Principles of immunodetection

by Martin Loignon Ph.D.

Lady Davis Institute for Cancer Research Jewish General Hospital

Immunodetection
Antibody-based methods allowing the specific:
Detection Quantification Localisation

Of antigens by means of antibody binding

Aims and Objectives

Basis of antibody production and antigen interaction Conceptualise the different analytical techniques based on this interaction Examples of clinical application Research problems requiring immunoanalyses Troubleshooting of some common problems

Discovery of antibodies

1899 *Jules Bordet, Complement and antibody activity in bacteriolysis

1900 *Paul Erlich, Antibody formation theory 1926 Lloyd Felton & GH Bailey, Isolation of pure antibody preparation 1934-8 John Marrack, Antigen-antibody binding hypothesis

1941 Albert Coons, Immunofluorescence technique

1948 Astrid Fagraeus, Demonstration of antibody production in plasma B cells 1959-62 *Rodney Porter et al., Discovery of antibody structure 1963 Jaques Oudin et al., antibody idiotypes 1964-8 Anthony Davis et al., T and B cell cooperation in immune response 1965 Thomas Tomasi et al., Secretory immunoglobulin antibodies 1975 *Kohler and Milstein, Monoclonal antibodies used in genetic analysis 1985 *Tonegawa, Hood et al., Identification of immunoglobulin genes

Generation of an antibody: antigen processing

B cell activation

Structure of an antibody

Antibody and VDJ recombination

Classes of antibodies
Isotype Structure Placenta transfert
No

Activates Additional features complement

Yes First Ab in development and response

IgM

IgD

No

No

B-cell receptor

IgG

Yes

Yes

Involved in opsonization and ADCC. Four subclasses; IgG1, IgG2, IgG3, IgG4

IgE

No

No

Involved in allergic responses

IgA

No

No

Two subclasses; IgA1, IgA2. Also found as dimer (sIgA) in secretions.

Commercial production of antibodies: polyclonal vs monoclonal

Host animals ca be used to raise antibodies against a given antigen Slected clones from a polyclonal each recognizing a single epitope can be fused to a tumor cell (hybridoma) to proliferate indefinitely

Antigen-antibody interaction
Antigen: foreign molecules that generate antibodies or any substance that can be bound specifically by an antibody molecule
Proteins, sugars, lipids or nucleic acids Natural or synthetic

Antibody: molecules (protein) responsible for specific recognition and elimination (neutralization) of antigens
Different structures (7-8 classes in mammals) Powefull research tools for basic research, clinical applications and drug design

Antigenic determinants
An antibody will recognize
Epitope: defined segment of an antigen Immunoreactivity of epitopes may depend on primary, secondary, tertiary or quaternary structure of an antigen Define the possible applications Variability of epitopes depends on the species

Antibodies are antigen themselves

Nature of binding forces

Hydrogen bonding
Results from the formation of hydrogen bridges between appropriate atoms

Electrostatic forces
Are due to the attraction of oppositely charged groups located on two protein side chains

Van der Waals bonds

Are generated by the interaction between electron clouds (oscillating dipoles)

Hydrophobic bonds
Rely upon the association of non-polar, hydrophobic groups so that contact with water molecules is minimized (may contribute up to half the total strength of the antigen-antibody bond)

Antigen-antibody binding

Antigen-antibody affinity

The affinity with which antibody binds antigen results from a balance
between the attractive and repulsive forces. A high affinity antibody implies a good fit and conversely, a low affinity antibody implies a poor fit and a lower affinity constant

Antigen-antibody interaction: concentration dependence

Concentration of unknown samples are determined from a standard curve STD concentration values are obtained when the interaction between

Non specific binding

Saturation radioligand binding experiments measure specific radioligand binding at equilibrium at various concentrations of the radioligand. These experiments are performed to determine receptor number and affinity on cells but also between radiolabeled antigen and Ab.

This can take anywhere from a few minutes to many hours, depending on the ligand, receptor, To, and other experimental conditions.
The lowest concentration of radioligand will take the longest to equilibrate. When testing equilibration time, therefore, use a low concentration of radioligand (perhaps 10-20% of the KD). Nonspecific binding is almost always a linear function of ligand concentration. The analyses depend on the assumption that you have allowed the incubation to proceed to equilibrium.

Dissociation off rate experiments

Variable Meaning
X Y Time Total binding

Comment
Usually expressed in units of sec. or min. Usually expressed in units of cpm, mol/mg, sites/cell Specific binding (same units as Y)

Span

Difference between binding at time zero and plateau

Plateau

Binding that Nonspecific binding doesn't dissociate (same units as Y). Dissociation rate Expressed In units of constant inverse time (inverse of units of X-axis) Half-life

T1/2

0.69302/k

Each ligand-receptor complex dissociates at a random time, so the amount of specific binding follows an exponential dissociation.

Sigmoidal dose response curve

General equation for a dose response curve It shows response as a function of the logarithm of concentration X is the logarithm of agonist concentration and Y is the response Log EC50 is the logarithm of the EC50 (effective concentration, 50% of maximal response) IC50 (inhibitory conc.)

90%

10%

Doses response curves

Ligand receptor interaction
Growth factors Hormones

Antibody antigen interaction

RIA, ELISA

Activity of chemotherapeutics Enzymatic activators/inhibitors

Cross reactivity

One and two sites competition

Laboratory use of antibodies

Quantitation of an antigen RIA, Elisa Identification and characterization of protein antigens Immunoprecipitation Western blotting Cell surface labelling and separation Localisation of antigens within tissues or cells Expression librairies Phage display

Detection principles
Radiolabelled isotopes (antigen)

125I, 32P, 35S

Enzymes (Ab)
Peroxydase

Chromophores (Ab)
Fluorogenic probes (UV, visible or IR)

Peroxydase reaction

RIA: radio immuno assay

Typical RIA standard curve

RIA interference

Elisa: Enzyme-linked immunosorbent assay

Sandwich Elisa

Western blotting

Two dimensional electrophoresis

1st dimension 2nd dimension

Stable pH gradient

Molecular weight kDa

pH

Immunoprecipitation

Proteomics Western Blotting

Immunohistochemistry

Phosphospecific antibodies to study cellular signaling

Phosphorylation and dephosphorylation affect the structure and activity of proteins Cellular signalling is characterized by cascades of phosphorylation Kinases and phosphatases maintain phosphorylated/dephosphorylated state of proteins Phospho/Tyrosine/Threonine/ Serine

DNA damage inducible cascades

Phosphospecific detections
Phospho Ser, Thr, Tyr Sequence specific (a-Ser18 p53)

Antibodies against other posttranslational modifications

Ubiquitination Sumoylation Acetylation Methylation Geranylation Etc...

Antibodies against non-protein antigens

Specific DNA damage (CPD, 6-4PP) Sugars Lipids Vitamins (vit D) Iodine

Research requiring immunoanalyses

Identification of signaling pathways
Protein modifications Signaling partners

Activity of drugs (lead compounds) Lack of specific molecules

Specific ligands (side effects) New antibodies

dsDNA breaks

UV, Inflammator MMS y cytokines Tpl-2 MEK K2

Kinases and signal transduction

MEK K3

ATM

SHPT P1

Cdc42 Hs

Rac1 TAK 1 MEK K4

c-Abl Pyk2

Ly n
MLK s

ASK 1

MAP3Ks
TAO s Inhibited by PD98059 (MEK2) Inhibited by CSAIDS (CytokineSuppressive AntiInflammatory Drugs) RAF 1

MEKK 1

SEK MK 1 K7 Synergize in SAPK activation SAPK s

MK K3 a M3/ 6 MK P1 MK P5 p38 s

MK K6 a

ME K5

MEK 1/2 MK P2 MK P4 MK P3

MEKs
MAPKs

Pac 1

ERK 5

ERK1/ Pac (Hematopoi 2 etic 1 only)

a
MAX CHOP/ GADD1 53 MEF2 A-C p53 ELK 1/T CF

eg SB203580
MAPKAPK2/3

PRAK

MSK1/2

MNK1/2

RSKs

c-jun

ATF2

NFAT4 , NFAT c1

CDC2 5B

Transcription Factors
HSP25/27 WIP PP2B/ CDC2 1 Calcineurin Inhibits nuclear transloca tion Cytoskelet on

Effector Kinases
eIF4E CREB, Histone H3, HMG14 Chromatin Remodelli ng

Translati on

Phage display

Bacteriophage structure

Production of recombinant phages

cDNA librairies

Phage display: Ab production

Originally developped to produce monoclonal antibodies, phage display is a simple yet powerful technology that is used to rapidly characterize protein-protein interactions from amongst billions of candidates. This widely practiced technique is used to map antibody epitopes, create vaccines and to engineer peptides, antibodies and other proteins as both diagnostic tools and as human therapeutics

Alternatives to specific antibodies

Fluoresent proteins
CFP GFP

TAGS
GST His Myc Strep Flag

Gene of interest

YFP
RFP

Affinity

a-Tag Ab

a-FP Ab Direct visualisation

FRET:
Fluorescence resonance energy transfer

Localization of CEBP by FRET

Localization of BFP- and RFP-C/EBP protein expressed in mouse 3T3 cells using 2p-FRET microscopy. The doubly expressed cells (BFP-RFP-C/EBP) were excited by 740 nm and the donor (A) and acceptor (B) images of proteins localized in the nucleus of a single living cell were acquired by single scan

Clinical use of antibodies

Diagnostic
Detection of peptides and other molecules in various diseases
Endocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidism Tumor antigens (p53 tumor suppressor, PSA, a-foetoprotein) Antibodies against viral proteins (AIDS, hepatitis)

Therapeutic
Neutralizing antibodies
Anti-ErbB2 for breast and ovarian cancer Anti-CD20 for B-cell non-Hodgkin's lymphoma Antisera and antidotes (viruses and venoms)

Drug discovery
Identification of therapeutic targets (phage display)

Therapeutic applications
Neutralizing antibodies
Antidotes and antivenin (snake & spider bites) Tumor antigens ErbB-2, melanoma and T-cell leukemia, antibodies coupled to toxins Autoimmune antibodies, cytokines TNF-a Antisera aigainst virus, bateria and toxins (vaccine) Anti IgE and IgM for allegies (experimental) Quantitation of blood peptides (hormones metabolites)

Activating antibodies
Complement activating for uncontrolled bleeding (hemophilia)

Concentration of serum peptides

Blood levels of:
Hormones Antibodies Enzymes Metabolites

Detection of HIV proteins by WB

gp160 viral envelope precursor (env) gp120 viral envelope protein (env) binds to CD4

p31 Reverse Transcriptase (pol) p24 viral core protein (gag)

Immunodiffusion

Zone of equivalence: formation of large complexes

The problems of chemotherapy

Chemotherapy/ radiotherapy DNA Damage Drug resistance arising from altered drug delivery to target

Sensors

Transducers

Drug resistance arising from sensor/transducer defects

Cytoplasmic/Nuclear effectors

Drug resistance arising from effector defects

Chromatin Structure Transcription

DNA repair Cell cycle checkpoints

Apoptosis

Physiological roles of antibodies

Protect against
Viral infections Bacterial infections Foreign bodies
Antigens

Deleterious in
Autoimmune diseases
Reumathoid arthritis Type 1 diabetes Lupus Crohn disease

Graft rejection and hypersensitivity responses

Health care perspectives

Ab against antigens could lead to diagnostic test or vaccine for several diseases
BSE (mad cow disease) or human variant Creutzfeldt Jakob.
Paramithiotis et al. A prion protein epitope selective for the pathologically misfolded conformation. Nat Med. 2003 Jul;9(7):893-9 Caprion Pharmaceuticals Inc., St-Laurent, Quebec, Canada.

Vaccine against HIV

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science. 2001 Aug 10;293(5532):1155-9.

SARS Nil virus

Antidotes

Lacking an antibody for your protein or antigen of interest is limiting the progression of your research!

Expression librairies