Principles of

immunodetection

Aims and Objectives

• Basis of antigen-antibody interaction

• Conceptualise the different techniques

based on this interaction

• Examples of clinical application

• Research problems requiring

immunoanalyses

Role of antibodies

• Protect against – Viral infections

– Bacterial infections

– Foreign bodies

• Antigens

• Deleterious in – Autoimmune diseases

• Reumathoid arthritis Lupus

• Type 1 diabetes Croh’n disease

– Graft rejection

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

– 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

Structure of an antibody

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

Generation of an antibody:

antigen processing

B cell activation

Antibody and VDJ recombination

Generation 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

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

Antigen-antibody interaction:

concentration dependence

Concentration of unknown samples are determined from a standard 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%)

• IC50 (inhibitory conc.)

Sigmoidal dose response curve

10%

90%

• Antibody antigen interaction

– RIA, ELISA

– Ligand receptor interaction

– Growth factors

– Hormones

• Activity of chemotherapeutics

• Enzymatic inhibitors

Doses response curves

Cross reactivity

One and two sites competition

Detection principles

• Radiolabelled isotopes

– 125I, 14C, 32P, 35S

• Enzymes

– Peroxydase

• Chromophores

– Fluorogenic probes, fluorescent proteins

Peroxydase reaction

RIA: radio immuno assay

RIA interference

Elisa: Enzyme-linked immunosorbent assay

Sandwich Elisa

Western blotting

Two dimensional electrophoresis

pH

Mo

lecu

lar

wei

gh

t k

Da

1st dimension 2nd dimension

Immunoprecipitation

Western Blotting

Immunohistochemistry

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

• Experimental

– Drug screening (phage display)

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)

• 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

Phosphospecific antibodies to study

cellular signaling

DNA damage inducible cascades

Phosphospecific detections

Cytoskelet

on Translati

on

ERK

5

ELK

1/T

CF

MEF2

A-C

ATF2 NFAT4

, NFAT

c1

MAX CHOP/

GADD1

53

Transcription Factors

c-jun

SAPK

s

Inhibits

nuclear

transloca

tion

Effector

Kinases

MAPKAP-

K2/3

PRAK MSK1/2 MNK1/2 RSKs

p38

s

HSP25/27 CREB, Histone

H3, HMG14

eIF4E

Chromatin

Remodelli

ng

ASK

1

Tpl-2 MEK

K2

MEK

K3

MEKK

1

RAF

1

SEK

1

MK

K7 a

MK

K3 a

MK

K6

ME

K5

MEK

1/2

ERK1/

2

MAP3Ks

MAPKs

MEKs

a

Inhibited by

CSAIDS

(Cytokine-

Suppressive

Anti-

Inflammatory

Drugs)

eg SB203580

Synergize in

SAPK

activation

p53

PP2B/

Calcineurin

MK

P1

CDC2

5B

CDC2 WIP

1

Pac

1

Pac

1

MK

P5

MK

P4

MK

P2

MK

P3 M3/

6

(Hematopoi

etic only)

Inhibited by

PD98059

(MEK2)

c-Abl

Rac1

dsDNA

breaks

Inflammator

y cytokines

ATM

MEK

K4

TAK

1

TAO

s

MLK

s

UV,

MMS

Pyk2 Ly

n

SHPT

P1

Cdc42

Hs

Kinases and signal transduction

FRET: Fluorescence resonance energy transfer

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

Localization of CEBP by FRET

cDNA librairies

Expression librairies

Phage display

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

Clinical 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

• Identification of signaling pathways – Protein modifications

– Signaling partners

• Activity of drugs (lead compounds)

• Lack of specific molecules – Specific ligands (side effects)

– New antibodies

Research problems requiring

immunoanalyses

The problems of chemotherapy

Chemotherapy/

radiotherapy

Sensors

Transducers

Cytoplasmic/Nuclear effectors

Chromatin

Structure

Transcription

DNA repair

Cell cycle

checkpoints Apoptosis

Drug resistance arising

from sensor/transducer

defects

Drug resistance arising

from effector defects

DNA Damage

Drug resistance arising

from altered drug

delivery to target