Development of a Membrane-Based Immuno-PCR

Assay for Enhancing the Sensitivity of Lateral Flow

Detection

Development of a Membrane-Based Immuno-PCR

Assay for Enhancing the Sensitivity of Lateral Flow
Detection
By
Roni Nugraha

A thesis submitted in partial fulfillment

of the Requirements for the Degree
Masters of Science in Engineering

Department of Chemical Engineering

Chung Yuan Christian University
2008

ii

Abstract

Membrane-based immuno-PCR procedure was developed by applying

the immuno-PCR technique on membrane nitrocellulose to increase the sensitivity
of the lateral flow immunochromatography assay. The test procedure followed the
traditional lateral flow immunochromatography assay. But instead the gold
nanoparticles-conjugated antibody, this study employed DNA-conjugated
antibody as the signal reporter. The DNA reporter was amplified by PCR to
enhance the detection sensitivity.

The detector antibody was optimized its conjugation method for the
DNA-streptavidin complex. Two methodologies were tested the effect of the ratio
of DNA to streptavidin and the ratio antibody to DNA-streptavidin complex. The
1:1 ratio of antibody to DNA-streptavidin complex was found to give the best
result and therefore selected for further test.

Premixing of the detector antibody and DNA, 5 minutes waiting time,

and 0.5 mg/ml spraying concentration of capture antibody were concluded the
best condition to give the best signal. This condition was demonstrated on the
membrane-based immuno-PCR procedure to detected human IL-6 and reported
the detection sensitivity is <0.001 pg/ml of human IL-6. Further improvement of

reagents and assay formats is necessary to reduce the high background problem in
this study.

ii


PCR

DNA DNA
PCR
DNA streptavidin DNA-Streptavidin
DNA-Streptavidin
DNA-Streptavidin 11

DNA 5 0.5
mg/ml human IL6 <0.001 pg/ml

iii

Acknowledgement

My foremost thank goes to my thesis adviser Dr. Jui Chuang Wu.

Without him, this thesis would not have been possible. I thank him for his
patience and encouragement that carried me on through difficult times, and for his
insights and suggestions that helped to shape my research skills. His valuable
feedback contributed greatly to this thesis.

I am grateful to the Head of Chemical Engineering Dept, Dr Tsair Wang

Chung, who introduced and helped me to start my graduate student life in
Chemical Engineering. I thank the rest of my thesis committee members: Dr.
Tzong-Yuan Wu and Dr. Hung-Ju Richard Su. Their valuable feedback helped me
to improve the thesis in many ways.

I thank all the students and staffs in Chemical Engineering department,

whose made my time was very enjoyable. I am grateful for time spent with
roommates and friends, Wetra, Irdham, Iqbal, Yusuf, Ratno, Osmand and all
international students in Chung Yuan Christian University. Also to my labmates,
Dan, Carol, Annie, Peter, Kevin, Syin, Anan and Vani, that had helped me a lot
and gave me support during my study in CYCU.

iv

Last but not least, I thank to my parents, my sister, my brother and my

lovely wife for their understanding, endless patience and encouragement when it
was most required.
Chung Li, Taiwan, July 2008
Roni Nugraha

Contents
Abstract

................................................................................................................ i

.............................................................................................................. iii
Acknowledgement.................................................................................................. iv
Contents .............................................................................................................. vi
List of Figure........................................................................................................ viii
List of Table ........................................................................................................... ix
Chapter 1. Introduction ........................................................................................... 1
1.1 Antibody-antigen interaction ...................................................................... 1
1.2 Immuno-PCR .............................................................................................. 9
1.3 Lateral flow immunochromatography assay............................................. 13
1.4 Motivation ................................................................................................. 19
1.5 Objective ................................................................................................... 21
Chapter 2. Principle .............................................................................................. 22
2.1 Antibody-DNA conjugation...................................................................... 22
2.2 Lateral flow ............................................................................................... 24
2.3 Membrane-based immuno-PCR................................................................ 26
Chapter 3. Experimental ....................................................................................... 29
3.1 Materials.................................................................................................... 29
3.1.1 Polymerase Chain Reaction ................................................................ 29
3.1.2 Agarose gel electrophoresis ................................................................ 30
3.1.3 Lateral flow immuno-PCR.................................................................. 30
3.1.4 Buffer Solution.................................................................................... 31
3.2 Instruments................................................................................................ 33
3.3 Methods..................................................................................................... 35
3.3.1 Antibody-DNA conjugation................................................................ 35
3.3.2 Preparation of immunochromatographic test strips ............................ 38
3.3.3 Optimization of lateral flow-immuno-PCR assay............................... 40
3.3.4 Determination of the optimal concentration of capture antibody ....... 43
3.3.5 Sensitivity test of lateral flow immuno-PCR for human IL-6 ............ 44

vi

3.3.6 Analysis of gel image using ImageJ software..................................... 44

Chapter 4. Result and Discussion ......................................................................... 47
4.1 Preparation of antibody-DNA conjugate .................................................. 47
4.2 Optimization of lateral flow immuno-PCR............................................... 50
4.3 Sensitivity test of lateral-flow immuno-PCR for human IL-6 .................. 58
Chapter 5. Conclusion .......................................................................................... 60
Reference ............................................................................................................. 61
Appendix ............................................................................................................. 66

vii

List of Figure
Figure 1-3 Three proposed models described antibody-antigen interaction........... 8
Figure 1-4 The format of immuno-PCR and ELISA.. .......................................... 10
Figure 1-5 The methods employed to conjugate DNA onto antibody.. ................ 12
Figure 1-6 Two formats of the lateral-flow immunochromatography assay. ....... 18
Figure 2-1 Formation of the DNA-conjugated antibody....................................... 22
Figure 2-2 Band distribution of DNA-streptavidin complex on agarose gel
electrophoresis. ..................................................................................... 23
Figure 2-3 Basic principle of the lateral flow immunochromatography assay ..... 26
Figure 2-4 The principle of membrane-based immuno-PCR................................ 28
Figure 3-2 DNA-conjugated antibody formation.................................................. 37
Figure 3-3 Preparation of immunochromatography strips. ................................... 39
Figure 3-4 Optimization of lateral flow immuno-PCR in order to get low
background with two different methods.. ............................................. 42
Figure 4-1 Analysis of DNA-streptavidin complex in 2% agarose gel ................ 48
Figure 4-2 Conjugation of biotinylated antibody with DNA-streptavidin
complex presented in 2% agarose gel electrophoresis. ........................ 49
Figure 4-3 Agarose gel electrophoresis of optimization of lateral flow
immuno-PCR. ....................................................................................... 52
Figure 4-4 Optimization of sample pre-treatment................................................. 52
Figure 4-5 Optimization of waiting time. ............................................................. 54
Figure 4-6 Optimization of test waiting time........................................................ 54
Figure 4-7 Optimization of spraying concentration of capture antibody.............. 56
Figure 4-8 Optimization of spraying concentration of the capture antibody ........ 56
Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human
IL-6. ...................................................................................................... 59
Figure 4-10 The sensitivity test of the lateral-flow immuno-PCR on human
IL-6. ...................................................................................................... 59

viii

List of Table
Table 1-1 Overview of applications of lateral-flow immunochromatography
assay...................................................................................................... 14
Table 3-1 Design of the concentration ratio of biotinylated DNA to
streptavidin.. ......................................................................................... 35
Table Appendix 1 The Result of ImageJ measurement on the electrophoresis
band of antibody-antigen interaction. ................................................... 66
Table Appendix 2 The Result of ImageJ measurement on the electrophoresis
band of waiting time optimization........................................................ 66
Table Appendix 3The Result of ImageJ measurement on the electrophoresis
band of the spraying concentration of capture antibody....................... 67
Table Appendix 4 The Result of ImageJ measurement on the electrophoresis
band of the sensitivity test of the lateral-flow immuno-PCR ............... 67

ix

Chapter 1. Introduction
1.1

Antibody-antigen interaction

Bodies normally develop immune reactions to protect organisms from

pathogens and tumors that can endanger their lives by identifying and killing the
pathogens and tumors cells. Antibody is one part of the immune system that
identifies and neutralizes foreign molecules such as bacteria and viruses.
Antibodies contribute to immunity in three main ways: they prevent pathogens
from entering or damaging cells by binding with them; they can stimulate removal
of a pathogen by macrophages and other cells by coating the pathogen; and they
can trigger direct pathogen destruction by stimulating other immune responses
such as the complement pathway [1].

Immune response can be stimulated when a body is invaded by foreign

molecules, called antigen. Substances that trigger an immune response generally
meet several criterias: the heterogeneity (different sequence); foreignness
(different from host); and the size (relatively large, e.g. 100 kDalton). Hapten
(small molecules) acts as an antigenic agent if conjugated with carrier proteins.
Most antigens are protein or polysaccharide, but only a small region of that
structure is recognized by an antibody. This small region called an antigenic

determinant or epitope. Nucleic acids and lipids are antigenic when these two
molecules conjugated with protein or polysaccharide.

Antibody has a four chain structure as the basic structure of antibody. It

contains two identical heavy chains and two identical light chains. The heavy
chain and the light chain are connected together by inter-disulfide bonds and noncovalent interactions. The heavy and light chains can be separated in two domains,
variable and constant domains. Each antibody has a specific amino acid sequence
in variable domain. Most variability of sequences found in the variable region
called the hypervariable regions or the complementaritys determining regions.
This region determines the specificity of antibody to bind antigen (Figure 1-1).

Based on structure of heavy chain, antibody can be divided to five class

of immunoglobulin; immunoglobulin M (IgM), immunoglobulin D (IgD),
immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgG).
Their heavy chains are denoted by the corresponding lower-case Greek letter (, ,
, , and , respectively).

H2
N

NH2

5
3

H2N

NH2

1
6
S

S
S

S
S

HOOC

COOH

HOOC

COOH

Figure 1-1 Basic structure of an antibody. 1. Fab region 2. Fc region 3.

Heavy chain with one variable (VH) domain followed by a constant domain
(CH1), a hinge region, and two constant (CH2 and CH3) domains. 4. Light
chain with one variable (VL) and one constant (CL) domain 5. Antigen
binding site (paratope) 6. Hinge regions.

Based on function relationship of immunoglobulin, an antibody can be

divided to two parts: Fab and Fc regions. The Fab fragment contains antigen
binding sites or called paratopes at the end of the region. Each Fab fragment
consists of one constant and one variable domain and each domain has heavy and
light chains. The Fc fragment is easily to be crystallized and has effectors function
of antibody. Both Fab and Fc fragments can be separated using papain enzyme.
This enzyme digests the immunoglobulin molecules from the hinge region. The
enzyme pepsin cleaves heavy chain of immunoglobulin at the inter-chain disulfide
bonds or below hinge region, resulting in a fragment that contains both antigen
binding sites. This fragment called F(ab')2 because it was divalent and the two
antigen binding sites remain linked together. The remaining of heavy chain was
digested by this enzyme into small part of peptide [2]. How an antibody can be
cleaved into parts is shown in Figure 1-2.

Figure 1-2 The Y-shaped immunoglobulin molecule dissected by partial

digestion with proteases. Papain cleaves the immunoglobulin molecule into
three pieces, two Fab fragments and one Fc fragment (upper panels). The Fab
fragment contains the V regions and binds antigen. The Fc fragment is
crystallizable and contains C regions. Pepsin cleaves immunoglobulin to yield
one F(ab)2 fragment and many small pieces of the Fc fragment, the largest of
which is called the pFc fragment (lower panels). F(ab)2 is written with a
prime because it contains a few more amino acids than Fab, including the
cysteines that form the disulfide bonds [2].

The most important characteristics of an antibody response are the

specificity, isotype or class, and affinity to its antigen. Antibody molecules are
very specific to their corresponding antigen. The binding strength of an antibody
to its antigen, in a single antigen-binding site binding to a monovalent antigen, is
termed its affinity; whereas the total binding strength of a molecule with more
than one binding site is called its avidity.

There are three models proposed to describe the interaction between

antibody and antigen; lock and key, induced fit models and preexisting
equilibrium/conformational selection model [3] as shown in Figure 1-3. Lock
and key model was first proposed 120 years ago by Fischer to describe the
interaction between enzymes and their respective substrates. This model then was
applied to explain the interaction between antibody and antigen. According to this
model, the antigen binding site of antibody was very rigid and the antigen had to
fit to the binding site. Unfortunately, this model couldnt explain the anomaly of
protein that can react with different shape of substrates [4]. The induced fit model,
proposed by Koshland in 1958 [4], try to solve this anomaly. Koshland introduced
postulate to describe his model that the binding site of protein must have precise
orientation and the substrate may generate the conformational change at the active
site so it would bring the proper orientation for interaction. This model is actually

similar in the principle that the protein must have precise orientation to fit with the
substrate except that the fitting only occurs after the changes induced by the
substrate itself. Study performed by Betts and Sternberg [5] on the conformational
changes for a set of 39 complexes and predominantly enzyme inhibitors, leading
to the conclusion that proteinprotein recognition occurs by the mechanism of
induced fit. However, Bosshard [6] noted that induced fit is possible only if the
match between the interacting sites is strong enough to provide the initial complex
strength and longevity so that induced fit takes place within a reasonable time. He
also pointed that there is an alternative mechanism to induced fit, called the
preexisting equilibrium/conformational selection model [6]. According to this
model, the antigen binding site on antibody doesnt need to make some change on
conformational to fit with the antigen. The antibody just simply selects the antigen
that has epitope with precise conformation with its binding site or in reciprocal
term, the antigen selects the antibody that has complementary conformation [7].
Studies conducted by Berger [8] and Foote & Milstein [9] validate this model.
However, Bosshard [6] stated that even the conformational selection model was
valuable and alternative for induced fit, it didnt mean that induced fit didnt occur
in protein-protein interaction. Actually, combination of these two models seems to
be the best to describe the interaction between molecules that apparently do not
actually fit to begin with.

antigen

Antigen binding site

Figure 1-3 Three proposed models described antibody-antigen interaction.

(A) Lock and key model proposed by Fischer in 1894, (B) induced fit model
proposed by Koshland to solve the anomaly that occurs in protein-protein
interaction,

(C)

proposed

by

Bosshard,

is

called

the

preexisting

equilibrium/conformational selection model. Based on this model, the antibody

has several conformational that can bind different antigen conformation.

1.2

Immuno-PCR
The development of immunoassay has grown rapidly since its first

establishment. After the introduction of radioimmunoassay (RIA), the first

quantitative immunoanalytical technique was proposed by Yalow and Barson [10]
in 1959, the methodologies of immunoassay using antibody have still continued to
expand to improve the sensitivity, shorten the assay time and make usage ease for
signal-generated molecules and alternative nonradioactive molecules.

Immuno-PCR, the immunoassay term introduced by Sano et al in 1992

[11], is one of ultimate quantitative immunoanalytical technique for protein
detection. This immunoassay technique utilizes the ability of DNA that can be
amplified exponentially from a single molecule. Immuno-PCR was similar with
ELISA in the basic working principle, but immuno-PCR uses DNA as target
marker instead of enzyme in ELISA (Figure 1-4). Because it possesses the
amplification ability of DNA, immuno-PCR allowed enhancement in sensitivity in
a 1000-10000 fold number of magnitude. But the sensitivity of immuno-PCR is
still limited by the sensitivity of the interaction of antibody and antigen and the
selectivity of the antibody to the target antigen [12].

Substrate
Enzyme

DNA

2nd antibody

2nd antibody
PCR
Product

1st antibody

Immuno-PCR

1st antibody

ELISA

Figure 1-4 The format of immuno-PCR and ELISA. In immuno-PCR, a DNA

is used as the target market instead of an enzyme in ELISA.

The immuno-PCR technique is constructed with three basic components;

the antigen recognition system, the DNA marker amplification system and the
signal manifestation [13]. The antigen recognition system involves the selection
of antigen-antibody pair with a high sensitivity and selectivity and an indirectly
DNA-marker with the antibody.

Linking the DNA marker to antibody is the most cumbersome step in

immuno-PCR. The early work of immuno-PCR, DNA was conjugated to antibody
via streptavidin-protein A chimera (Figure 1-5.A). The chimera has two
independent and specific binding sites: one is to biotin, derived from the

10

streptavidin moiety, and the other is to the Fc portion of the immunoglobulin G

(IgG) molecule, derived from the protein A moiety. However, the use of this
chimera is limited to direct Immuno-PCR formats, which lack capture antibodies
with the same affinity as the detection antibody. Later, Ruzicka et al [14],
developed a new method to solve this problem. They conjugated biotinylated
dsDNA with biotinylated IgG by avidin. These three components were assembled
by the simple mixing of stoichiometric ratios (Figure 1-5.B). However, this
methodology has a potential drawback. That is the lack of homogeneity of the
avidin-DNA conjugates, which would reduce the accuracy and reproducibility of
the system. Besides, in situ assembly could create variable stoichiometry in the
reaction components. As well, extra steps are normally required for addition of
biotinylated reagents with binding proteins. Numerous wash steps are also needed
to remove excess reagents to free assay components from nonspecifically bound
reagents. Consequently, immuno-PCR assays become procedurally complex and
require considerable hands-on time [15]. Many researchers tried to reduce the
complexity of immuno-PCR by presynthesized antibody-DNA conjugates. Some
researchers [15], [16], [17] used direct covalent coupling to presynthesize
antibody-DNA conjugate (Figure 1-5.C), while others used biotin-streptavidin
coupling [18], [19].

11

protein A chimera

DNA

DNA

DNA

chemical crosslinker

Figure 1-5 The methods employed to conjugate DNA onto antibody. (A)
ProteinA-strevtavidin chimera is used to connect biotinylated DNA and the Fc
region of IgG. (B) Streptavidin conjugate biotinylated DNA and biotinylated
antibody. (C). DNA covalenty linked to antibody.

Since its first demonstration, immuno-PCR has been applied in

laboratory analysis and medical diagnosis. There are numerous examples of such
immuno-PCR applications, including detection of bacteria such as Clostridium
botulinum neurotoxin A [20] and Streptococcus pyogenes [21]. Immuno-PCR also
becomes major immunoanalytical method for analysis of viral infection. Barletta
reported on an improved sensitivity of real-time IPCR as compared with reverse
transcriptase-PCR (RT-PCR) for detection of HIV [22]. Low level of tumor and
disease-associated antigens [23], [24] also can be detected by immuno-PCR with
a good sensitivity.

12

1.3

Lateral flow immunochromatography assay

Lateral flow immunochromatography is the most widely used membranebased immunoassay application. Compare to other methods, lateral flow gives an
easy of use and short assay time. The application of membrane on immunoassay
was started in 1979 by Towbin et al. in demonstrating that protein can be
transferred to microporous membrane nitrocellulose and detected using antibodies
[25].

Since that year, the application of membrane on immunoassay was

proliferated. The first test of lateral flow was made for detection of human
chorionic gonadotropin (GDP). Nowadays, the lateral flow has been used for
monitoring ovulation, detecting infectious disease organisms, analyzing drugs of
abuse, and measuring other analytes important to human physiology. Table 1-1
provides an overview on the variety of biomedical applications realized so far.

The application of membrane in immunoassay can be sorted in three

categories, antigen or antibody immobilized in specific location on a solid support
(ex. Western blot and dot blot immunoassay), a miniature of thin-layer
chromatography where the analyte flows transversely as the mobile phase and the
membrane as the stationary phase (ex. Lateral flow immunochromatography
assay), and the analyte flowing-through the porous membrane.

13

Table 1-1 Overview of applications of lateral-flow immunochromatography assay

Protein

Antigen

Year

Bacteria

Vibrio harveyi

2007

Virus

Canine distemper

2008

Hormone

19-Nortestosterone

2007

class

Bacteria

Cryptosporidium
parvum.

2000

Remarks

Ref.

Non-

Sithigorngul P

competitive

et al. [26]

Noncompetitive

An DJ, et al. [27]

Competitive

Liu L, et al. [28]

Non-

Kozwich et al.

competitive

[29]

Cardiac

Human heart-type fatty

marker

acid-binding protein

Toxin

Aflatoxin B1

2005

Competitive

Hormone

Clenbuterol

2006

Competitive

Zhang et al. [32]

Antibiotic

Sulfonamides

2007

Competitive

Wang et al. [33]

Bacteria
Toxin
Insecticide
Nucleic
acid
Enzyme
Food
additive
Cells
protein

Legionella
pneumophila
Microcystins
Carbaryl and
endosulfan
Single stranded DNA
Canine Trypsin-like
immunoreactivity

2003

2006

Noncompetitive

Noncompetitive

Chan et al.[30]
Delmulle et al.
[31]

Horng et al. [34]

2003

Competitive

Kim et al. [35]

2006

Competitive

Zhang et al. [36]

Nucleic acid

Corstjens et al.

lateral flow

[37]

Non-

Waritani et al.

competitive

[39]

2003

2007

Glycyrrhizin

2005

Frataxin

2008

Noncompetitive
Noncompetitive

Putalun et al.[38]

Willis et al. [40]

14

Membrane-based immunoassay has been widely used, primarily because

it has high analyte-binding surface. The performance and capability of membranebased immunoassay are influenced by specific characteristics that build membrane
properties. Polymer type, porosity, surfactant content, and strength are several
key membrane characteristics [41]. The choice of membrane was considered
mostly based on the porosity of the membrane, capacity of protein-binding and
strength. Because the application of membrane for detection of protein uses
reactant that must flow through the membrane matrix, the porosity becomes an
important point in the selection of membrane type. Capacity of protein-binding
surface is corresponding with the ability of membrane immobilizing the protein.

A number of forces specifically hydrophobic interactions, hydrogen

bonding, and electrostatic force are known involved in the binding mechanism
of proteins, but the exact mechanism still not fully understood. There are two
proposed models to describe the mechanism of protein-binding on membrane. The
first model suggests that the electrostatic force involved in initial binding, while in
the long-term attachment, the binding is accomplished by combination between
hydrogen bonding and hydrophobic interaction. The second model suggests that
the initial binding is accomplished by hydrophobic interaction while the long-term
is caused by electrostatic force [42].

15

The lateral-flow immunochromatography assay is made up of a number

of components, including reagents, a sample pad, a conjugate pad, an aborbant
pad, and a porous membrane on which the reaction occurs. These components
interact together to affect many different aspects that influence the final
performance of lateral flow assay.

Generally there are two formats of lateral flow immunoassay,

competitive and non-competitive formats. The competitive format is mostly used
for testing small molecules with a single antigenic determinant that cannot be
bound with two antibodies simultaneously. While the non-competitive format is
used for testing the analyte with multiantigenic sites likes big molecules such as
hormone, bacteria or virus.

In the competitive format, the target analytes bind either to the reporter
particles or to the immobilized ligands. In the first case, the target analytes bind to
the ligands and block the ligands from binding to the reporters. This format was
used by Ho and Wauchope [43] to detect aflatoxin B1 (AFB1). The target analytes
was conjugated with liposome and the AFB1 antibody is immobilized at the
capture zone, where the competition occurs between AFB1-conjugated liposome
and AFB1. In the second format of competitive assay, the target analytes was
bound to the reporters and block these reporters from binding to the immobilized

16

ligand. Esch et al [44] used this format to detect water-borne Cryptosporidium

parvum oocysts. In both cases, the presences of the target analytes interfered the
binding of reporter to the test ligand and resulted in nonexisting signal which
indicated the presence of the target analytes.

The non-competitive assay or sandwich format uses two antibodies to

bind the analyte in between. The first antibody acts as a reporter, while the second
antibody captures the analytes at the test line. Different with the competitive assay,
which was interpreted analyte presence for a nonexistent signal; the single
intensity of the test line is equivalent with the concentration of the analyte for the
non-competitive assay.

17

Figure 1-6 Two formats of the lateral-flow immunochromatography assay. In

the competitive format, the protein-conjugated antigen is immobilized at test line,
while in the non-competitive assay, anti-antigen antibody is immobilized.

18

1.4

Motivation

Even though the lateral flow immunochromatography gives so many

advantages when it is applied on detection of proteomic substances in a userfriendly format, very short time to get test result, a long-term stability over a wide
range of climates, and relatively inexpensive to make, the lateral flow
immunochromatography still has limitations in the sensitivity and quantification.
Currently, the lateral flow immunochromatography assay uses colloidal gold
nanoparticles or latex particles as the signal-generated molecule. These molecules
generate visual detection without additional readers but they also pay the cost that
a comparatively high sensitivity is not easy to achieve.

Polymerase chain reaction (PCR) has found a multitude of commercial

applications in the life sciences and in vitro diagnostics. In the PCR, the
researchers are enabled to produce millions of copies of a specific DNA sequence
in

approximately two

hours.

The

application

of

PCR

technique

in

immunoanalytical field has been proved increasing the detection limit of the assay.
On the other hand, immuno-PCR, immunoassay based PCR as the signalgenerated method, has been reported amplifying signal with 100-10000 folds
greater than that by ELISA.

19

By combining the lateral flow immunochromatography assay with a PCR

technique, this study hopes to increase the sensitivity of lateral flow detection.
The signal-generated molecules such as colloidal gold or latex particles are
changed with a nucleic acid in the immuno-PCR assay. By this assay the nucleic
acid on the membrane can be amplified to generate million copies of nucleic acid
and analyzed by gel electrophoresis. The intensity of the gel bands can be
quantified using available commercial software such as ImageJ or Photoshop.
This labeling method can therefore overcome the limitations mentioned above of
the lateral flow immunochromatography assay.

The membrane-based immuno-PCR also offers several advantages

compare with other immunoassay. Utilizing non-hazardous material on its
application makes membrane-based immunoassay can be applied at a laboratory
with population-dense environment. The first step of the assay, the presence of the
gene product is detected; whereas the second amplifies the single gene. Both steps
can be conducted on the case scene. A portable PCR machine makes its
application feasible. Employing immuno-PCR on the lateral flow assay besides
makes short assay time is achieved also bring the sensitivity higher than
conventional lateral flow.

20

1.5

Objective
The objective of this study was to develop a membrane-based immuno-

PCR to enhance the sensitivity of lateral flow immunochromatography assay. The

membrane-based immuno-PCR not only was expected to screen out the desired
antigen by the antibody-antigen interaction as the conventional lateral-flow
immunochromatography, but also provided a signal amplification methodology.
The specific detector of DNA-conjugated antibody could immunospecifically bind
the desired antigen from the sample, and the conjugated DNA could be amplified
using PCR technique. Its amplified intensity was considered to be relative with
the signal antigen.

21

Chapter 2. Principle
2.1

Antibody-DNA conjugation

In this study, DNA was conjugated with antibody using streptavidin as

the bridge. The conjugation was conducted by two steps. The first step,
biotinylated DNA was conjugated with streptavidin to form DNA-streptavidin
complex. The second step, biotinylated antibody was added to the previously
prepared DNA-streptavidin complex and formed DNA-conjugated antibody.

Figure 2-1 Formation of the DNA-conjugated antibody. Biotinylated DNA is

conjugated first with streptavidin to form DNA-streptavidin complex. The
complex is further conjugated with biotinylated antibody to form DNAconjugated antibody.

22

The DNA-streptavidin complex can be monovalent, divalent trivalent or

tetravalent and detectable from the band of gel electrophoresis. Each complex
shows a band at different position for different molecular weight level. The
monovalent complex shows a band on low molecular weight level but a little
higher than free DNA, whereas the divalent one shows the band about twice
higher than monovalent (Figure 2-2). Biotin-modified antibody can be added to
the DNA-streptavidin complex when the gel shows monovalent, divalent or
trivalent form, because there still have vacant sites on streptavidin available for
biotinylated antibody. As tetravalent form doesnt have any vacant site for biotinmodified antibody, this form cannot be used to make DNA-conjugated antibody.

Figure 2-2 Band distribution of DNA-streptavidin complex on agarose gel

electrophoresis. A = marker, B = free DNA, C = monovalent DNA on
streptavidin, D = divalent, E= trivalent and F= tetravalent. Since the tetravalent
conformation has the highest molecular weight, it moves slower and its band
locates closer to the sample well than other conformations.

23

2.2

Lateral flow

The innovation of immunoanalytical method for diagnostic is more

evident than in therapeutics drug monitoring. Over the past decade, single-use
lateral flow immunoassays have been extremely successful in the laboratory and
in outpatient clinic and primary care environments.

In the lateral flow

immunoassay, the reaction occurs in a porous solid phase materials such as

nitrocellulose membrane. All reaction components are impregnated and
immobilized on membrane and the sample is brought to contact with the
components by flowing its diluents.

In this study, the lateral flow immunochromatography assay is made

from four main components: sample pad, membrane, absorbent pad, and backing
pad. The sample pad is functionalized as a sample collector and made from glass
fiber. The membrane material is made from nitrocellulose. The membrane
provides the testing zone. The capture antibody is deposited as narrow band on
membrane and dried. The absorbent pad functions to absorb the overflow of the
sample to make the capillary flow continuous. The plastic backing pad with the
pressure-sensitive adhesive serves as a structural support for the layers above.

24

The basic working principle of lateral flow immunoassay was shown in

Figure 2-3. The antibody was immobilized on the membrane by the forces
hydrogen bond, hydrophobic interaction, and electrostatic force. With the
presences of high anti-chaotropic salt concentration, only antibody can bind to the
membrane, while other molecules are washed away. The analyte solution then
flows through the membrane and is captured by antibody.

The capillary migration is used to distribute antigen and detector

antibody on a rectangular membrane. The strip absorbs a finite volume of the
detector antibody/sample antigen mixture. As the detector antibody and sample
antigen migrate past the immobilized antibody at the membrane, they are
immunospecifically bound to the surface of the membrane. The migration process
is normally complete within 5 minutes and results in test strips in the form of bars
at the zone of immobilized antibody. The intensity of the zone is directly related
to the antigen concentration of the original sample.

25

Figure 2-3 Basic principle of the lateral flow immunochromatography assay.

The antigen is collected on sample pad and flows to the conjugate pad and catch
by labeled antibody. The antigen/antibody complecx flow through the membrane
and immunospecifically bind with the immobilized antibody.

2.3

Membrane-based immuno-PCR

The principle of membrane-based immuno-PCR is described in Figure 24. The format of membrane-based immuno-PCR is similar with the indirect assay
of immuno-PCR. There are two antibodies used to capture the antigen. The first
antibody is immobilized on membrane nitrocellulose by spraying it onto the
surface of membrane. The membrane binds the antibody with several forces,
mainly hydrophobic interaction and electrostatic forces. The second antibody is
premixed first with the antigen. Premixing can reduce the stoichiometry

26

variability of the reaction between antigen and DNA-conjugated antibody when

the in situ reaction is applied.

The antigen/antibody mixture flows onto the membrane by the capillary

force and migrates through the immobilized capturing antibody on the membrane.
At this step, the antigen is immunospecifically bound with the capturing antibody
and the reaction is then complete.

For the amplification of DNA, the membrane piece, where the antibody
is immobilized, is cut off and put into a PCR tube to mix with PCR cocktail
containing primer, Taq DNA polymerase, dNTP, and water. The mixture is heated
to release the DNA from the membrane so that the amplification process will be
effective. The final product of PCR is then analyzed on gel electrophoresis.
.

27

Figure 2-4 The principle of membrane-based immuno-PCR. The principle of

membrane-based immuno-PCR is similar with the lateral flow assay. DNAconjugated antibody is used instead of gold nanoparticles-conjugated antibody.
The signal is analyzed from the product of PCR.

28

Chapter 3. Experimental

3.1

Materials

The following materials were used in the experiment:

3.1.1

Polymerase Chain Reaction

Double strands DNA 80 bp (5-biotin-TAG CAC GGA CAT ATA TGA
TGG TAC CGC AGT ATG AGT ATC TCC TAT GAC TAC TAA GTG
GAA GAA ATG TAA CTG TTT CCT TC-3) [45] as the target marker in
immuno-PCR was purchased from Purigo Biotech, Inc (Taiwan)

Forward primer 20 mer (TAG CAC GGT CAT ATA TGA TG) was
purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to
anneal to the template strand of DNA and allow the Taq Polymerase to
synthesize a strand complementary from the template strand.

Reverse primer 20 mer (GAA GGA AAC AGT TAC ATT TC) was
purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to
anneal to the template strand of DNA and allow the Taq Polymerase to
synthesize a strand complementary from the template strand.

dNTPs from Amersham (USA), for incorporating onto newly synthesized

DNA strands during the extension step of PCR

29

Taq Polymerase from New England Biolab (England) for synthesizing

new strand DNA during PCR.

PCR standard buffer from New England Biolab (England), to promote Taq
usage and maintain the pH of solution during PCR

3.1.2

Agarose gel electrophoresis

Molecular weight DNA ladder were purchased from New England Biolab
(England). It was used as a molecular weight marker in gel electrophoresis

5X TBE buffer was from Biobasic (Canada) as a buffer for electrophoresis

gel.

Agarose was from Biobasic (Canada) to make agarose gel to analyze DNA.

Ethidium bromide was from BioBasic (Canada) and was used to stain the
agarose gel, so that the gel bands can show on the Gel Imaging System.

3.1.3

Lateral flow immuno-PCR

Streptavidin was from Jackson ImmunoResearch Lab, Inc (USA). Used
for as a bridge to conjugate antibody and DNA

Human Interleukin-6 was from USBiological, as an antigen

Biotinylated mouse anti-human IL-6 antibody was from USBiological, for

capturing human IL-6 on lateral flow

30

Rabbit anti-mouse IgG was from USBiological, printed as control line on

lateral flow immuno-PCR to capture the mouse anti-human IL-6 antibody.

Nitrocellulose membrane 5 m AE98 was from Sartorius, on which the

reactions in lateral flow immuno-PCR took place.

Absorbent pad CS6 was from Whatman to keep the capillary force in the
lateral flow immuno-PCR assay.

Plastic backing pad was from Adhesive Research, Inc as a backing for
lateral flow materials.

Sample pad 33 Glass was from S&S, used to hold the sample of lateral
flow immuno-PCR.

3.1.4

Buffer Solution
Sodium chloride was from Bio Basic Inc, as material for making PBS
solution and protein lateral flow buffer

Potassium chloride was from Bio Basic Inc, as material for making PBS
solution

Sodium phosphate dibasic anhydrous was from Mallinckrodt, as material

for making PBS solution

Potassium phosphate was from Mallinckrodt, as material for making PBS

solution

31

Phosphate buffer saline (PBS)

Into a baker, 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4
were added with 800 ml of distilled H2O. The pH was adjusted to 7.4 with
HCl then added ddH2O to 1 liter. PBS buffer was used for routine
immunohistochemical staining and also used for diluting antibodies and
antigen.

Tween 20 was from Bio Basic Inc, as material for making protein lateral
flow buffer.

Bovine serum albumin was from Bio Basic Inc, as material for making
protein lateral flow buffer.

HEPES was from Calbiochem, as material for making protein lateral flow
buffer.

Tris (base) was from Bio Basic Inc, as material for making protein lateral
flow wash buffer.

Magnesium chloride was from Bio Basic Inc, as material for making
protein lateral flow wash buffer.

32

Protein lateral flow buffer

The lateral flow buffer was made by mixing 15 ml of Tween 20, 10 g BSA,
2.383 g HEPES, and 7.8894 g NaCl into 800 ml ddH20. The pH was
adjusted to 7.5 then added ddH20 until the final volume was 1 L.

Protein lateral flow wash buffer

The buffer was made to wash the nitrocellulose membrane. The
composition of this buffer was 6.057 g Tris, 2.922 g NaCl, and 5.0825 g
MgCl2. The materials were dissolved with ddH2O until the final volume
was 500 ml.

3.2

Instruments

The following instrument was used in the experiment:

TLC Linomat 5 (CAMAG Switzerland) for spraying antibody onto

nitrocellulose membrane.

Gel electrophoresis apparatus (Mupid Advance, Japan).

Microcentrifuge

(Mikro

120

Hettich

Zentrifugen,

Germany)

for

centrifuging protein and DNA.

PCR machine (Astec) for setting up temperature during PCR.

Biohazard safety hood was from High Ten Scientific Corp, for preparing
PCR and protein dilution.

33

Microwave for dissolving agarose powder into solution.

Balancer Precise 205A, for measuring weight of materials.

pH meter 330A from Orion, for measuring pH of solutions.

GeneFlash (Syngene, USA), as the gel imaging system.

Oven (Deng Yng, Taiwan) as an incubator.

The parameter input dialog

N2 Gas supply
Syringe

Globals

Enter

Syringe volume (100 l)

Dosage Speed (70 nl/s)

Plate

Predosage Volume (0.1 l)

Parameters

Enter

Plate Size (20 x 20 cm)

Number of tracks (1)
Number of Samples (1)
Band Length (25 mm)
First position X (21.5 mm)
Track Distance (5 mm)

Display and function key

App. Position Y

Track
Assignment

Enter

Track No 1
Track Volume ( 1l)
Sample ID

Save Method

Enter

Default method
Method No. 3

Figure 3-1 Structure and the example of parameter input dialog of Linomat 5
TLC machine for a typical spraying procedure. Following the procedure shown
above to key in appropriate parameters, the TLC machine sprayed the desired
strips for the lateral flow test.

34

3.3

3.3.1

Methods

Antibody-DNA conjugation
The conjugation between antibody and DNA principally follow the

method of Niemeyer et al. [46]. The antibody was conjugated with DNA using
streptavidin (SA) as a bridge, utilizing the high affinity nature between biotin and
streptavidin (Figure 3-2). First, DNA was conjugated with streptavidin to formed
DNA-streptavidin complex. Conjugates of streptavidin and biotinylated dsDNA
fragment were typically prepared by adding DNA (3 M in ddH2O) to PBS and
subsequently added streptavidin (100 g/ml in PBS) until the total volume was 15
l (Table 3-1).

Table 3-1 Design of the concentration ratio of biotinylated DNA to

streptavidin. Several ratios was designed and tested to get the appropriate
concentration of DNA and streptavidin that give better result.
Molar ratio
(DNA:SA)

5:1

4:1

3:1

2:1

1:1

1:2

1:3

1:4

1:5

DNA 3 M
(l)

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

0.5

0.65

0.9

1.35

2.7

5.4

8.1

10.8

13.5

13

12.85

12.6

12.15

10.8

8.1

5.4

2.7

Streptavidin
100 g/ml
(l)
PBS buffer
(l)

35

The mixture of DNA and streptavidin was then incubated for 1 h at 37 C.

After DNA-streptavidin complex were prepared, the biotinylated mouse anti
human IL-6 antibody (7.5 l, 90 g/ml) was conjugated with the DNAstreptavidin complex. The mixture was incubated for 30 min at 37 C. After
incubation, the product was stored at 4 C.

36

+
Biotinylated DNA

Streptavidin

DNA-Streptavidin Complex

Biotinylated Antibody

Antibody-DNA conjugate

Figure 3-2 DNA-conjugated antibody formation. DNA-conjugated antibody

was formed using streptavidin as the bridge. DNA was first conjugated with
streptavidin to form the DNA-streptavidin complex. The antibody was then added
to the complex and formed the DNA-conjugated antibody.

37

3.3.2

Preparation of immunochromatographic test strips

The immunochromatographic strips was prepared with the following

procedures. First, membrane strips were sprayed with 0.5 mg/ml control capture
antibody, rabbit anti-mouse IgG, and the test capture antibody, mouse anti human
IL-6 antibody, using Linomat 5 machine with dosage speed 70 nl/s and N2
pressure 70 psi. The control line and test line were separately sprayed at two
different places near the top of the 50 x 25 mm membrane sheet, leaving a 5-mm
space in between. The membrane was stored in desiccators at room temperature
and 40% relative humidity for a day and then cut into 4 mm x 25 mm strips using
scissor. An absorbent pad was cut in sections of 4 x 15 mm and fixed on the
backing pad at the far end of the strip in the direction of the flow. The sample pad
with dimension 4 x 10 mm was fixed at the starting end of the strip. The
immunochromatographic strip was ready for use. The preparation procedure and
strip assemble were illustrated in Figure 3-3.

38

Figure 3-3 Preparation of immunochromatography strips. (A) The membrane

was sprayed with antibody at two separated line using Linomat 5. (B) the test line,
contained mouse anti-human IL-6 antibody and the control line contain rabbit anti
mouse IgG. (C) The sample pad and the absorbent pad were adhesed at the ends
of the device. The conjugate antibody was premixed with the sample; therefore
the conjugate pad was unnecessary.

39

3.3.3

Optimization of lateral flow-immuno-PCR assay

Lateral flow immuno-PCR was optimized the following two parameters

in order to get a low background: the conjugation condition between human IL-6
and DNA-conjugated anti human IL-6 antibody and the lateral flow test waiting
time.

Conjugation between antigen and its corresponding antibody was tested

in two different methods. The first method was premixing antigen with its
corresponding antibody. 10 l of 100 ng/ml antigen and 10 l DNA-conjugated
antibody (1:100 dilution) were mixed together and incubated for 5 min at 37 C.
After incubation, the mixture was added with 20 l lateral flow buffer then
applied onto the immunochromatography test strips. The second method was
adding the antigen onto the membrane first. After 5 min, the DNA-conjugated
antibody was added onto the membrane. After another 5 min the membrane was
washed using 1 ml lateral flow wash buffer. The result indicated that the first
method gave the better performance; therefore it was chosen to optimize the
second parameter.

The second parameter was the test waiting time for reducing background
after the immunochromatography strip was applied with a sample. The waiting

40

time was set at 2 min, 5 min, and 7 min. After applying the wash buffer, the
membrane around the test line was cut into about a 2mm piece. This membrane
piece was mixed with the PCR cocktail containing 10 l of 1 M of each primer,
10 l of 1 mM of dNTPs, 5 l of 10x standard Taq buffer and 0.4 l of Taq DNA
polymerase. The mixture was then subjected to PCR with the thermal cycling
conditions 95 C for 5 min as the initial denaturation, then 42 cycles of 95 C for
20 s, 53 C for 30 s and 72 C for 20 s. In the last cycle, 72 C was extended to 5
min and cooled to 4 C for 30 min. The PCR products were finally loaded in a 2%
agarose gel electrophoresis and the intensity of each band was measured using
ImageJ software.

41

Figure 3-4 Optimization of lateral flow immuno-PCR in order to get low

background with two different methods. Method A was premixing antigen and
corresponding antibody, whereas method B was with a step by step addition of
antigen and corresponding antibody. The result indicated method A was better.

42

3.3.4

Determination of the optimal concentration of capture antibody

The optimal concentration of capture antibody was determined using the

parameter from the previous optimization section. Four concentrations of capture
antibody: 1 mg/ml, 0.5 mg/ml, 100 g/ml, and 10 g/ml, were sprayed onto
nitrocellulose membrane. The membrane was stored in desiccators at room
temperature and 40% relative humidity for a day. The absorbent pad and sample
pad were assembled as described in section 3.3.2.

Ten microliters of antigen were mixed with 10 l DNA-conjugated anti

human IL-6 antibody and incubated for 5 min at 37 C. This premixed solution
then was mixed with 20 l lateral flow buffer and applied onto the membrane strip
and waited for 5 min. The strip was then washed by flowing 1 ml lateral flow
washing buffer and dried in room temperature for 5 min. The test line and the
control line were cut off using a knife and the membrane piece was moved into a
PCR tube and mixed with the PCR cocktail. The mixture was heated at 95 C for
5 min then subjected to PCR described in section 3.3.3. The PCR product was run
on 2% electrophoresis gel. The intensity of the band was measured using ImageJ
software.

43

3.3.5

Sensitivity test of lateral flow immuno-PCR for human IL-6

The sequential dilution of human IL-6, 10 pg/ml to 0.001 pg/ml, was

tested using lateral flow immuno-PCR to see its sensitivity. Ten microliters of
antigen were mixed with 10 l DNA-conjugated anti human IL-6 antibody and
incubated for 5 min at 37 C. This premixed solution then was mixed with 20 l
lateral flow buffer and applied onto the membrane strip and waited for 5 min. The
strip was then washed by flowing 1 ml lateral flow washing buffer and dried in
room temperature for 5 min. The test line and the control line were cut off using a
knife and the membrane piece was moved into a PCR tube and mixed with the
PCR cocktail. The mixture was heated at 95 C for 5 min then subjected to PCR.
The PCR product was run on an electrophoresis gel. The intensity of the band was
measured using ImageJ software.

3.3.6

Analysis of gel image using ImageJ software

ImageJ software was used to analyze the intensity of agarose gel image.
The software can be obtained freely from NIH website (http://rsb.info.nih.gov/ij/).
The tutorial for 1D gel image analysis using Image J can be loaded from internet.
In briefly, the band was selected using the rectangular selection tool by outlining
the first lane. In the ImageJ toolbar, selected Analyze>Gels>Select First Lane (or

44

press "1") and "Lane 1 selected" will be displayed in the status bar (Figure 3-5.A).
Analyze>Gels>Plot Lanes (or press "3") was selected to generate the lane profile
plots. The base lane was drawn in each peak using the straight line selection tool
(Figure 3-5.B) . The size for each peak was measured by clicking inside with the
wand tool (Figure 3-5.C). The data was saved and analyze further using Microsoft
Excel.

45

A)

B)

C)

D)

Figure 3-5 Analysis of gel image using ImageJ software. (A) Outlining the band using the rectangular selection tools. (B) Plotting
the line. (C) Measuring the area of each peak. (D) Saving the data.

46

Chapter 4. Result and Discussion

4.1

Preparation of antibody-DNA conjugate

The conjugation between antibody and DNA was made using

streptavidin as a bridge. In a first set of experiment, DNA was preconjugated with
streptavidin. The preconjugation was prepared by mixing DNA and streptavidin in
several combination starts from 1:5 molar ratios to 5:1 molar ratio as described in
Table 3-1. The result of combination between DNA and streptavidin was
characterized using 2% agarose gel electrophoresis in the presence of standard
molecular weight as presented in Figure 4-1.

Characterization on gel electrophoresis of DNA-streptavidin complex

showed two bands at 80 bp and 300 bp for an equal or lower ratio of DNA to
streptavidin. The single band shown in the gel represented free DNA from the
excess of DNA used for the combination. Two bands at 80 bp and 300 bp shown
in the gel represented free DNA and tri-adduct of DNA-streptavidin complex,
respectively. The intensity of the band at 300 bp was overall higher than that at 80
bp. This proved that mostly DNA was conjugated with streptavidin in tri- adduct
of DNA-streptavidin complex and left small amount of free DNA in the solution.
One streptavidin molecule binds three molecules of DNA indicate that mostly

47

streptavidin behave trivalent linker molecule despite it has tetravalent binding

capacity. In addition, no appearance of other band at gel showed that streptavidin
only bind at trivalent format. The trivalent format left one free binding site that
can be occupied by biotinylated antibody so that antibody-DNA conjugate can be
made by the streptavidin bridge.

Lane
DNA ratio
SA ratio

Free
DNA

1
5
1

2
4
1

3
3
1

4
2
1

5
1
1

6
1
2

7
1
3

8
1
4

9
1
5

Figure 4-1 Analysis of DNA-streptavidin complex in 2% agarose gel. The

result indicated that the complex of DNA-streptavidin was formed when an
equal or lower ratio of DNA to streptavidin was used.

48

The DNA-streptavidin complex was further conjugated with the work

antibody to form the antibody-DNA conjugates. In the previous experiment the
DNA-streptavidin complex was successfully produced only when one or higher
molar excess of streptavidin was added. The resulting complex was in the agarose
gel with the band shown in 300 bp. The biotinylated antibody was then mixed
with each successful complex at equimolar ratio between antibody and DNA. The
result of conjugation was presented in the Figure 4-2.

Lane
DNA-SA
ratio
Antibody
ratio

1
M

Free

1-1 1-1 1-2 1-2 1-3 1-3 1-4 1-4 1-5

10
1-5

DNA

Figure 4-2 Conjugation of biotinylated antibody with DNA-streptavidin

complex presented in 2% agarose gel electrophoresis. The conjugation of the
antibody to DNA-streptavidin complex will result in the decreasing of the
intensity for the band at 300 bp.

49

The introduction of biotinylated antibody to the DNA-streptavidin

complex solution leaded the appearance of novel band with a high molecular
weight and reduction of the intensity of the band from DNA-streptavidin complex.
The reduction was clearly visible at equimolar ratio between DNA and
streptavidin. It was because one free binding site at DNA-streptavidin complex
was filled already by biotinylated antibody, formed antibody-DNA conjugate.
Antibody-DNA conjugate has high molecular weight so that it couldnt pass
through the pore of agarose gel. The intensity of the band was therefore reduced at
300 bp.

4.2

Optimization of lateral flow immuno-PCR

Antibody-DNA conjugate was applied to lateral flow immuno-PCR.

Lateral flow immuno-PCR term was referred to application of immuno-PCR on
lateral flow. The format of lateral flow immuno-PCR was similar with general
lateral flow, but the antibody-DNA conjugate was used instead of the antibodygold nanoparticles conjugate in general applications.

To get a high signal to noise ratio, the lateral flow immuno-PCR was
optimized. Two approaches were conducted for this optimization. In the first
approach, the reactions between antigen and antibody-DNA conjugate was

50

premixed at a tube; whereas in the second approach, they were step by step added
on membrane. Their immuno-PCR products were run on a 2% gel electrophoresis
as shown in Figure 4-3. In this Figure 4-3, the premixing approach gave a lower
background and higher test intensity than the step-by-step approach. In order to
more clearly indicate the optimization result, the intensity of each band was
further measured by ImageJ software and presented in signal to noise ratio as
shown in Figure 4-4. In the figure, premixing method gave about twice higher
ratio compared with the approach of step by step addition.

51

1a

1b

2a

2b

Figure 4-3 Agarose gel electrophoresis of optimization of lateral flow

immuno-PCR. The antigen and antibody-DNA conjugate reaction was 1 = step
by step addition or 2 = premixing. a = negative control , b = human IL6 sample

2.5
signal to noise ratio

2
1.5
1
0.5
0
sequential

Pre-mixed

Type of reaction between antibody and antigen

Figure 4-4 Optimization of sample pre-treatment. The intensity was presented

in signal to noise ratio

52

The high background at the step-by-step method was believed caused

from a low capillary force. After addition of antigen onto the membrane, the
membrane became saturated with solution. Next addition of antibody-DNA
conjugate therefore flew slower by a lower capillary force. A low capillary force
made migration of antibody-DNA conjugate slow, such that the antibody-DNA
conjugate was trapped on the membrane to cause a high background.

Premixing method ensured the reaction between antigen and antibody

and also prevented from the reaction create stoichiometry variables. Since
molecules were not trapped on membrane, an additional wash to remove excess
reagent also can be avoided.

The second variable to be optimized to minimize background was the test

waiting time. The waiting time was set in 2 min, 5 min, and 7 min. After the
waiting time, the membrane was washed by the lateral flow wash buffer to
remove the excess reagent that could cause background. The membrane was then
cut around the test line and mixed with PCR cocktail and subjected to the PCR.
Sample and background signals were determined on a 2% agarose gel
electrophoresis as the previous optimization. The result was shown in
Figure 4-5 and Figure 4-6.

53

1a

1b

2a

2b

3a

3b

Figure 4-5 Optimization of waiting time. The immuno-PCR products were run
on a 2% gel electrophoresis. The waiting time set at (1) 7 min, (2) 5 min and (3) 2
min. Signals from (a) sample and (b) background were compared with each other.

signal to noise ratio

2.5
2
1.5
1
0.5
0
2 min
o

5 min
Waiting time

7 min

Figure 4-6 Optimization of test waiting time. Analyis of 2% agarose gel

electrophoresis of immuno-PCR products. The intensity was reported as signal to
noise ratio. The highest value of signal to noise ratio was further chosen for the
next experiment.

54

The graphic of signal to noise ratio showed that 5 min waiting time was
the best choice for the next experiment. It should give enough time for antibody
and antigen to reach the stoichiometry for reaction. In other case, 2 minute waiting
time was not long enough for antigen and antibody to reach the stoichiometry for
reaction, such that when a wash was applied, the capture antibody was not strong
enough to bind with the antigen and therefore washed away. Two minutes were
also possibly not long enough to allow all samples to flow through the test line
and thus the residual samples were carried by the wash buffer to cause a
background. For the case of 7-minute waiting time, its sample signal was lower
than the 5-minute one, but its background signal was also higher. The higher
background was possibly caused by unwashed antibody-DNA conjugate on
nitrocellulose membrane. The antibody-DNA conjugate already bound strongly to
the membrane for a long waiting time, such that it couldnt be removed when the
wash was applied.

To obtain the best assay response and sensitivity, the spraying

concentration of capture antibody was optimized in this experiment. An optimal
spraying concentration supposedly offered a highest signal to noise ratio and also
saved raw material. The result of this test was presented in Figure 4-7 and Figure
4-8.

55

a
b
1 mg/ml

b
a b
b
a
a
0.5 mg/ml 100 g/ml 10 g/ml

Figure 4-7 Optimization of spraying concentration of capture antibody. (a)

sample signal. (b) background

2.5
signal to noise ratio

2
1.5
1
0.5
0
0.01 mg/ml

0.1 mg/ml

0.5 mg/ml

1 mg/ml

mass of capture antibody

Figure 4-8 Optimization of spraying concentration of the capture antibody.

The result in figure 4-7 was shown in signal to noise. 0.5 mg/ml obviously gave a
better result.

56

Figure 4-7 and Figure 4-8 optimized the spraying concentration of

capture antibody. The figure showed that the 0.5 mg/ml of capture antibody gave
the highest ratio. Concentrations higher or lower than 0.5 mg/ml didnt give a
better result, possibly due to the labeling efficiency of antigen to the immobilized
antibody and steric constraints of capture antibody. As the antibody concentration
was lower than the optimal one, the antibody/antigen interaction likely decreased
and resulted in the decreasing of the ratio. Furthermore, decreasing in the
concentration of the capture antibody also increased the empty spaces that can be
occupied by the reporter DNA-conjugated antibody. That was why the
background increased as the concentration of the capture antibody increased. For
the concentrations of the capture antibody above the optimal concentration, the
steric constraint of capture antibody was the main reason for ratio decreasing. Due
to random orientation of the capture antibody, overcrowding antibody in the
capture line will result in the decreasing of antibody/antigen contacts. The
background of 1 mg/ml obviously didnt contribute to the diminishment of the
ratio, because it was very low already.

57

4.3

Sensitivity test of lateral-flow immuno-PCR for human IL-6

The lateral-flow immuno-PCR was tested for human IL-6 detection

sensitivity. The conditions previously optimized were applied in this test. A serial
of ten fold dilution from 10 pg/ml to 0.001 pg/ml of human IL-6 was detected by
the membrane-based immuno-PCR methodologies. The result of the sensitivity
test was presented in Figure 4-9 and Figure 4-10. From the result of gel
electrophoresis analysis, membrane-based immuno-PCR was showed to be able to
detect <0.001 pg/ml of human IL-6. The signal of membrane-based immuno-PCR
in this research was not linear. The critical point of this technique was on how to
cut the membrane correctly. The dimension of the membrane from each antigen
concentration should be cut in the uniform dimension. High variability in the
dimension will result in non linear signal.

58

Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human
IL-6. The lateral-flow immuno-PCR was able to detect 0.001 pg/ml of humanIL6. M = marker, 1-5) Human IL-6 with ten fold serial dilution from 10 pg/ml to
0.001 pg/ml, 6) = negative control,

Final gel read outs

3000
2500
2000
1500
1000
500
0
0

0.001

0.01

0.1

10

Original concentration of Human IL-6 (pg/ml)

Figure 4-10 The sensitivity test of the lateral-flow immuno-PCR on human
IL-6. membrane-based immuno-PCR was able to detect as low as 0.001 pg/ml
human IL-6

59

Chapter 5. Conclusion
A membrane-based immuno-PCR was developed in this study to increase
the sensitivity of the lateral flow immunochromatography assay. The method was
similar with the conventional lateral flow immunochromatography assay. But
instead the gold nanoparticles-conjugated antibody, this study employed a DNAconjugated antibody as the signal reporter. By the traditional PCR procedure, the
reporter DNA on membrane was amplified to improve the detection sensitivity.
The optimal condition for sample preparation was premixing antigen and
DNA-conjugated antibody and waiting for five minutes after sample application.
Premixing ensured the reaction between antigen and antibody and also prevented
the reaction from creating variable stoichiometry. Five minutes gave enough time
for antibody to react with antigen but did not trap the DNA-antibody in the pore
of the membrane. The concentration of receptor antibody was optimized as 0.5
mg/ml to achieve the highest signal to noise ratio. In addition, the membranebased immuno-PCR can detect <0.001 pg/ml of human IL-6. Further development
on the reagent and method is necessary to reduce the background and to increase
the linearity of the signal.

60

Reference
[1]

Ravetch J and Bolland S (2001). IgG Fc receptors. Annu Rev Immunol 19:
275290.

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Appendix
Table Appendix 1 The Result of ImageJ measurement on the electrophoresis
band of antibody-antigen interaction for Figure 4-4.

Premixed
Step-by-step

Sample intensity

Background intensity

Signal to Noise ratio

12969.66
13692.34

5597.024

2.317242
1.247065

10979.65

Table Appendix 2 The Result of ImageJ measurement on the electrophoresis

band of waiting time optimization for Figure 4-6.
Waiting time

Sample intensity

Background intensity

Signal to Noise ratio

2 min

5521.225

4315.773

5 min

7503.125

3222.468

1.279313
2.328378

7 min

4453.225

2843.276

1.56623

66

Table Appendix 3 The Result of ImageJ measurement on the electrophoresis

band of the spraying concentration of capture antibody for Figure 4-8.
Concentration
of capture

Sample intensity

Background intensity

Signal to Noise ratio

1 mg/ml

3975.912

2447.912

1.624205

0.5 mg/ml

6583.912

3154.397

2.087217

0.1 mg/ml

4789.962

3722.79

1.286659

0.01 mg/ml

5537.175

4015.347

1.379003

antibody

Table Appendix 4 The Result of ImageJ measurement on the electrophoresis

band of the sensitivity test of the lateral-flow immuno-PCR for Figure 4-10.
Concentration
of human IL-6

Intensity

10 pg/ml

2441.397

1 pg/ml

1539.548

0.1 pg/ml

1534.598

0.01 pg/ml

2166.426

0.001 pg/ml

1749.669

0 pg/ml

962.012

67