments reveals differences in the DNA
sequence therefore indicating that the clones represent diverse antibody sequences.
4.4 Discussion
Eight RNA samples containing antibody genes were combined into two sample pools
(donor and patient) and taken through the repertoire cloning process to generate four
high quality recombinant Fab libraries. Size and diversity of the libraries compared
favourably to published libraries.
4.4.1 Polymerase Chain Reactions
During optimisation and generation of sufficient product for library construction, over
1100 PCRs were performed. This was due to two major setbacks encountered. The
first was that some of the PCRs, especially the lambda chain specific reactions, were
difficult to optimise and resulted in low product yield. The other problem was that
initially QIAquick Gel Extraction Kit (Qiagen) was used for purification of primary
PCR products and large losses of DNA occurred. Unfortunately several rounds of PCR
and purification were carried out before the problem was identified. Despite careful
troubleshooting of the gel extraction process, the cause could not be isolated since the
kit worked with other unrelated samples. All subsequent gel extractions were
performed using Wizard SV Gel Clean-Up System (Promega) which resulted in good
DNA recoveries.
The immune library in this project was constructed according to the methods of de
Haard et al. (1999), who generated a large naive library containing 3.7 x 10^^ Fab clones.
The input for re-amplification to introduce restriction sites (secondary PCR) was 100-
200 ng purified DNA per 100 |iL reaction. This large input was used in order to ensure
maintenance of diversity. For many of the PCR reactions performed in this project, this
amount of DNA was not available due to inefficient PCR amplification, and an input of
20-25 ng was used. Since the aim of this project was to produce an immune library
which doesn't need to be so large (typically -10^ clones) this smaller input should be
sufficient as 25 ng of a 650 bp DNA fragment will contain over 10^ molecules. An
experiment was performed (results not shown) where varying amounts of template (5 to
200 ng) were used for secondary PCR which indicated that product yield was the same
regardless of input.
4.4.2 Trouble Shooting of the Transformation Process
Early attempts at single chain library construction resulted in a total number of

transformants not exceeding 1 c f u . Ligated samples and vector controls (vector
ligated in the absence of insert) gave a similar number of transformants.
Q
Transformation of unmodified pCESl gave efficiencies of approximately 10 cfu/|xg

DNA indicating that the transformation process was working efficiently. Testing the
effects of variables within the digested vector production process revealed that exposure
of digested vector to UV light resulted in loss of transformation efficiency despite the
fact that the time of exposure was minimal. Unfortunately, this was one of the last
variables to be tested as other researchers in the same laboratory had used the same light
box during library construction and experienced no such problems. UV damage of
restricted DNA has been reported in the literature as capable of causing transformation
efficiencies to drop by 2 to 3 orders of magnitude as compared to control DNA
(Grundemann and Schomig, 1996). This is similar to results obtained in this project
where exposure of digested vector to 100 % UV intensity resulted in a drop in number
of transformants of 4 orders of magnitude as compared to a non-UV exposed control.
Since transformation efficiency was always reduced to a background level rather than
abolished entirely, this was indicative of damage to the single stranded ends of DNA
rather than intact plasmid. This increased sensitivity to denaturation of ss DNA as
compared to ds DNA has been shown (Becker and Wang, 1989) and is also affected by
DNA sequence. This might explain why these particular samples were sensitive to UV
damage where others were not. Use of guanosine or cytidine in the electrophoresis
buffer has been shown to protect DNA from damage (Grundemann and Schomig, 1996)
but an effective technique was developed to excise DNA bands without any UV
exposure. Using this technique the total number of transformants was increased to a
level (10^ to 10^) which, according to published results (Aujame et al., 1997), would
allow the isolation of high affinity antibodies.
Ligation and transformation of undamaged DNA immediately revealed the next

problem. Although transformation efficiencies of ligated vector and insert were now
increased, so too was the vector background. Vector background is the number of
transformants obtained when the vector is ligated in the absence of insert and in some
samples was as high as 1 w h i c h is impractically high for Hbrary construction. This
background was shown by PGR screening to consist of re-Ugated singly digested
pCESl and double digested pCESl re-ligated without the CK region (results not shown).
The re-ligation of non-complementary DNA overhangs was addressed by the use of calf
intestinal alkaline phosphatase (CIP) treatment of the vector. CIP removes the
phosphate group from both 5' ends of the DNA thereby enabling ligation only with
insert sample which has phosphate groups present (Sambrook et al., 1989).
Re-ligation of singly digested pCESl was also addressed by the use of further cleavage
sites (Xhol and Ncol) within a non-essential region of vector DNA. Information from
the V BASE database of human antibody genes (MRC Centre for Protein Engineering,
1997) was checked for the occurrence of these restriction sites in human variable
antibody germline genes (VH, VK, VL, D, JH, JK and JL segments). Ncol showed 1
functional segment restriction site and Xhol showed 3 as compared to 4, 0, 18 and 0 for
the cloning sites ApaLI, AscI, BstEII and Sfil respectively (BstEII cloning takes
advantage of a naturally occurring restriction site at the start of the VH region) out of a
total of 162 segments. In addition, the NCBI Entrez Nucleotide database (National
Center for Biotechnology Information, 2004) was searched for human immunoglobulin
variable region sequences. Approximately 50 sequences were chosen at random from
the several thousand available and searched for Ncol and Xhol restriction sites using
'All in One' software (Karreman, 2002). None of the sequences analysed had a
recognition site for either enzyme. Use of these strategies in addition to an extra gel
extraction step after the first vector digest successfully reduced vector backgrounds to
approximately 10^ transformants.
4.4.3 Librarv Qualitv
The main considerations when judging library quality are size, percentage of full length
insert and diversity. Single chain library sizes compared favourably with those obtained
by de Haard et al. (1991) at approximately 10^ transformants. The proportion of full
length insert (55 to 90 %) was also comparable to published values of 65 to 100 %.
Unfortunately Fab library sizes were not as high, at a total of approximately 10^
transformants. This appeared to be due to an overall drop in transformation efficiency
Q
as early experiments routinely showed 10 transformants per |j,g unmodified pCESl

whereas in later experiments this had dropped to 10 . Several strategies were employed
to reverse this trend including the use of different media and culture conditions when
making electrocompetent cells (lower growth temperature and harvesting at different
optical densities) but higher efficiencies could not be obtained. Library size is very
important when constructing a naïve library as it has been shown that the affinity of
antibodies isolated correlates to library size; the larger the library the better affinity
antibodies obtained (Aujame et al, 1997). However this is not necessarily true for
immune libraries as the intention is to recover the antibody chain heavy and light pairs
as were present in the original sample. There are many examples of immune library
construction in the literature with sizes ranging fi-om 10^ to 10^ clones (Aujame et al.,
1997; Mao et al., 1999; Wu et al., 2001; He et al., 2002; Dantas-Barbosa et al., 2005)
from which antibodies with good affinity have been isolated. The percentage of full
length insert in the four optimal Fab libraries obtained were comparable to published
values at 65 to 90 % (as compared to 86 % in the de Haard naive library) and the
libraries were diverse.
4.4.4 In Summary
All RNA samples derived from LCLs were reverse transcribed and amplified before
being combined into two pooled samples (donor and patient) for library construction.
Donor and patient PGR products were cloned separately due to a high representation of
anti-D antibodies in the donor samples which may complicate selection of lower
incidence clones during biopanning. In total, eight high quality Fab libraries were
constructed and the four with the highest fiill length insert ratios were selected for use in
biopanning experiments. The libraries were sufficiently large for the isolation of high
affinity antibodies, as compared to published repertoires, had a high proportion of fiill
length insert and were diverse.
5 Antibody Selection from Immune and Naïve Phage Display
Libraries
5.1 Aim
The aim of this section of research work was the selection of Fab clones reactive against
red blood cell (RBC) surface antigens from constructed immune libraries and also a
large naive library via phage display. Various methods of biopanning were employed
including whole cell biopanning (recombinant HEK cells and RBCs) and biopanning
against immobilised, recombinant antigen.
5.2 Introduction
The Fab libraries constructed using the lymphocytes of donors, based on analysis of
serum samples from the original donors, would be expected to contain antibodies that
bind to various RBC surface antigens belonging to several different blood group
systems. These include Fya and Fyb from the Duffy system, K from the Kell system,
Jka from the Kidd system and D, C and E from the Rhesus system. Phage displaying
Fab fragments (phage antibodies) specific for these antigens have to be separated from
the >10^ other phage antibodies present in the phage antibody libraries in a process
termed biopanning.
kDs Figure 5.1 SDS-PAGE analysis of the

105 1 -fc- Fyb/GPA (Duffy b/Glycophorin A)
59.8
43.3
recombinant protein.
A-Coomassie Blue staining. B-Immunoblotting
28.3
with an anti-Fy6 antibody.
18.1 SDS-PAGE analysis of Fya/GPA recombinant
15.4
protein used in this project was similar.
^ (Figure from Wasniowska et al., 2000).
In this process, the phage particles displaying a Fab fragment on their surface are
exposed to the antigen of interest. Non-specific binding phage are washed away and the
specific binding phage are recovered. The clones of interest are then characterised and
can be expressed as soluble Fab fragments.
One of the main requirements for biopanning is a source of antigen for selection of the
phage antibodies. In the case of Fy^ antigen, a recombinant version of the extracellular
domain of the Duffy membrane protein (Figure 5.1) was a gift for use in this project
from Wasniowska (Wroclaw, Poland). The Fy^ recombinant protein includes the
amino-terminal extra cellular domain of the Duffy antigen from amino acids 3 to 60. It
also contains part of the Glycophorin A sequence and a hexahistidine tag to facilitate
affinity purification of the protein. This protein can subsequently be adsorbed on to a
solid surface, followed by biopanning of a phage antibody library against the
immobilised antigen, whereby antigen-specific phage antibodies will bind and non-
specific phage antibodies may be removed through extensive washing.
Soluble, recombinant proteins were however not available for any of the other antigens
included in this study. For these antigens, it was necessary to use whole cell biopanning
processes for which there were two types of cells available. Transfected human
embryonic kidney (HEK) cells expressing membrane-bound antigens were a gift from
Marion Reid at the New York Blood Centre. These cells lines were stably transfected
with various RBC surface antigens including Duffy a, Duffy b and Kell proteins. Also
supplied was a cell line transfected with an empty cloning vector to allow for
subtraction biopanning. Exposing the phage antibodies to essentially the same (host)
cells except for the absence of the antigen of interest, results in depletion of phage
antibodies which would co-elute with antigen specific phage during the biopanning
process. These 'non-specific' phage antibodies would not be removed from the antigen
positive cells during washing steps if they were bound specifically to an irrelevant cell
surface molecule. Therefore subtraction biopanning improves the chances of isolating a
positive clone since phage antibodies against other host cell membrane proteins are
removed from the phage antibody library before antigen-driven selection.
As the recombinant HEK cells were to be used as the vehicle for display of membrane-
bound antigen, it was necessary to firstly confirm the presence of antigen on the surface
of the cells, and also to determine the stability of expression of antigen over time. As
the time taken for rounds of biopanning (up to four) can extend into periods of weeks, it
was necessary to ensure that the recombinant HEK cells continued to express the
recombinant antigen over the period. The transfected cells were tested for the presence
and maintenance of the red cell antigen during cell culture by flow cytometry. In this
technique, fluorescently labelled cells in suspension are passed through a flow chamber,
one cell at a time, where they are illuminated with a suitable wavelength of light (Figure
5.2). The fluorescent label attached to the cell is excited and emits light (e.g. forward
scatter, side scatter) which is detected and stored as digital information. This data
yields information on the size, shape and homogeneity of the cells passing through the
detector. The cell population of interest can be identified by a user defined 'gate' and
cell populations can be quantified. An example of data presentation from flow
cytometry analysis of blood cell populations is shown in Figure 5.3.
The other whole cell biopanning method employed RBCs selected from a Phenocell
panel (CSL; Figure 5.4). A cell suspension, positive for a particular antigen, was
chosen for biopanning and subsequently two other cell suspensions were chosen from
the panel that carried all the antigens present on the positive cells except for the antigen
of interest. For example, cell number 10 was chosen for Fy^ selection (Figure 5.4). As
well as being positive for Fy^, this red blood type was also positive for; c, e, k, Jk^, Jk'',
N, s and Le^. In order to minimise the possibility of isolating antibodies to these other
epitopes, two other cell types were chosen from the panel which were negative for Fy^
but positive for c, e, k, Jk^, Jk^, N, s and Le^ These were cell numbers 6 and 9. This
process of choosing cells for selection and subtraction purposes was repeated for all
antigens of interest in the panel; Fya, Fy^ K, Jk^ D, C and E. In two cases, it was not
possible to subtract for all of the antigens present on the selection cells. In biopanning
experiments for Jk^ antigen, it was not possible to subtract for Le^ antigen, and in
experiments with D antigen there was no subtraction for Kp^ antigen.
Figure 5.2 Basic diagram of flow cytometry.
Cells pass in 'single file' through a band of light at a suitable wavelength to excite
the fluorophore used to label the cells. Light is emitted, captured by various
detectors and digitally translated into graphical and numerical data by the
associated software.
(Figure adapted from Murphy, 1996)
I'' 10^ 10^ 10' 10"

Relative fluorescence- Intensify I 111 11 I I
255
FSG-H
Figure 5.3 Analysis of blood cell populations by flow cytometry.

Left: Histogram showing the number of events (cells measured) against the
fluorescence intensity.
Right: Dot plot (e.g. forward scatter against side scatter) from stained human
peripheral blood cells with selected cell populations (gates) shown as different
colours and outlined. The red, green and blue populations represent lymphocytes,
monocytes and granulocytes respectively, separated on the basis of size and
homogeneity.
(Figures adapted from Osbourne, 2000)
The other stage in the phage antibody selection and isolation procedure, where access to
antigen is crucial, is in screening of clones obtained after rounds of biopanning. Once
phage antibodies are eluted from antigen, they are re-infected into E. coli and each
eluted phage results in a colony on the selection plate. Phage antibodies are produced
from these single clones in a microtitre plate which is replicated and the original stored
as a master plate. Phage antibodies from the replica plate are tested in a phage ELISA
format (Figure 1.35) where each clone is exposed to antigen in a separate well of a
microtitre plate. Phage carrying a Fab molecule that is specific for the antigen bind to
the plate and are detected using an enzyme-linked conjugate that binds to the phage
particle. Any clone giving a positive signal in the ELISA can be recovered from the
master plate and cultured for further characterisation and eventual expression of the
antibody carried by the phage.
A screening ELISA requires a solid phase consisting of the antigen of interest, usually
the same as that used for biopanning. This is straightforward in the case of a purified
protein (such as Fy^ extracellular domain) as it is resuspended in a low ionic strength
buffer and incubated in the wells of a microtitre plate. Screening for an antigen which
cannot be obtained as a purified protein is more difficult and necessitates the use of cells
in solution or the immobilisation of cells on to the surface of microtitre wells. It is also
possible to screen for antibodies to RBC surface antigens by using an assay format that
identifies positive reactions by red cell agglutination.
it
1-1 nr'''-'
i i
5*
SI
Sli i1
Figure 5.4 Antigen Composition Sheet for Phenocell RBC Reagent Panel used in
biopanning.
Eleven RBC samples are included which have been typed for 22 different antigens
from the Rhesus, Kell, Duffy, Kidd, MNS, P, Lewis, Lutheran and Colton systems.
5.3 Methods
5.3.1 Characterisation of Transfected HEK293 Cells by Flow Cytometry
Transfected cells carrying antigens relevant to antibodies present in the constructed

libraries were characterised by binding with specific human antiserum. The following
cell lines were tested; 293T.Fya.4 carrying Fy^ antigen, 293T.Fyb.9wt carrying Fy^
antigen and 293T.K1.41 carrying K1 (Kell 1) antigen. Cells transfected with an empty
cloning yector (293T.Vector5) were used as a negative control for some samples.
Insufficient 293T.Vector5 cells were available for all controls, and so a cell line
carrying an irrelevant antigen was used (293T.Jsa.lO). Samples containing 10^ cells
were reacted with positive serum samples from one donor for each specificity along
with a negative control (Section 2.5.8). Human antibody bound to the cells was
detected with an anti-Human Immunoglobulin-FITC conjugate and samples were
analysed by flow cytometry. Samples were analysed on a Coulter EPICS Elite ESP
flow cytometer using wavelengths suitable for FITC detection and collecting forward
and side scatter data (experimentation carried out by ARCBS-NSW flow cytometrist).
Transfected HEK293 cells were also analysed using the same technique to test for
maintenance of transfected antigen over many passages. Several aliquots of an early
passage of cell line 293T.Fya.4 were frozen according to Section 2.5.6. Aliquots were
thawed (Section 2.5.2) and set up in culture at two week intervals (Section 2.5.7).
Cultures were maintained and passaged over a period of 2 to 3 months (Section 2.5.7)
to give a set of cultures at 9, 13, 17, 21 and 25 passages (14, 25, 39, 53, 67 days after
inoculation respectively) which were all analysed on the same day. Empty cloning
vector (293T.Vector5) cell line was used as a negative control.
5.3.2 Analysis of Recombinant Fy^ Protein by ELISA
The recombinant protein containing the extracellular Fy^ antigen domain was tested by
ELISA. The lyophilised Fy^ protein was resuspended in PBS to giye a 1 mg/mL stock,
which was aliquoted and stored at -20 °C. This sample was diluted to 5 }ig/mL in
carbonate binding buffer and added at 50 }iL per well to a Nunc Maxisorp microtitre
plate. The plate was coated at 4 °C oyemight before being washed in PBST. Serial
dilutions of Fy^ positive and Fy^ positive (as negative control) human antiserum were
carried out in PBST (1 in 4, 1 in 8 and 1 in 16) and added to the plate at 50 [xL per well
along with PBST alone as a control to check for non-specific binding of the conjugate.
The plate was incubated at RT for ~1 h before being washed in PBST. Binding of
human antiserum was detected by the addition of anti-Human Immunoglobulin Alkaline
Phosphatase conjugate at 1/5000 dilution in PBST. The plate was incubated and
washed as before and conjugate binding detected by the addition of PNPP substrate
(SIGMAi^^5'7^'^p-Nitrophenyl phosphate Tablets, Sigma), reconstituted according to
manufacturers instructions and added at 100 \xL per well. The developed plate was read
on a microplate reader at 405 nm.
5.3.3 Rescue of Phage Libraries for Biopanning
Library glycerol stocks were used to inoculate 2TYAG media for library rescue
according to Section 2.7.7. Donor and patient libraries were rescued separately. Donor
libraries KDHD (vector containing kappa light chain with heavy chain insert) and
HDLD (vector containing heavy chain with lambda light chain insert) were pooled and
rescued together (see Table 4.10 for library details) as were patient libraries HPKP and
HPLP (vector containing heavy chain with kappa or lambda insert). Phage rescue from
glycerol stocks for second and subsequent rounds of biopanning was performed
5.3.4 Biopanning of Immune and Naïve Libraries Against Recombinant Fy^ Protein
The donor library was selected for biopanning against the Fy^ recombinant protein as
three of the donor serum samples were shown to contain Fy^ antibodies whereas none of
the patient samples contained Fy^ antibodies. As well as the constructed immune
library, a naïve, scFv library (Vaughan et al., 1996) obtained from Cambridge Antibody
Technology was also biopanned against the Fy^ recombinant protein. The strategy of
biopanning both immune and naïve libraries was envisaged to maximise the opportunity
for isolation of a range of specific phage antibodies. A total of three rounds of
biopanning were carried out in each case according to Section 2.7.9.
5.3.5 Biopanning of Immune Libraries Against Whole Cells (Transfected HEK293

and RBCs)
As previously mentioned, for most antigens of interest a soluble antigen was not
available. This necessitated the use of whole cell biopanning strategies. Cells carrying
antigens relevant to antibodies present in the immune libraries were selected for
biopanning. The donor library was biopanned against Fy^ and K1 HEK293 cell lines,
and the patient library biopanned against Fy^ and K1 cell lines using Vector 5 cell line
for subtraction biopanning in both cases (Section 2.7.10). The donor library was also
biopanned against RBCs carrying the following antigens; Rhesus D, C and E, Fy^ and
K1 using antigen negative RBCs for subtraction biopanning (Section 2.7.11). The
patient library was biopanned against RBCs carrying the following antigens; Rhesus E,
Fy^, Kell 1 and Jk^. A total of three rounds of biopanning were carried out in each case.
5.3.6 Analysis of Fv^ Clones Isolated by Biopanning
Individual clones from Fy^ biopanning (soluble antigen or whole cell) output plates
were rescued according to Section 2.7.14 and the resulting phage screened for
specificity by Fy^ phage ELISA (Section 2.7.15). Several positive and negative clones
were selected from this screen to be used in development of a whole RBC screening
assay. These clones were rescued in the same way (Section 2.7.14) but instead of using
100 [iL volumes in a microtitre plate, selected clones were rescued in 5mL volumes in
50 mL tubes. These samples were titrated (1 in 2 to 1 in 128) and analysed by Fy^
phage ELISA (Section 2.7.15). Wells without any phage were included to check for
non-specific binding of conjugate and were used as a background absorbance value.
5.3.7 Development of a Screening Assay for RBC Antigen Specific Clones
Positive and negative clones selected during Fy^ biopanning were used in development
of a RBC based screening assay which could then be used to screen other RBC
antibodies for which purified antigen is not available (such as Fy*', Rhesus D and Kell
antigens). RBC screening assay formats are illustrated in Figure 5.5. In assay A, RBC
were immobilised using wheat germ agglutinin, a type of lectin, which binds to RBC
surface glycoproteins (Tamai and Mazda, 1999). In all assays, specific phage
antibodies are detected with anti-M13 followed by an enzyme linked secondary
antibody except for assay D where binding is detected utilising the presence of
endogenous RBC haemoglobin peroxidase (Moore and Conradie, 1982). Immobilised
RBC assays were carried out according to Section 2.7.16.
/Substrate (PNPP)
A / Colour development B
Substrate
Anti-Mouse Conjugate (PNPP) colour
development
Anti-Sheep
Conjugate
Antigen specific Fab
Solid phase assay Solution phase assay Solution phase assay
D
Wash rbc
and transfer
Substrate (ABTS) Colour

development from
endogenous rbc peroxidase
Antigen
Specific
Fab
Anti-Mi 3
Antibody
Solution phase
Figure 5.5 RBC screening assay formats.

A: Immobilised lectin binds RBC via carbohydrate groups and phage antibodies
specific for RBC antigens are detected using anti-M13 and alkaline phosphatase
(AP) conjugate (PNPP substrate, para-nitrophenyl phosphate). B: RBC in solution,
sensitised with specific phage antibodies, are cross linked (agglutinated) using anti-
M13 antibody. C: Assay format same as in A but RBC in solution rather than
immobilised. D: Washed RBC, sensitised with phage antibodies are captured on
an anti-M13 solid phase and bound RBC detected due to the presence of
endogenous peroxidase (ABTS substrate: 2,2'-azino-bis(3-ethylbenzthiazoline-6-
sulphonic acid).
5.4 Results
5.4.1 Characterisation of Transfected HEK293 Cells by Flow Cytometry
Flow cytometry data was collected from the main cell population which was isolated by
a user defined gate based on a dot plot of side versus forward scatter fluorescence data.
Cells falling outside the main cell population were excluded from analysis (Figures 5.7
to 5.9). Data was also expressed as a histogram of cell count against fluorescence
intensity (Figure 5.6). Between 7000 and 11,000 cells were analysed from each sample.
For all serum samples tested, average and peak fluorescence values were higher for
antigen positive cells than for cells with no antigen present (Table 5.1 and Figure 5.6),
The cell count at peak fluorescence was similar for all cell suspensions indicating that
sampling was consistent across the experiment (Table 5.1).
In addition, maintenance of Fy^ antigen expression on transfected HEK293 cells was

monitored by flow cytometry over a period of ~9 weeks (25 passages; Table 5.3). Data
was collected in the same way as for the previous flow cytometry experiments on Fy^
and Fy^, and Kell antigen transfected cells. Results from the time course experiment
show that the Fy^ signal is maintained over the course of the experiment (Table 5.2 and
Figure 5.10) and all time course samples gave higher average fluorescence values than
the negative control (293T.Vector5 cell line).
K1
-V6
Anti-Fya + FyaCells Anti-Fya + Qrl Cells Anti-Fyb + Fyb Cells Anti-Fyb + Qrl Cells Anti-K + K1 Cells Anti-K + Qrl C
Figure 5.6 Characterisation of transfected HEK293 cells by flow cytometry.

Average fluorescence values for detection of specific human antiserum against
antigen positive and control HEK293 transformed cell lines. For all serum
samples tested, average fluorescence values were higher for antigen positive cells
than for cells with no antigen present. See Table 5.1 for data.
Table 5.1 Characterisation of transfected HEK293 cells by flow cytometry.
Average fluorescence represents the total fluorescence intensity divided by the
number of cells measured. The peak fluorescence represents the most commonly
occurring value for fluorescence intensity within the gated population (mode), and
the cell count is the number of cells having the modal fluorescence value. See
Figures 5.7 to 5.9.
Sample Antibody Cell Line Average Peak Cell count at
Fluorescence Fluorescence Peak
Fluorescence
Positive Anti-Fya 293T.Fya.4 6.09 7.0 27
Control Anti-Fya 293T.Vector5 1.79 1.5 29
Positive Anti-Fyb 293T.Fyb.9wt 5.87 5.0 36
Control Anti-Fyb 293T.Jsa.lO 2.06 2.0 24
Positive Anti-K 293T.K1.41 9.31 10.0 17
Control Anti-K 293T.Jsa.lO 1.79 2.0 31

G: A
SCATTER 5000/5000
C 3_
•vO
O-
ID
CO.
_<E
J O.
0-)
LJ_ C
KI
o.
(N
O" —T" m 1 I I n I III 1 I VII Ifir

10 20 40 50 60 I I III
90LS
1 10 100 1000
FITC LOG
G: A
SCATTER
11
- 1 ||
• /
1.1 ,1
r . \
—'fill inn ' 1 1 1 mill—^tS^îV'Itt- "I I I 111II
30 40
90LS FITC LOG
Figure 5.7 Flow cytometry dot plots (left) and histograms (right) for Fy^ specific
human antiserum binding to HEK cells carrying Fy^ antigen.
Top: 293T.Fya.4 HEK293 cells reacted with anti-Fy^ positive human antiserum
and labelled with anti-human Ig FITC conjugate.
Bottom: 293T.Vector5 HEK293 cells with no antigen, reacted with anti- Fy^
positive human antiserum and labelled with anti-human Ig FITC conjugate.
G: A
SCATTER 5000/5000
30 40 10 100 1000
90LS FITC LOG
6: A
SCATTER 5000/5000
I 111 III mri

10 100 1000
FITC LOG
Figure 5.8 Flow cytometry dot plots (left) and histograms (right) for Fy specific
human antiserum binding to HEK cells carrying Fy*' antigen.
Top: 293T.Fyb.9wt HEK293 cells reacted with anti- Fy^ positive human antiserum
and labelled with anti-human Ig FITC conjugate.
Bottom: 293T.Jsa.lO HEK293 cells carrying Kell Jsa antigen (used as a negative
control in the absence of sufficient 293T.Vector5 cells), reacted with anti- Fy^
G: A
SCATTER 5000/5000
• 1!M
!
• /
/ 1
I1 ,
30 40
90LS FITC LOG
G: A
SCATTER 5000-5000
FITC LOG
Figure 5.9 Flow cytometry dot plots (left) and histograms (right) for Kell (Kl
antigen) specific human antiserum binding to HEK cells carrying Kell antigen.
Top: 293T.K1.41 HEK293 cells carrying K1 (Kell 1) antigen, reacted with anti-K
Bottom: 293T.Jsa.lO HEK293 cells carrying Kell Jsa antigen (used as a negative
control in the absence of sufficient 293T.Vector5 cells), reacted with anti-K positive
human antiserum and labelled with anti-human Ig FITC conjugate.
2.5
8c 2
(1)
CO
0)
o
¡1 1.5
(D
O)
(U
5 1
0.5
Control 13 17 21 25
Passage Number
Figure 5.10 Time course flow cytometry data for Fy^ specific human antiserum
binding to cells carrying Fy^ antigen over a time course of 9 to 25 passages.
Average fluorescence values for detection of specific human antiserum against
antigen positive and control HEK293 transformed cell lines over a time course of 9
to 25 passages. See Table 5.2 for data.
Table 5.2 Flow cytometry results for Fy^ specific human antiserum binding
to293T.Fya.4 HEK293 cells carrying Fy^ antigen over a time course of 9 to 25
passages.
Average fluorescence represents the total fluorescence intensity divided by the
number of cells measured. The peak fluorescence represents the most commonly
occurring value for fluorescence intensity within the gated population (mode), and
the cell count is the number of cells having the modal fluorescence value.
Sample Cell Line Passage Average Peak Cell count at
Fluorescence Fluorescence Peak
Fluorescence
Control 293T.Vector5 9 0.6 0.5 32
Positive 293T.Fya.4 9 2.18 1.2 30
Positive 293T.Fya.4 13 2.51 1.2 28
Positive 293T.Fya.4 17 2.17 1.1 28
Positive 293T.Fya.4 21 2.32 1.1 28
Positive 293T.Fya.4 25 1.46 1.1 25

Table 5.3 Time course flow cytometry analysis of HEK cells carrying Fy^ antigen.
Flow cytometry dot plots (left) and histograms (right) for Fy^ specific human
antiserum binding to293T.Fya.4 HEK293 cells over 9 to 25 passages.
Negative Control ^ I
Cell line: Vector 5
Passage 9 •i
a,. -
Sample 1
Cell line: Fya.4
Passage 9
it. - ——rzt
Sample 2
Cell line: Fya.4 '
f1
I
Passage 13 I
3t. " Mr-*. J rz—=r
Sample 3
Cell line: Fya.4 A
Passage 17
- .Wil
Sample 4
Cell line: Fya.4 ,1
Passage 21 i >
' 'i
Sample 5
Cell line: Fya.4 A-
Passage 25 'i
4. -
Ik
5.4.2 Analysis of Recombinant Fy^ Protein by ELISA
Recombinant Fy^ protein immobilised on a microtitre plate was tested by reaction with
human anti-Fy^ serum. Human anti-Fy"^ serum was used as a negative control and
detection was carried out with anti-Human IgG alkaline phosphatase conjugate plus
chromogenic substrate (Figure 5.11). The signal from anti- Fy^ serum binding to
recombinant Fy^ protein was significantly higher than the anti- Fy^ serum control. The
anti- Fy^ serum control showed slight non-specific binding at higher concentration but
signals were no higher than would be expected from a whole, unfractionated serum
sample. Results indicated that the recombinant protein was suitable for use in
biopanning experiments to isolate specific anti-Fy^ phage antibodies.
07
0.6
0.5
0.4
"Anti-Fya
Anti-Fyb
0.3
0.2 1
0.1
1 in 16 1 in 8 1 in 4
Antibody Concentration
Figure 5.11 ELISA to test the recognition of Fy^ recombinant protein by specific
human antiserum (anti-Fy^).
Human anti- Fy'' serum was used as a negative control. See Table 5.4 for data.
Table 5.4 ELISA to test the recognition of Fy® recombinant protein by specific
human antiserum (anti-Fy^).
Human anti- Fy*' serum was used as a negative control.
Human antiserum ELISA Signal from ELISA Signal from Anti-
Dilution Factor Anti-Fy^ serum Fy^ serum
0 0.081 0.083
1 in 16 0.242 0.1115
1 in 8 0.387 0.133
1 in 4 0.602 0.167
5.4.3 Rescue of Phage Libraries for Biopanning
Phage antibodies were recovered from phagemid stocks in E. coli by infection with
helper phage. The presence of phagemid within the bacterium confers ampiciUin
resistance and successful helper phage infection is indicated by the acquisition of
kanamycin resistance. Thus, library rescue can be monitored by comparing the
proportion of ampicillin-resistant colonies to the number with conferred kanamycin
resistance. A successful rescue is indicated by the number of colonies on the
ampicillin selection plates (Bradbury and Marks, 2004b). Efficient helper infection for
the patient library (100 %) and acceptable levels for the donor library (40 %) were
achieved as shown in Table 5.5.
Table 5.5 Antibiotic selection of phagemid libraries post helper phage infection.
A 1 ^L sample was plated on to LBAmp and LBKan agar plates and incubated at
37 °C overnight.
Donor Library Patient Library
Number of colonies on ampicillin selection plates. 150 120
Number of colonies on kanamycin selection plates. 60 140
Efficiency of helper infection. 40% 100%
5.4.4 Biopanning of Immune and Naive Libraries Against Recombinant Fy^ Protein
The donor library was biopanned against the recombinant Fy^ protein over a series of
three rounds (Figure 5.12). A decrease in the phage input: output ratio is an indication
of enrichment of antigen-specific phage antibodies. This is because the phage titre of
the output vîll increase over rounds of biopanning due to amplification of antigen-
specific phage antibodies in E. coli, while input phage titre remains constant. The
phage output titre from round 2 of the biopanning process was very low so the second
round was repeated but no increase in output was observed. The output from the third
round was lower, making it unlikely that enrichment for Fy^ positive clones was
occurring. This lack of enrichment was also reflected in the input: output ratio which
increased over the four rounds.
Biopanning of the Vaughan naive library (Vaughan et al., 1996) using the same antigen
and the same method showed very different results (Figure 5.12). Biopanning was
carried out over four rounds, and after an initial drop in output at the second round,
outputs increased in the third and fourth rounds. The input: output ratio decreased over
rounds two to four, which may be indicative of a successful selection.
Table 5.6 Phage input and output titres for donor and naïve libraries over three
and four rounds respectively.
Aliquots of phage, pre- and post-exposure to antigen were reinfected into TGI and
plated onto selection media to give the phage titre. Ratio is phage input divided by
phage output.
Phage Input Phage Output Ratio
Donor Library Round 1 1.2 X 10^ 5.0 X 10^ 24
Donor Library Round 2 1.2 X 10^ 5.0 X 10^ 2.4 X 10^
Donor Library Round 2 Repeat 2.5 X 10^ 1.5 X 10^ 1.7 X 10^
Donor Library Round 3 5.0 X 10^ 5.0 X 10' 1.0 X 10^
Naive Library Round 1 5.0 X 10^ 2.0 X 10^ 2.5
Naive Library Round 2 4.4 X 10^ 3.0 X 10^ 1.5x10^
Naive Library Round 3 1.2 X 10^ 2.5 X 10^ 0.5 X 10"^
Naive Library Round 4 2.5 X 10^ 7.5 X 10^ 0.3 X 10^
1.E+10 1
1.E+09
• Input
1.E+08
• Output
1.E+07
1.E+06
I-
® 1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
DLR1 DLR2 DLR2 Rpt DLR3 NLR1 NLR2 NLR3 NLR4
Figure 5.12 Phage input and output titres for donor (DL) and naïve (NL) libraries
over three (R1 to R3) and four rounds respectively (R1 to R4).
Round 2 was repeated for the donor library. Aliquots of phage, pre- and post-
exposure to antigen were reinfected into TGI and plated onto selection media to
determine the phage titre. See Table 5.6 for data.
5.4.5 Biopanning of Immune Libraries Against Whole Cells (Transfected HEK293

and RBCs)
All cell based biopanning experiments were carried out over three rounds. Three
HEK293 cell lines were used for biopanning which carried the following antigens; Fy^,
Fy^ and K1. The donor library was biopanned against Fy^ and K1 cell lines and the
patient library was biopanned against Fy'^ and K1 cell lines (Figure 5.13). In all cases
the initial phage output titres were relatively low but had increased by the third round.
For RBC biopanning, donor and patient phage libraries were pooled and exposed to the
following antigens; Fy^ and Fy^, Kl, Jk^ and D, C and E (Figure 5.14). The first round
output titres for RBC biopanning were low but increased over the second and third
rounds. However, there were no significant decreases in input: output ratios over the
three rounds of biopanning suggesting that antigen-driven selection was not occurring.
Table 5.7 Phage input and output titres of biopanning against HEK 293 cells over
three rounds.
Donor and patient libraries were biopanned separately. Aliquots of phage, pre-
and post-exposure to antigen were reinfected into TGI and plated onto selection
media for determining the phage titre. Ratio is phage input divided by phage
output.
Input Output Ratio

HEK Fya Donor Library Round 1 2.0 X 10^ 3.5 X 10^ 0.6 X 10^
HEK Fya Donor Library Round 2 4.0 X 10^ 6.0 X 10^ 0.7 X 10^
HEK Fya Donor Library Round 3 4.0 X 10'^ 1.3 X 10^ 3.1 X 10^
HEK Fyb Patient Library Round 1 5.0 X 10^ 7.0 X 10^ 0.7 X 10^
HEK Fyb Patient Library Round 2 1.5x10'^ 1.2 X 10^ 1.3 X 10^
HEK Fyb Patient Library Round 3 8.0 X 10'^ 1.6 X 10^ 5.0 X 10^
HEK Kell Donor Library Round 1 2.0 X 10^ 2.0 X 10^ 1.0 X 10^
HEK Kell Donor Library Round 2 8.0 X 10^ 1.2 X 10^ 6.7 X 10^
HEK Kell Donor Library Round 3 1.0x10^' 1.4 X 10^ 0.7 X 10^
HEK Kell Patient Library Round 1 5.0x10^ 4.0 X 10^ 1.3 X 10^
HEK Kell Patient Library Round 2 6.5x10^ 1.4 X 10^ 4.6 X 10^
HEK Kell Patient Library Round 3 5.0 X 10^ 2.4 X 10^ 2.1 X 10^
Table 5.8 Phage input and output titres of biopanning against human RBCs over
three rounds.
Donor and patient libraries were pooled and biopanned together. Aliquots of
phage, pre and post exposure to antigen were reinfected into TGI and plated onto
selection media to determine the phage titre. Ratio is phage input divided by
phage output.
R1 Input R1 Output R2 Input R2 Output R3 Input R3 Output

RBC
1.0 X 2.1 X 10^ 1.0 X 10'^ 1.0 X 10^ 3.2 X 10'^ 3.6 X 10^
Fya
R1 Ratio 0.5 X 10^ R2 Ratio 1.0x10^ R3 Ratio 0.9x10^
RBC
5.0 X 10^ 2.3 X 10' 6.0 X 10^ 8.0 X 10' 2.3 X 10'^ 2.1x10^
Fyb
R1 Ratio 2.2 x 10^ R2 Ratio 0.8x10^ R3 Ratio 1.1x10^
RBC
4.0 X 10^ 3.4 X 10' 4.0 X 10^ 2.2 X 10^ 3.4 X lO'^ 2.8 X lO''
Kell
R1 Ratio 1.2x10^ R2 Ratio 1.8x10^ R3 Ratio 1.2x10^
RBC
2.3 X 10^ 3.2 X 10' 5.0 X 10^ 7.5 X 10' 2.7 X 10'^ 1.5 X 10^
Kidd
RBC
2.0 X 10^ 2.4 X 10' 1.0 X 10'^ 2.3 X 10^ 1.4 X 10'^ 1.6 X 10^
D
RBC
3.4 X 10^ 3.2 X 10' 5.0 X 10^ 8.0 X 10^ 2.0 X 10'^ 4.5 X 10^
C
RBC
1.4 X 10^ 8.1 X 10' 3.0 X 10^ 5.0 X 10' 1.5x10'^ 1.3 X lO''
E
1Ef11
1E+10
1&-09
1&08
1Ef07
pi
S 1&-06
0G))
£ 100000
Q.
10000
1000
100
10
HEKFya HEK Fya HEK Fya HEK Fyb HEK Fyb HEK Fyb HEK Kell HEK Kell HEK Kell HEK Kell HEK Kell HEK Kell
DLR1 DLR2 DLR3 PLR1 PLR2 PLR3 DLR1 DLR2 DLR3 PLR1 PLR2 PLR3
Figure 5.13 Phage input and output titres of biopanning against HEK 293 cells over three rounds.
Phage input (light coloured bars) and output titres (dark coloured bars) for donor and patient libraries over three (R1 to R3) rounds of
biopanning against HEK293 cells carrying antigen are shown. See Table 5.7 for data. Colours represent different panning experiments.
1&-11
1&-10
m R1 Input
1&-09 • R1 Output
1Ef08 • R2 Input
1&-07 • R2 Output
I 1&06
• R3 Input
0O))
5100000
Q. • R3 Output
10000
1000
it
100
10
RBCFya RBC Fyb RBC Kel RBC Kidd RBCD RBCC RBCE
Figure 5.14 Phage input and output data for RBC whole cell biopanning over three rounds.
Phage titres for pooled donor and patient libraries over three rounds (R1 to R3) of biopanning against RBCs carrying antigen are
shown. See Table 5.8 for data
5.4.6 Analysis of Positive Clones Isolated by Biopanning
Individual clones isolated by biopanning of the donor and naive libraries against
recombinant Fy^ antigen were rescued and tested for specific binding activity. A
control was set up for each sample to show non-specifically binding phage (skim-milk
coated solid phase). For the donor and naïve library round 2 biopanning, 24 clones
were selected for analysis and 48 clones from naive library round 3 biopanning. Results
for binding to positive and control antigen were plotted (Figure 5.15). No positive
clones were identified from the donor library but several positive signals were obtained
from the naive library. The same clones from the naive library were rescued and re-
tested by phage ELIS A in order to confirm the results (Figure 5.16). Many of the
samples were positive on both occasions suggesting that these samples contain phage
carrying scFv which are specific for Fy^ antigen.
Round 3 clones isolated by whole cell biopanning against HEK293 or RBC carrying the
Fy^ antigen were also tested for binding to Fy^ recombinant protein by phage ELIS A.
Some positive signals from antibody phage clones were identified in these assays
including H4 and H6 from biopanning against RBC and Dl, H3 and G6 from
biopanning against HEK cells (Figure 5.17 and Table 5.9). This experiment was
repeated (results not shown) and further positive signals from antibody phage clones
were obtained.
Table 5.9 Raw phage ELISA data results from third round biopanning of pooled
donor and patient libraries against recombinant HEK293 or RBCs carrying the
Fy^ antigen.
ELISA data was first blanked against substrate only signal (~0.12 absorbance units
at 450 nm) and subsequently the signal from the negative control plate was
subtracted from the positive signal. Any samples showing an absorbance value
greater than or equal to the substrate background value after this process are
highlighted in bold; those giving a negative value are italicised.
RBC 1 2 3 4 5 6 7 8 9 10 11 12
Row A 0.01 0.00 0.01 0.00 0.01 0.01 0.00 0.00 0.01 0.00 0.00 0.01
Row B 0.00 0.01 0.00 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.00 0.00
RowC 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.00 0.01
Row D 0.02 0.11 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.00 0.08 0.02
Row E 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.00
Row F 0.02 0.01 0.03 0.00 0.01 0.01 0.04 0.01 0.02 0.00 0.02 0.01
RowG 0.01 0.00 0.03 0.00 0.01 0.02 0.02 0.00 0.00 0.00 0.01 0.00
Row H 0.01 0.01 0.01 0.16 0.00 0.18 0.01 0.00 0.01 0.00 0.00 0.00
HEK 1 2 3 4 5 6 7 8 9 10 11 12
Row A 0.01 0.01 0.03 0.02 0.03 0.03 0.03 0.03 0.04 0.02 0.01 0.02
Row B 0.01 0.08 0.02 0.21 0.17 0.02 0.03 0.00 0.01 0.05 0.00 0.06
RowC 0.01 0.04 0.02 0.02 0.02 0.03 0.03 0.02 0.05 0.02 0.02 0.03
Row D 0.14 0.02 0.03 0.02 0.02 0.00 0.04 0.02 0.02 0.02 0.10 0.02
Row E 0.27 0.02 0.03 0.05 0.04 0.01 0.02 0.03 0.04 0.00 0.01 0.01
Row F 0.31 0.02 0.05 0.02 0.01 0.02 0.02 0.03 0.02 0.02 0.00 0.02
RowG 0.01 0.02 0.08 0.01 0.02 0.18 0.03 0.00 0.01 0.02 0.00 0.00
Row H 0.00 0.02 0.41 0.02 0.02 0.01 0.02 0.01 0.00 0.01 0.00 0.02
0.8
Samples 49 to 88 - Naïve Library Round 3

< — •
0.7
• Negative Control (Skim milk solid phase)

0.6
D Positive Signal (Recombinant Duffy a solid phase)
0.5
Samples 25 to 48
«3
C
O)
Naïve Library Round 2
'¿Ô
< 0.4
W
-J
Ul
0.3
Samples 1 to 24
Donor Library Round 2
0.2 ^^ •
0.1
oOODUllllkiOOOnDDDnODifl Hljifijinfijilllinnnrtliyaftftuiftfi
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91
Sample number
Figure 5.15 Phage ELISA results for individual clones from second and third round biopanning of donor and naïve libraries.
Samples were tested for specific (red bars) and non-specific (blue bars) binding to Fy^ recombinant antigen via phage ELISA.
0.9 • First ELISA
• Second ELISA
0.8
0.7
0.6
re
c
en
w
< 0.5
CO
LU
0.4
0.3
0.2
0.1
I
\ "b ^ \ ^ N^ N^ q> ^ ^ ^ ^ ^^ ^ ^ 4 Ix^ li^ ^
Sample number
Figure 5.16 Repeat phage ELISA results from third round biopanning of naïve library against Fy® recombinant antigen.
Blue bars show signals from the first ELISA and red bars show signals from the repeated ELISA
1 12 1 12
< <
o C
CL
CO 00
C7) p
CM CL
€ ^ O
• X X . S
q:
1 12 1 12 ^
' <
D)
(D t
CNJ
o
LU -p CÛ
X ^ ^
Figure 5.17 Photograph of substrate developed phage ELISA plates from third
round biopanning samples of pooled donor and patient libraries against Fy^
antigen positive HEK293 or RBC.
Pos: Plate coated with recombinant Fy^ antigen. Neg: Plate coated with skim milk
solution as a negative control. A total of 96 colonies were rescued for each cell ty pe
and the phage samples replicated between positive and control plates, i.e. Well A1
on the 'RBC Pos' plate has the same phage sample as well A1 on the 'RBC Neg'
plate. See Table 5.9 for raw data.
5.4.7 ELISA of Selected Positive Clones
Positive clones identified in the screening assay of third round naïve library clones were
cultured and rescued in a larger volume to allow further testing. Negative clones were
also rescued for use as negative controls. Four positive (numbered 9, 23, 27 and 36
based on their position in the microtitre plate) and two negative clones were selected (4,
6) and assayed by phage ELISA (Figure 5.18). In addition, these samples were diluted
in series and assayed for binding to Fy^ antigen. The positive samples gave a titratable
response and the negative sample responses were at background levels (Figure 5.19).
1.8
1.6
1.4
1.2
< 1
«2 0.8
0.6
0.4
0.2
23 27 36 43 BIO B12 kADII rADII F3 G3 G6 Conj only Substrate

only
Clone number
Figure 5.18 ELISA of positive Fy^ clones isolated from naïve and immune libraries.
Naïve library (9, 23, 27, 36 and 43) and immune library (BIO, B12, k A D l l , r A D l l ,
F3, G3 and G6) rescued phage antibodies were bound to Fy^ antigen and detected
using anti-M13 conjugate. See Table 5.10 for data.
Table 5.10 Raw data from ELISA of positive Fy^ clones isolated from naïve and
immune libraries.
Naïve (9, 23, 27, 36 and 43) and immune (BIO, B12, kADll, rADll, F3, G3 and G6)
library samples were analysed in duplicate and the average taken.
Clone Number Absorbance in duplicate Average
9 1.5335 1.4875 1.5105
23 1.1474 1.5188 1.3331
27 1.4852 1.4353 1.46025
36 1.5401 1.5188 1.52945
43 1.4629 1.4444 1.45365
BIO 0.0667 0.0871 0.0769
B12 0.077 0.0805 0.07875
kADll 0.0656 0.0681 0.06685
rADll 0.0764 0.0736 0.075
F3 0.0788 0.0755 0.07715
G3 0.0624 0.071 0.0667
G6 0.0662 0.0806 0.0734
Conj only 0.0772 0.0872 0.0822
Substrate only 0.0593 0.0587 0.059
Table 5.11 Raw data from ELISA titration of positive and negative Fy® clones
isolated from the naïve library.
Fy^ positive (9, 23, 27 and 36) and negative (4 and 6) samples were analysed in
duplicate and the average taken.
Dilution
Clone 9 Clone 23 Clone 27 Clone 36 Clone 4 Clone 6
Factor
No sample 0.0539 0.0524 0.0548 0.0556 0.052 0.0522
1 in 128 0.0635 0.0608 0.0652 0.0566 0.0521 0.0526
1 in 64 0.0725 0.0744 0.0712 0.0701 0.0515 0.0505
1 in 32 0.1224 0.1272 0.1297 0.1342 0.0528 0.0531
1 in 16 0.3039 0.3124 0.3122 0.3203 0.0555 0.0501
1 in 8 0.6387 0.6107 0.6001 0.6237 0.0526 0.0543
1 in 4 0.828 0.826 0.7198 0.7321 0.0556 0.0552
1 in 2 0.878 0.8702 0.7603 0.8424 0.0561 0.0484
1 1
0.9
—•—Clone 9 - • - C l o n e 23
0.8
Clone 27 — C l o n e 36
0.7 -
Clone 4 -"•—Clones
0.6
ë 0.5
CO
€
o
(A
$ 0.4
0.3
0,2
0.1
1 in 128 l i n 64 1 in 32 1 in 16 1 in 8 1 in 4 1 in 2
Phage antibody titration
Figure 5.19 ELIS A titration of positive and negative Fy^ clones isolated from the
naïve library.
Fy^ positive (9, 23,27 and 36) and negative (4 and 6) rescued phage antibodies
were bound to Fy^ antigen and detected using anti-M13 conjugate. See Table 5.11
for data.
5.4.8 Screening Assay for RBC Antigen-Specific Clones
Phage antibodies specific for Fy^ antigen were isolated from the naive library and
cultured to provide sufficient sample for use in the development of a whole RBC
ELISA. Negative clones were also generated for use as controls. In assay A, RBCs
were captured in microtitre wells using lectin to form an antigen solid phase for binding
of phage antibodies. A primary, unconjugated anti-M13 antibody was used to detect
bound phage, followed by a secondary alkaline phosphatase conjugated antibody as the
available anti-M13 horseradish peroxidase (HRP) conjugate could not be used due to
the presence of endogenous peroxidase in RBCs.
The integrity of the RBC solid phase was monitored by absorbance after immobilisation
(after 3 plate washes) and just before substrate addition (after 9 plate washes; Figure
5.20). Despite the fact that a significant number of RBCs remained on the solid phase
up to the final step, no positive signal was obtained from the assay. All wells showed
an absorbance of approximately 0.05 to 0.1 at 405 nm which is the level expected for a
background reading or negative control (results not shown). Similarly, readings from
the other three assay formats did not show any difference between positive and negative
wells. Assay B did not show agglutination in any wells and results from assay C were
similar to those from assay A. Assay D gave higher levels of absorbance at 450 nm but
all values were almost the same suggesting that some form of non-specific binding was
occurring (results not shown).
0.18
0.16
0.14
0.12
S 0.1
0.08
0.06
0.04
0.02
After 3 washes After 9 washes No RBC
Figure 5.20 Monitoring by absorbance of RBC adherence to solid phase during

ELISA.
See Table 5.12 for data.
Absorbance after Absorbance after
Average Average
3 washes 9 washes
0.165 0.173 0.077 0.107
0.170 0.173 0.116 0.097
Immobilised 0.175 0.168 0.115 0.114
red blood 0.172 0.164 0.169 0.115 0.107 0.108
cells 0.163 0.170 0.109 0.110
0.166 0.171 0.101 0.109
0.165 0.171 0.115 0.126
No cells 0.037 0.039 0.038 0.039 0.039 0.039
5.5 Discussion
Biopanning of two immune libraries (donor and patient) and a naive library (Vaughan et
al., 1996) was carried out using soluble and cell based antigens. Soluble antigen and
transfected cell antigens were tested for their suitability for use in biopanning.
Biopanning was carried out against a wide range of clinically significant RBC antigens
and several antibody clones were isolated with potential for use as phenotyping reagents.
5.5.1 Characterisation of Transfected Cells by Flow Cytometry
In order to isolate antibodies useful for blood typing, it was necessary to show that
expressed antigens on cells used for biopanning were the same as those found on RBCs.
This was achieved by flow cytometry analysis of transfected cell lines. All samples
tested yielded favourable results indicating that the RBC surface antigens of interest
were being expressed. Antibody-positive human donor antisera were able to recognise
the expressed antigens thereby indicating that the same epitopes were present on the
recombinant cells as those on human RBCs carrying the same antigen.
Multiple rounds of biopanning for the isolation of antigen-specific phage antibodies

may span up to a two week period, so it was crucial to determine whether the expression
of membrane-bound antigens was stably maintained during cell culturing. Time course
cell culture sampling, followed by analysis of membrane-bound antigen by flow
cytometry, showed that the cell lines were stably transfected and expressed the antigens
over many weeks and passages. This was of practical importance as it meant cultures
could be maintained and harvested as and when needed for biopanning, rather than
repeatedly initiating cultures from early passage stocks. Average fluorescence values
from flow cytometry of Fy^ positive cells, dropped by approximately 40 % between 21
and 25 passages. This may indicate that a decrease in antigen expression was occurring;
however the 21 passage time point represented over 7 weeks of culturing which gave
sufficient time for the completion of an experiment and did not impact on the HEK cell
biopanning process.
In addition to biopanning against cell based antigens, a recombinant protein containing
the extracellular domain of the Fy^ protein was used. The recombinant Fy^ protein was
also tested prior to use in biopaiming experiments. Human antiserum containing anti-
Fy^ antibodies was shown to bind to the immobilised Fy^ antigen. There was no
significant cross reactivity with Fy^ specific antiserum whose antigen binding site
differs from Duffy a by only one amino acid. This indicated that the same epitopes
were present on the recombinant protein as those on human RBCs, and thus validated
the use of the recombinant Fy® protein as a reagent in biopanning experiments. The
availability of recombinant Fy^ protein was advantageous as biopanning against a solid
phase antigen is more straightforward than whole cell biopanning. Whole cell
biopanning is inherently more difficult due to the complexity of whole cells and issues
of phage binding to irrelevant proteins and the lipid bilayer.
5.5.2 Biopanning and Screening
Two immune libraries were generated in this project referred to as 'donor' and 'patient'.
The differentiation of donor from patient is defined by the source of lymphocytes for
library construction, whereby the starting material for the donor library originated from
plasmapheresis donors, who had been deliberately immunised with RBC carrying the D
antigen. During this process donors also became sensitised to other antigens of interest
in this project and it is likely that antibodies to D predominate and may hamper attempts
to isolate the antibodies occurring in lower abundance (Marks et al., 1993). The patient
library however, was generated from individuals who had undergone a transfusion
reaction and therefore was not biased towards any antigen other than those of interest in
this project. The donor samples were shown at the time of collection to contain
antibodies to D, C, E, Cw, K, M and Fy® and the patient samples Fy^, Jk^ and C.
During library rescue, the conversion from phagemid to phage antibody particles was
far more efficient for the patient library than for the donor library. Donor library rescue
was repeated several times with different batches of helper phage but no improvement
could be obtained. Although the efficiency obtained was regarded as acceptable
(Bradbury and Marks, 2004b), this may be a reason why biopanning of the donor library
against Fy^ recombinant antigen was not successful. Biopanning output dropped over
successive rounds to very low levels and screening of the second round output did not
identify any positive clones. The low output titres may be related to the library not
containing specific phage antibodies rather than to the biopanning technique as the
naive library was biopanned alongside the donor library using exactly the same cultures
and reagents and appeared to be a lot more successful. In naive library biopanning,
after an increase from the first to second rounds, phage input: output ratios decreased in
subsequent rounds suggesting enrichment of antigen specific clones. The isolation of
antigen-specific phage antibodies was confirmed in the screening assay, where several
positive clones were identified. Whole cell biopanning of donor and patient libraries
against transfected cell lines resulted in fairly low output for the first rounds which
increased over the next two rounds. However, no significant decrease was observed in
phage input: output ratios suggesting that enrichment of positive clones was not
occurring.
Donor and patient libraries were pooled for biopanning against RBCs. This approach
was taken for practical reasons as time and resources were not available to biopan the
libraries separately. Input: output ratios from RBC biopanning were possibly more
indicative of enrichment of positive clones than those from the transfected cell lines.
This may be due to the fact that RBC were much easier to resuspend during washing
steps and consequently may have been washed more efficiently. It has been reported
that RBC are more robust compared to other mammalian cells, and thus are more suited
to whole cell biopanning (Vera et al., 2005). Human embryonic kidney cells were
inclined to aggregate and not resuspend completely after centrifugation and therefore
may have retained more non-specifically bound phage due to ineffective washing.
Screening assays of round three output from Fy^ positive HEK293 cells and RBCs
yielded several positive signals for binding to recombinant Fy^ antigen but a repeat
assay of 'positive' clones for antigen specificity showed that these phage antibodies
were in fact non-specific.
Overall, no positive clones were obtained from biopanning of the immune libraries
against Fy^ antigen displayed either as a recombinant protein immobilised on plastic
Immunotubes, as a membrane-bound protein on a transfected cell or on a RBC. Since
Fy^ specific clones were isolated from the naïve library, these results suggest that there
may not be any Fy^ specific clones present in the immune libraries. Two of the donor
239
blood samples (1 and 2) used to construct the immune library were shown to contain Fy^
specific antibodies so it is possible that these clones were lost during library
construction. Starting with antibody producing B cells and finishing with a phage
display library containing the B cell immunoglobulin gene repertoire is a complicated
process. There are several steps which can be inefficient and a clone represented at a
low level in the original antibody repertoire may not be retained.
The first step in library construction is EBV transformation of peripheral blood

mononuclear cells (PBMCs) which contain antibody producing B cells.
Immortalisation of B cells by EBV transformation has been reported to give
transformation efficiencies as low as 1 % (Bird et. al., 1981) up to 33 % (Stein and Sigal,
1983). In addition, resulting lymphocyte cell lines can be unstable with respect to
antibody secretion or cell growth (Goosens et al., 1987). For example patient 4
lymphocyte cell line dropped from a cell count of 10 million (86 % viability) to less
than a million cells (10 % viability). The addition of macrophages recovered cell counts
to 30 million and allowed RNA extraction for library construction but it is likely that
clones were lost during this process.
There are several examples in the literature demonstrating that antibody phage
repertoires can be selected on whole RBCs. Marks et al. (1993) expressed a single
chain Fv which agglutinated B positive RBCs after the addition of a cross linking
antibody. This assay was attempted with positive clones isolated from the naive scFv
library which had been shown to be positive for Fy^ antigen (RBC screening assay B).
After sensitising Fy^ positive RBC with positive phage, anti-M13 antibody was added as
a cross linker but no agglutination was observed. It is possible that the presence of the
phage particle affected agglutination and expression of soluble scFv is required for the
agglutination assay.
Moore and Conradie (1982) detailed an assay in which RBCs were sensitised with
human antiserum and then captured on a solid phase by anti-human globulin. The assay
was demonstrated to be effective with several antibody specificities including Fy^.
This assay format was applied to Fy^ specific phage antibodies isolated from the naive
library where sensitised RBC were transferred to an anti-M13 solid phase, but no
difference was in signal was observed between positive and negative samples (RBC
screening assay D). No meaningful results were obtained from a further two screening
assay formats (A and C) which possibly indicates that the assay format was not at fault
but that there was a more fundamental problem.
The isolated phage antibodies used in development of a whole RBC screening assay
were selected on a recombinant fusion protein containing the Fy^ extracellular domain.
It is possible that Fy^ antigen in its native form on the surface of an RBC may not be as
accessible as in the recombinant protein. In addition, the use of phage antibodies in all
of these assays may have prevented antibody binding since the phage particle is much
larger than an antibody molecule. An M l 3 phage is close to 1 \xm in length which is
large compared to an antibody of - 5 0 nm in size and as such may be too bulky to allow
access of the attached antibody to the RBC surface antigen. It is possible that in order
to develop an effective whole cell screening assay, it would be necessary to recover
soluble antibody from the screening plates rather than antibody phage particles. This is
straightforward from an experimental point of view but time constraints prevented a
repeat of this work with soluble antibodies.
Although extensive studies and experiments were performed using whole cell
biopanning, the optimal method for isolating antigen-specific phage antibodies
employed purified recombinant Fy^ protein. The only clones that could be analysed
from this work were selected against Fy^ RBCs which were tested using Fy^
recombinant protein. Biopanning against other RBC antigens, especially Rhesus D,
could have been successful as anti-D secreting cells would have been represented in
higher numbers in the original B cell antibody repertoire. This was indicated by the
high titres of anti-D antibody in donor serum and the fact that anti-D was the only
antibody detected upon testing of lymphocyte cell line culture supematants.
5.5.3 In Summary
Biopanning of two immune (donor and patient) and one naive library (Vaughan library)
was carried out using soluble (recombinant Fy^ antigen) and whole cell antigens
(transfected HEK cells and human RBC). No specific clones were isolated from the
immune libraries but several clones specific for Fy^ antigen were isolated from the naive
library. Biopanning was carried out on a range of RBC antigens in addition to Fy^
antigen but the resulting clones could not be screened due to the absence of a whole
RBC screening assay. These isolated clones against D, C, and E antigens from the
Rhesus blood group; Fy'' from the Duffy blood group; K1 from the Kell blood group
and Jk^ from the Kidd blood group have the potential to contain antibodies useful for
RBC phenotyping.
6 Discussion
Blood transfusion is an essential part of modem medicine and saves many lives. Last
year in Australia, the Australian Red Cross Blood Service (ARCBS) collected over a
million blood donations. The ARCBS supplies hospitals and other health providers
with approximately three quarters of a million units of red cells alone, not to mention
the many other vital blood products derived from donations. All of these donations are
carefully screened to establish their blood type based on the presence of clinically
significant RBC surface antigens, a process referred to as phenotyping. This allows the
blood to be used safely to treat patients and reduces the risk of severe reactions due to
transfusion of incompatible blood. Phenotyping is carried out by reacting red cells with
specific antibodies from human or animal sources. Antisera for identifying many of the
blood types are available but some are rare and expensive to obtain. Monoclonal
antibodies represent a renewable resource for phenotyping, and have advantages over
the use of polyclonal antisera. The aim of this project was to isolate these antibodies
using phage display technology to provide an inexpensive, consistent and unlimited
reagent supply for use in routine blood grouping assays.
Conventional monoclonal antibody production using murine hybridoma technology is

frequently not effective for producing antibodies reactive with RBC surface antigens.
The immune response in animals is less complex than that in humans, and the resulting
repertoire of antibodies, although specific for the required antigens, may not recognise
the same epitopes as the human antibody repertoire and is therefore not always suitable
for blood grouping. In addition, murine monoclonal antibodies can give spurious
phenotyping results in some cases (Beck et al., 1993), and in this situation a conclusive
result can only be obtained by the use of human polyclonal reagents. At present, some
phenotyping assays are still performed using antibody positive serum which is harvested
from human donors and pooled to produce a blood grouping reagent. This is a costly
and time consuming process, with reagents often in limited supply and showing
considerable batch to batch variation.
In recent years, murine monoclonal antibodies have been developed, along with some
human monoclonal antibodies that are suitable reagents for blood typing. The most
successful technique for producing human monoclonal antibodies using cell biology has
been to combine EBV transformation of peripheral lymphocytes with fusion to an
immortal cell line. However, there are difficulties with the technique; fusion
efficiencies can be low and the resulting cell lines may be unstable (Siegel and
Silberstein, 1994). A viable alternative is the use of phage display technology to
produce human antibodies. Phage display is an inherently powerful technique, and
since its inception in the early 1990's, has been used to generate recombinant antibodies
against a myriad of antigens. The technique involves the construction of libraries of
immunoglobulin genes captured in phage particles capable of displaying the gene
product (antibody fragments) on their surface. Used as an alternative to hybridoma
technology and tissue culture methods for cloning antibodies, phage display can be
thought of as a technique to 'immortalize expressed antibody genes rather than the cells
from which the genes were derived' (Siegel, 2001).
Although no recombinant antibodies are currently in routine use for blood typing, they
have been utilised in other fields. The first commercially available antibody of this type
is 'Humira', an antibody specific for human tumour necrosis factor approved for the
treatment of rheumatoid arthritis (Salfeld et al., 2000). Many other therapeutic
antibodies are being tested in clinical trials including treatments for other autoimmune
diseases and cancer (Holliger and Hudson, 2005). Recombinant antibodies have been
isolated by phage display for the purpose of blood grouping. Many of these are anti-D
antibodies as these are useful in HDN prophylaxis as well as RBC phenotyping (Siegel
et al., 1997; Miescher et al., 1998; Oudin et al., 2000). Recombinant antibodies which
bind to other blood group antigens have also been generated including A and B (from
ABO system; Chang and Siegel, 2001; Santos-Esteban and Curiel-Quesada, 2001) , E
from the Rhesus system (Marks et al., 1993), M and N from the MNS system (murine
library; Czerwinski et al., 1999), Lu^ from the Lutheran system (Richard et al., 2006)
and Kp^ from the Kell system and also antibodies to D, B and HI (the latter being from
the I blood group system; Marks et al., 1993). Recombinant antibodies to the D antigen
have also been isolated from a synthetic library generated from germ-line gene
segments (Hughes-Jones et al., 1999).
Library construction methods used in the production of these recombinant antibodies to
blood group antigens involved the extraction of RNA from PBMC isolated from human
blood samples o f - 5 0 mL volumes (Marks et aL, 1991; Siegel et al., 1997). This
amount of blood contains - 2 x 1 0 ^ antibody-producing B cells which yield sufficient
RNA for recombinant antibody library construction. In the work described in this thesis,
B cell samples producing antibodies of interest were limited; therefore the B cell
populations were expanded to give sufficient cells for RNA extraction. This was
achieved by EBV transformation of PBMC samples to yield 'immortal' lymphocyte cell
lines (LCL). The generation of LCL also allowed frozen stocks and cell banks to be
generated to provide a renewable supply of cells for mRNA extraction and
immunoglobulin gene library construction.
Modified versions of this process have been published which enrich for B cells of
interest. This selection step can be performed either before or after EBV transformation
of lymphocytes. EBV transformation of a PBMC sample to form an LCL presents the
opportunity to select for cells producing the antibody of interest and discard irrelevant B
cell subpopulations (Kempf et al., 2001). This is achieved by diluting cells into separate
wells and selecting wells that are shown by immunoassay of the supernatant to contain
relevant B cells. An enrichment step of this nature may make it more likely that the
required antibodies will be isolated from the resulting phage display library.
Pre-EBV transformation enrichment techniques involve the selection of single antigen-

specific B cells. B cells of interest are selected by rosetting with antigen-coated
magnetic beads (Lagerkvist et al., 1995) and expanded by culture with feeder cells
before RNA extraction. Kozbor et al. (1979), selected B cells producing antibodies to
trinitrophenyl (TNP) by rosetting with antigen coated RBC in a similar process.
Agglutination assays of LCL supematants generated in this project did not indicate the
presence of any antibodies of interest from the Duffy, Kell and Kidd blood groups. The
only antibody present was anti-D as demonstrated by strong agglutination of D-positive
RBC by lymphocyte cell culture supernatant. This may indicate that the other RBC
antigen specific clones were at a very low level or that they had been lost during
transformation or culture.
Samples of B cell RNA from several human donors were obtained via EBV
transformation and LCL culture for use in this project. These samples had been shown
to contain antibodies to several clinically significant RBC surface antigens including D,
C and E from the Rhesus system; Fy^ and Fy^ from the Duffy system; K from the Kell
system and Jk^ from the Kidd system. Antibody genes were amplified from B cell
cDNA and cloned into a bacteriophage vector in a two stage cloning process designed to
maximise efficiency (de Haard et al., 1999). Research into parameters affecting Fab
phage display found that 'the use of a bicistronic vector design with an fd-fragment-pIII
fusion', as used in this project, was optimal with regard to 'phage yield, display of Fabs
on the phage and expression of soluble Fabs' (Kirsch et al., 2005). This resulted in the
random recombination of antibody genes to produce a combinatorial immune library of
Fab antibody fragments. The Fab antibody format was chosen to circumvent problems
of multimerisation encountered with expression of the smaller scFv fragments. Phage-
displayed scFv fragments have a tendency to form dimers and higher order multimers
which impart higher apparent affinities and can lead to isolation of fragments with low
affinities (Griffiths et al., 1993). Recent work has expanded the choice of antibody
formats further with the production of a single chain Fab in which the CHI and CL
domains are connected by a polypeptide linker (Hust et al., 2007).
The Fab antibody libraries constructed in this project were of high quality based on their
size, percentage of full length insert and sequence diversity. Fab library sizes
(approximately 3x10^ clones) were comparable to published results for other immune
libraries (10^-10^ clones) from which antibodies of high affinity have been isolated
(Mao et al., 1999; Wu et al., 2001; Zwick et al., 2001; He et al., 2002; Dantas-Barbosa
et al., 2005). The percentage of clones within the library containing a full complement
of insert for Fab expression was also comparable to published methods utilising a
similar cloning system with the same pCESl cloning vector (de Haard et al., 1999).
The clones within the libraries were shown to be diverse based on restriction enzyme
analysis with results indicating that an average of less that one in ten of the clones was
likely to have a DNA sequence identical to that of another clone in the library.
In addition to the libraries generated in this project, a large naive library was obtained
from Cambridge Antibody Technology in the UK under a Materials Transfer
Agreement (Vaughan et al., 1996). Although an immune library would be expected to
contain antibodies reactive with the target antigen, the diversity of antibodies may be
restricted. A large, naive library may contain antibodies not present in the natural
human immunoglobulin gene repertoires, as the shuffling of heavy and light V genes
may create new specificities. Biopanning using both an immune and naive library is
likely to yield a wider range of antibody specificities than the immune library alone.
Research comparing isolation of scFvs from immune and naive libraries suggests that
naive library biopanning yields antibodies against all areas of an antigen whereas
immune library biopanning against the same antigen results in the isolation of
antibodies to a few immunodominant epitopes (Amersdorfer et al., 2002). Thus
biopanning against RBC antigens of interest was performed using both immune and
naïve libraries to optimise the opportunity for isolating diverse panels of antibodies with
the required antigen specificity. The naïve single chain Fv library used in this study
was constructed using variable gene segments from 43 non-immunised human donors,
and a number of high affinity antibodies to a range of antigens have been previously
isolated from this library (Vaughan et al., 1996). Furthermore, the library was used for
the development of biopanning and screening methodologies and strategies for isolating
antibodies that would be useful for RBC phenotyping.
There are a number of modes and formats for antigen-driven phage display selections
(Hoogenboom, 2005). These include the use of purified recombinant antigen, whole
cells displaying antigen (mammalian cells, yeast, bacteria etc.) or selections using
antigen conjugated to magnetic beads or other types of microbeads. In this project,
combinations of whole cell biopanning and biopanning against a recombinant antigen
were performed. Cells used for whole cell biopanning included RBCs and recombinant
human HEK cells expressing recombinant RBC surface antigens.
The recombinant Fab libraries generated in this project were biopanned against several
RBC antigens from the Duffy, Kell, Kidd and Rhesus systems. These antigens are
integral proteins embedded in the RBC membrane with the majority having multiple
transmembrane domains (the exception is the Kell antigen which has a single
transmembrane domain). As such, antigens isolated from the RBC membrane would be
unlikely to retain their native conformation. Phage antibody selection against antigen
which is identical to native antigen is crucial if the resulting antibodies are to be useful
in RBC phenotyping. Many RBC antigen polymorphisms are very subtle; for example,
the difference between Fy^ and Fy^ antigens is a single amino acid substitution.
Antibodies that are able to discriminate between polymorphic epitopes with fine
specificity are required to correctly phenotype RBC and avoid the hazards of incorrectly
matched blood. Therefore, whole red cell biopanning was used as one of the library
selection strategies.
RBCs have complex cell surface structures with each cell carrying approximately 250
different antigens. Biopanning against a single RBC antigen in this situation
necessitates the use of subtraction biopanning. This involves pre-exposing antibody
phage to RBC suspensions that carry all the antigens present on antigen positive cells
except for the antigen of interest. This strategy depletes non-specific clones in the
phage preparation and improves the chances of isolating a positive clone during antigen-
driven selection.
The immune libraries used in biopanning contain a polyclonal mix of antibodies to RBC
antigens. Thus biopanning against RBC, which themselves carry a mixture of RBC
antigens, is a complex antibody selection system. An alternative cell-based biopanning
method was employed in an attempt to simplify the process. This involved the use of
human embryonic kidney (HEK) cells which had been stably transfected with RBC
antigens (Yazdanbakhsh et al., 2000). The cell surface consists of many complex
glycoprotein structures, and an immune library derived from lymphocytes of donors
would be unlikely to have many phage antibody binders to HEK cell surface antigens.
In addition, round(s) of subtractive biopanning may be performed using non-transfected
HEK cells to remove phage antibodies that react with these HEK cell surface antigens.
The use of soluble antigen, immobilised on a solid phase, is the most successful
approach for recovering specific antibodies in phage display biopanning (Bradbury and
Marks, 2004a). Soluble antigen is rarely available in the case of membrane proteins but
was obtained under a Materials Transfer Agreement in the case of the Duffy a blood
group. Both the naive and immune libraries were selected against recombinant Fy^
extracellular domain (Wasniowska et al., 2000) and several Fy^ positive clones were
isolated from the naive library. Surprisingly, no Fy^ clones were apparent in biopaiming
of the immune library despite the library having been constructed fi-om samples shown
to be expressing anti- Fy^ antibody. This may have been due to the loss of Fy® antibody
producing clones during LCL culture. Both of the samples containing Fy^ specific B
cell clones showed a 50% drop in viability between EBV immortalisation and
harvesting for RNA extraction.
In addition, it is possible for diversity to be reduced during library construction resulting

in the loss of rare transcripts. Amplification and cloning strategies used in this project
were chosen to maximise clonal diversity. However, assembling linear vector and
insert into a circular molecule (ligation) can be inefficient (Legerski and Robberson,
1985) as can transformation of the products of ligation into bacteria (Dower et al., 1988).
Recent research employed direct amplification of circularised recombinant vector from
ligation reactions using bacteriophage Phi 29 polymerase (Christ et al., 2006). The
polymerase preferentially amplified circular DNA over linear DNA fragments by
rolling-circle replication and the resulting concatamers were digested and then re-
circularised. This allowed the generation of a large recombinant DNA library (>1 x 10^
clones) with 10^ fold more transformants than the library constructed with non-
amplified cDNA, and allowed the isolation of specific antibodies. Specific antibodies
could not be isolated from the un-amplified library suggesting that the clones had been
lost during library construction.
Another factor which may affect the recovery of clones of interest is the size of the
immune library. Each individual has ~1 x 10^ unique antibodies consisting of a pair of
heavy and light chains which, when combined at random, results in ~1 X 10'^ possible
molecules (Lemer, 2006). However, there are only around 55000 heavy chain and 3000
light chain genes that encode the antibody repertoire. Thus, Winter and Milstein (1991)
postulated that a library of 10^ clones would be required to give a reasonable chance of
recovering the heavy and light chain pairings of the original antibodies in the B cell
sample, and to isolate other high affinity antibodies. However, this theory is disputed
by Burton et al. (1991) who stated that immune libraries are 'rich in functional chains
that are recombined with each other relatively frequently to generate high affinity
antibodies'. An assertion supported by Caton and Koprowski (1990) who isolated
antibodies to influenza virus haemagglutinin from a murine combinatorial library (with
2.5 X 10^ members) which had very similar heavy and light chain combinations as
haemagglutinin-specific antibodies of hybridoma origin. The libraries constructed in
this project contained approximately 3x10^ clones which is within the clonal size range
249
of other immune libraries that have been used successfully to isolate antibodies with
affinities typically in the nM range (reviewed by Aujame et al., 1997).
Immune libraries generated in this project were biopanned against several RBC surface
antigens but only screened against the one antigen which was available in a soluble
form. Screening of the other selected clones was hampered by the lack of an effective
whole RBC screening assay. Antibody phage specific for Fy^ antigen (as shown by
specific binding to recombinant Fy^ antigen) did not bring about agglutination of Fy^
positive RBCs either alone or when cross-linked using an anti-phage antibody. This
may be due to the accessibility of RBC surface antigen to the bulky phage (~ 1.6 x 10^
Da; Berkowitz and Day, 1976) since the polymorphism determining the Duffy a/b
epitope in the Duffy protein is in the section of extracellular domain relatively close to
the membrane surface (position 44 of the 65 amino acid extracellular domain; Hadley
and Peiper, 1997). However Marks et al. (1993) showed agglutination of Rhesus D
positive RBCs with D specific antibody phage. This was despite the fact that the
Rhesus membrane protein is buried in the membrane and the extracellular domains
carrying the epitopes are short (not exceeding 20 amino acids in length; Cherif-Zahar et
al., 1990). Another possibility for the failure of specific phage antibodies to agglutinate
antigen positive RBC is the stability of the antigen itself on the RBC. The Duffy
glycoprotein is sensitive to proteolysis and the antigens may be difficult to detect on
prolonged storage of RBCs (Issitt and Anstee, 1998). Fresh Fy^ positive cells were not
always available during this project as their cost was prohibitive.
Ideal antibodies for use in RBC phenotyping are multivalent (i.e. IgM which is
pentameric) which can bring about RBC agglutination without the addition of a
secondary cross linking reagent. IgG antibodies, having only two antigen binding sites,
do not agglutinate RBC until cross linked with anti-human globulin in a technique
referred to as indirect agglutination. This is more costly to perform in terms of
resources than a direct agglutination assay. In order for the Fy^ specific antibodies
isolated in this project to be usefiil blood grouping reagents, further genetic engineering
would be required in order to express the fi-agments as higher order multimers.
There are several methods by which this could be achieved. Fusion of a protein to
truncated IgM heavy chain (constant domains 2 to 4) results in multimerisation although
a range of sizes are generated (Vie et al., 1992). Fusion proteins containing the C-
terminal fragment of human C4-binding protein alpha chain (a component of the
complement system) seem to result in more consistent multimers containing seven
subunits (Shinya et al., 1999). The native C4-binding protein consists of seven alpha
chains and one beta chain that are C-terminally disulphide-bonded to form a 'spider-like
shape' (Dählback et al., 1983). Alternatively, tetrameric antibody molecules can be
generated by fusion of the antibody fragment to a biotin mimetic sequence (Luo et al.,
1998). When the fusion protein is added to streptavidin, which has four biotin binding
sites, a tetrameric antibody/streptavidin complex is formed. In a related technique,
Dübel et al. (1995) fused single chain Fv fragments to core-streptavidin (a truncated
form of streptavidin) which, upon expression, associated into tetramers although dimers
and larger aggregates were also formed.
More recent multimerisation techniques include the formation of pentameric antibody

molecules by fusion to a B-subunit monomer of E. coli verotoxin (Zhang et al., 2004).
Verotoxin consists of an A-subunit, which is the toxic moiety, and five B-subunits
formed into a 'doughnut-shaped structure' which are involved in host cell binding. This
technique was used to pentamerise a single domain antibody from a llama naive library
which had binding affinities for parathyroid hormone (used in the treatment of
osteoporosis) that were lO^-lO"^ fold higher than the monomeric fragment. This work
was extended to generate a decavalent antibody molecule by fusing a single domain
antibody to both the C- and N-termini of the verotoxin B-subunit (Stone et al., 2007).
The method of choice for future work involving multimerisation of the antibodies
generated in this project would be linkage via a leucine zipper. This is a helical
structure so called because it has a leucine amino acid residue at every seventh position
over a distance of eight helical turns and the leucine side chains of one helix
'interdigitate' with those of another to form stable, electrostatically bonded pairs
(Landschulz et al., 1988). This structure has been used to produce dimeric scFv
antibodies which are referred to as 'miniantibodies' so named as their flexible hinged
structure is similar to that of whole antibody (Pack and Plückthun, 1992). Tetrameric
antibody fragments were also generated by fusion with tetrameric alpha-helical bundles
(based on the work of Eisenberg et al., 1986).
Antibody phage libraries biopanned against D positive RBC have the potential to
contain high affinity antibodies to the Rhesus D antigen. The presence of potent anti-D
antibodies was indicated by complete agglutination of Rhesus positive RBC by
supematants from LCL cultures used in library construction. The technology is
available for these Fab antibodies to be rebuilt and expressed as fully assembled,
functional human antibodies (Mahler et al., 1997). These antibodies would then be
ideal for use in the prevention of haemolytic disease of the newborn (HDN). This is a
potentially fatal foetal or neonatal condition where maternal antibodies to RBC antigens
(generated as a result of RBC exposure during a previous pregnancy or blood
transfusion) cross the placenta and destroy foetal RBCs. This condition can be
prevented by the administration of anti-D antibody to mothers, pre- and post-natally,
who are at risk of HDN.
In summary, the recombinant antibody fragments to clinically significant blood group

antigens generated in this project present an opportunity to produce novel recombinant,
multivalent agglutinating antibodies. Further characterisation of these antibodies
associated with determining specificity and affinity is required. If suitable, and after
rebuilding as multivalent antibodies, these recombinant molecules would provide a cost
effective, reliable and consistent supply of reagents for use in routine RBC phenotyping.
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Appendices
Appendix 1 PGR Primer Sequences for Library Screening

scFv Forward:
TGT GGA ATT GTG AGC
ScFv Reverse:
ACA GAG AGC CCT CAT
Appendix 2 pCESl DNA Sequence
GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTT7U\ TGTCATGATA 50

ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGG7y\ ATGTGCGCGG 100
NIAITIPR Promoter Start
AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA 150
-l AmpR Promoter End
TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT 2 00
>lAmpicillin Resistance Gene Start
ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT 250
TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG 300
CTGAAGATCA GTTGGGTGCC CGAGTGGGTT ACATCGAACT GGATCTCAAC 350
AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT 4 00
GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG 4 50
CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 500
GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT 550
AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA 600
ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG 650
CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT 7 00
GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA 7 50
TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 8 00
TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC 8 50
AmpR Endi
ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG 900
GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT 950
GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC 1000
TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA 1050
AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT 1100
TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA 1150
TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 12 00
^pBR322 Origin Start
CAGACCCCGT AGAA^ÂGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG 1250
CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT 1300
TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT 1350
TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA 14 00
GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT 1450
AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 1500
GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA 1550
ACGGGGGGTT CGTGCATACA GCCCAGCTTG GAGCGAACGA CCTACACCGA 1600
ACTGAGATAC CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG 1650
GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG 17 00
CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT 17 50
CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 18 00
pBR322 Origin End>l
GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC 1850
CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG
CGTTATCCCC 1900
TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT
GATACCGCTC 1950
GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA
GGAAGCGGAA 2000
GAGCGCCCAA TACGC7V7\ACC GCCTCTCCCC GCGCGTTGGC
CGATTCATTA 2050
ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC
AGTGAGCGCA 2100
>lLac Prom Start
ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC 2150
Lac Prom EndNl IFCR Primer FORI
TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT 2200
TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTT TGGAGCCTTT 2250
^Gene III Leader Sequence Start
TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT 2300
^Gene III Leader Sequence End
i iApaLI ixhol
TGTTCCTTTC TATTCTCACA |GTGCAC|AGGT CCAACTGCAG GTCGAC|CTCG 2350
iHuman CK Gene Start
A^TCAAACG TGGAACTGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA 2400
TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA 2450
TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC 2500
TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC 2550
AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA 2600
GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGTTCAC 2650
Human CK Gene EndN^ i Asci i
CGGTGACAAA GAGCTTCAAC AGGGGAGAGT GTTAATAAGG CGCGCCAATT 2700
iPel B Leader Sequence Start
CTATTTCAAG GAGACAGTCA TAATGAAATA CCTATTGCCT ACGGCAGCCG 2750
Pel B Leader Sequence End>l
i Sfil i
4'Ncoi4'
CTGGATTGTT ATTACTCGCp GCCCAGCCGG |pCf\TGGpCCA GGTGCAGCTG 2800
^Human CHI Gamma Gene Start
i >|.BstEII
CAGGAGAGCG G3GTCACC CTCAAGCGCC TCCACCAAGG GCCCATCGGT 2850
CTTCCCCCTG GCACCCTCCT CCAAGAGCAC CTCTGGGGGC ACAGCGGCCC 2900
TGGGCTGCCT GGTCAAGGAC TACTTCCCCG AACCGGTGAC GGTGTCGTGG 2950
AACTCAGGCG CCCTGACCAG CGGCGTCCAC ACCTTCCCGG CTGTCCTACA 3000
GTCCTCAGGA CTCTACTCCC TCAGCAGCGT AGTGACCGTG CCCTCCAGCA 3050
GCTTGGGCAC CCAGACCTAC ATCTGCAACG TGAATCACAA GCCCAGCAAC 3100
Human CHI Gamma Gene Endi i His Tag Start
ACCAAGGTGG ACAAGAAAGT TGAGCCCAAA TCTTGTGCGG CCGCACATCA 3150
His Tag End>l ^Myc Tag Start
TCATCACCAT CACGGGGCCG CAGAACAAAA ACTCATCTCA GAAGAGGATC 3200
^^Myc Tag End >lGene III Start
TGAATGGGGC CGCATAGACT GTTGAAAGTT GTTTAGCAAA ACCTCATACA 3250
GAAAATTCAT TTACTAACGT CTGGAAAGAC GACAAAACTT TAGATCGTTA 3300
i PCR Primer REV i
CGCTAACTAT GAGGGCTGTC TGTGGAATGC TACAGGCGTT GTGGTTTGTA 3350
CTGGTGACGA AACTCAGTGT TACGGTACAT GGGTTCCTAT TGGGCTTGCT 34 00
ATCCCTGAAA ATGAGGGTGG TGGCTCTGAG GGTGGCGGTT CTGAGGGTGG 34 50
CGGTTCTGAG GGTGGCGGTA CTAAACCTCC TGAGTACGGT GATACACCTA 3500
TTCCGGGCTA TACTTATATC AACCCTCTCG ACGGCACTTA TCCGCCTGGT 3550
ACTGAGCAAA ACCCCGCTAA TCCTAATCCT TCTCTTGAGG AGTCTCAGCC 3600
TCTTAATACT TTCATGTTTC AGAATAATAG GTTCCGAAAT AGGCAGGGTG 3650
CATTAACTGT TTATACGGGC ACTGTTACTC AAGGCACTGA CCCCGTTAAA 37 00
ACTTATTACC AGTACACTCC TGTATCATCA AAAGCCATGT ATGACGCTTA 37 50
CTGGAACGGT AAATTCAGAG ACTGCGCTTT CCATTCTGGC TTTAATGAGG 38 00
ATCCATTCGT TTGTGAATAT CAAGGCCAAT CGTCTGACCT GCCTCAACCT 3850
CCTGTCAATG CTGGCGGCGG CTCTGGTGGT GGTTCTGGTG GCGGCTCTGA 3900
GGGTGGCGGC TCTGAGGGTG GCGGTTCTGA GGGTGGCGGC TCTGAGGGTG 3950
GCGGTTCCGG TGGCGGCTCC GGTTCCGGTG ATTTTGATTA TGAAAAAATG 4 000
GCAAACGCTA ATAAGGGGGC TATGACCGAA AATGCCGATG AAAACGCGCT 4 050
ACAGTCTGAC GCTAAAGGCA AACTTGATTC TGTCGCTACT GATTACGGTG 4100
CTGCTATCGA TGGTTTCATT GGTGACGTTT CCGGCCTTGC TAATGGTAAT 4150
GGTGCTACTG GTGATTTTGC TGGCTCTAAT TCCCAAATGG CTCAAGTCGG 4 2 00
TGACGGTGAT AATTCACCTT TAATGAATAA TTTCCGTCAA TATTTACCTT 4 250
CTTTGCCTCA GTCGGTTGAA TGTCGCCCTT ATGTCTTTGG CGCTGGTAAA 4 300
CCATATGAAT TTTCTATTGA TTGTGACAAA ATAAACTTAT TCCGTGGTGT 4 350
CTTTGCGTTT CTTTTATATG TTGCCACCTT TATGTATGTA TTTTCGACGT 4 4 00
Gene III EndN^ >l<LacZ Reporter Start
TTGCTAACAT ACTGCGTAAT AAGGAGTCTT AATAAGAATT CACTGGCCGT 4 4 50
CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTTAATC 4 500
GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC 4 550
LacZ Reporter End4
CGCACCGATC GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGCG 4 600
CCTGATGCGG TATTTTCTCC TTACGCATCT GTGCGGTATT TCACACCGCA 4 650
TATAAATTGT AAACGTTAAT ATTTTGTTAA AATTCGCGTT AAATTTTTGT 4700
TAAATCAGCT CATTTTTTAA CCAATAGGCC GAAATCGGCA AAATCCCTTA 4 7 50
ifl Origin Start
TAAATCAAAA GAATAGCCCG AGATAGGGTT GAGTGTTGTT CCAGTTTGGA 4 8 00
ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA 4 850
ACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC CCAAATCAAG 4 900
TTTTTTGGGG TCGAGGTGCC GTAAAGCACT AAATCGGAAC CCTAAAGGGA 4 950
GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT GGCGAGAAAG 5000
GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC 5050
fl Origin Endi
GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC 5100
AGGGCGCGTA CTATGGTTGC TTTGACGGGT GCAGTCTCAG TACAATCTGC 5150
TCTGATGCCG CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA 5200
CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC AGACAAGCTG 5250
TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG 5300
AAACGCGCGA 5310
Appendix 3 Primary PCR Primer Sequences
Primer Name =>nmer Seauence
IgGFOR 5' G T C C A C C T T G G T G T T G C T G G G C T T 3'
CkapFOR 5' A C A C T c T C C C C T G T T G A A G C T C T T 3'

Clam2F0R 5' T G A A C A T T C T G T A G G G G C C A C T G 3'
Clam7F0R 5' A G A G C A T T C T G C A G G G G C C A C T G 3'
VH1B/7ABACK 5' C A G R T G C A G C T G G T G C A R T C T G G 3'

VH1CBACK 5' S A G G T C C A G C T G G T R C A G T C T G G 3'
VH2BBACK 5' C A G R T C A C C T T G A A G G A G T C T G G 3'
VH3BBACK 5' S A G G T G C A G C T G G T G G A G T C T G G 3'
VH3CBACK 5' G A G G T G C A G C T G G T G G A G W C Y G G 3'
VH4BBACK 5' C A G G T G C A G C T A C A G C A G T G G G G 3'
VH4CBACK 5' C A G S T G C A G c T G C A G G A G T C S G G 3'
VH5BBACK 5' G A R G T G C A G c T G G T G C A G T C T G G 3'
VH6ABACK 5' C A G G T A C A G c T G C A G C A G T C A G G 3'
VkaplBBACK 5' G A C A T C C A G w T G A C C C A G T C T C C 3'

Vkap2BACK 5' G A T G T T G T G A T G A C T C A G T C T C C 3'
VkapSBBACK 5' G A A A T T G T G W T G A C R C A G T C T C C 3'
Vkap4BBACK 5' G A T A T T G T G A T G A C C C A C A C T C C 3'
Vkap5BACK 5' G A A A C G A C A C T C A C G C A G T C T C C 3'
Vkap6BACK 5' G A A A T T G T G C T G A C T C A G T C T C C 3'
Vlam1 ABACK 5' C A G T C T G T G C T G A C T C A G C C A C C 3'

VlamlBBACK 5' C A G T C T G T G Y T G A C G C A G C C G C C 3'
VlamlCBACK 5' C A G T C T G T C G T G A C G C A G C C G C C 3'
Vlam2BACK 5' C A R T c T G C C C T G A C T C A G c C T 3'
VlamSABACK 5' T C C T A T G W G C T G A C T C A G c C A C C 3'
VlamSBACK 5' T C T T C T G A G C T G A C T C A G G A C C C 3'
Vlam4BACK 5' C A C G T T A T A C T G A C T C A A C C G C C 3'
VlamSBACK 5' C A G G C T G T G C T G A c T C A G C C G T C 3'
Vlam6BACK 5' A A T T T T A T G C T G A C T C A G C C C C A 3'
Vlam7/8BACK 5' C A G R C T G T G G T G A c Y C A G G A G C C 3'
Vlam9BACK 5' C W G C C T G T G C T G A c T C A G C C M C C 3'
Appendix 4 Secondary PCR Primer Sequences
Primer Name Primer Sequence
BstEII Site
JH1/2F0R 5' T G A G G A G A C G G T G A C C A G G G T G C C 3'
JH3F0R 5' T G A A G A G A C G G T G A C C A T T G T C C C 3'
JH4/5FOR 5' T G A G G A G A C G G T G A C C A G G G T T C C 3'
JH6F0R 5' T G A G G A G A C G G T G A C c G T G G T C C C 3-
Sfi 1 Site Annealing region (same as sequence as for Primary PCR)
VH1B/7ABACKSFI 5' G T C C T C G C A A C T G C G G c C C A G C C G G C C G G C C C A G R T G C A G C T G G T G C A R T
A T C T G G 3'
VH1CBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C S A G G T C C A G C T G G T R C A G T
A T C T G G 3'
VH2BBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C C A G R T C A C C T T G A A G G A G T
A T C T G G 3'
VH3BBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C S A G G T G C A G C T G G T G G A G T
A T C T G G 3'
VH3CBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C GAG G T G C A G C T G G T G G A G W
A T C Y G G 3'
VH4BBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C C A G G T G C A G C T A C A G C A G T
A T G G G G 3'
VH4CBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C C A G S T G C A G C T G C A G G A G T
A T C S G G 3'
VH5BBACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C GAR G T G C A G C T G G T G C A G T
A T C T G G 3'
VH6ABACKSFI 5' G T C C T C G C A A C T G C G G C C C A G C C G G C C G G C C C A G G T A C A G C T G C A G C A G T
A T C A G G 3'
Asci Site Annealing region (same as sequence as for Primary PCR)
CkapFORASC 5' A C C G C C T C C A C C G G G C G C G C C T T A T T A A C A C T C T C C C C T G T T G A A G C T C T T 3'
Clam2F0RASC 5' A C C G C C T C C A C C G G G CGC G C C T T A T T A T G A A C A T T C T G T A G G G G C C A C T G 3'
Clam7F0RASC 5' A C C G C C T C C A C C G G G C G C G C C T T A T T A A G A G C A T T C T G C A G G G G C C A C T G 3'
AoaLI Site Annealing region (same as sequence as for Primary PCR)
Vkap1 BBACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A C A T C C A G W T G A C C C A G T C T C C 3'
Vkap2BACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A T G T T G T G A T G A C T C A G T C T C C 3'
VkapSBBACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A A A T T G T G W T G A C R C A G T C T C C 3'
Vkap4BBACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A T A T T G T G A T G A C C C A C A C T C C 3'
VkapSBACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A A A C G A C A C T C A C G C A G T C T C C 3'
Vkap6BACKAPA 5' A C C G C C T C C A C c A G T G C A C T T G A A A T T G T G C T G A C T C A G T C T C C 3'
Annealin g region (same as sequence as for Primary PCR)
VlamlABACKAPA 5' A C C G C C T C C A C c A G T G C A C A G T C T G T G C T G A C T C A G C C A C C 3'
Vlam1 BBACKAPA 5' A C C G C C T C C A C c A G T G C A C A G T C T G T G Y T G A C G C A G C C G C C 3'
VlamlCBACKAPA 5' A C C G C C T C C A C c A G T G C A C A G T C T G T C G T G A C G C A G C C G C C 3'
Vlam2BACKAPA 5' A C C G C C T C C A C c A G T G C A c A R T C T G C C C T G A C T C A G C C T 3'
VlamSABACKAPA 5' A C C G C C T C C A C c A G T G C A c T T T C C T A T G W G C T G A C T C A G C C A C C 3'
VlamSBACKAPA 5' A C C G C C T C C A C c A G T G C A c T T T C T T C T G A G C T G A C T C A G G A C C C 3'
Vlam4BACKAPA 5' A C C G C C T C C A C c A G T G C A c A C G T T A T A C T G A C T C A A C C G C C 3'
VlamSBACKAPA 5' A C C G C C T C C A C c A G T G C A c A G G C T G T G C T G A C T C A G C C G T C 3'
VlamSBACKAPA 5' A C C G C C T C C A C c A G T G C A c T T A A T T T T A T G C T G A C T C A G C C C C A 3'
Vlam7/8BACKAPA 5' A C C G C C T C C A C c A G T G C A c A G R C T G T G G T G A C Y C A G G A G C C 3'
Vlam9BACKAPA 5' A C C G C C T C C A C c A G T G C A c W G C C T G T G C T G A C T C A G C C M C C 3'
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THE UNIVERSITY OF NEW SOUTH WALES

Surname or Family name: Fiddes

First name: Jane Other name/s: Louise Sutton

Abbreviation for degree as given in the University calendar: PhD

School: BABS Faculty: Science

Title: Development of Recombinant Human Monoclonal

Abstract 350 words maximum: (PLEASE TYPE)

Declaration relating to disposition of project thesis/dissertation

FOR OFFICE USE ONLY Date of completion of requirements for Award:

THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS

Jane L. Sutton Fiddes

A thesis submitted in fulfillment of the requirements for the degree of

School of Biotechnology and Biomolecular Sciences

This work is dedicated to the memory of my father and grandfather.

John William George Sutton

March 1945 to January 2004

William George Sutton

March 1922 to 4'^ July 2004

May you both be at peace.

2 Materials and Methods 61

3 Antibody Genes from Human Blood Samples 109

4 Phage Display Fab Library Construction 149

1.1 A Short History of Blood Typing and Transfusion

No further advances in the field were

It was this published observation that prompted Landsteiner to investigate whether

Figure 1.7 ARCBS Headquarters, 1952-1974, at 'Petty's Hotel' York Street,

Figure 1.9 Cross-sectional diagram of the human RBC membrane.

Figure 1.10 Diagrammatic representation of SDS-PAGE analysis of red ceil

Table 1.1 The blood group systems and their symbols

025 —etc.* VLAN

Type A blood Type B blood

Results in destruction of rbc's ^ haemoiytic transfusion reaction.

Figure 1.12 An example of ABO incompatibility.

Another serious clinical problem associated with immune antibodies is haemolytic

Introduction of Anti-D Prophylaxis

1.3.1 The Rhesus System

Figure 1.15 Diagram of the proposed structure of the Rhesus polypeptide,

The occurrence of D+ individuals is high, at 85 % in the Caucasian population rising to

1.3.2 The Kell System

1.3.3 The Duffy System

nh2 S > Fy^/Fy^

The protein carrying the Duffy antigens is a glycoprotein of 35-43,000 Da molecular

Kidd antigens are regarded as relatively non-immunogenic compared to Rhesus, Kell

Kidd antigens are carried on a 46-60,000 Da molecular weight glycoprotein which is

Id) icjA <dimer) le) igM i(>ftn!amet)

Figure 1.19 Diagrams of the five classes of immunoglobulin.

Figure 1.20 Diagram of sequence variability in antibody molecules.

IEFO FrI ScOftl F fr3 iCOfOl

Rearranged D N A Rearranged DNA

Typically the primary immune response of humans to a foreign antigen is the

(STERILE RNA transcriptB|)

Figure 1.24 The class switching process resulting in antibodies of identical

As well as class switching, the antibody response undergoes a process of affinity

Heavy chain Light chain

Figure 1.25 Affinity maturation of antibody genes.

B c:e is Hyb-ndcma Myoioma

No growth Growth No growth

However disadvantages with monoclonal antibodies include;

Figure 1.27 Possible antibody formats used for cloning.

Just as hybridoma technology brought about a revolution in research and healthcare, so

Pre-B cell Beeil Plasma/memory celi

Synthetic Naive Immune