Principles of immunodetection

by

Martin Loignon Ph.D.Lady Davis Institute for Cancer Research

Jewish General Hospital

• Antibody-based methods allowing the specific: – Detection– Quantification– Localisation

• Of antigens by means of antibody binding

Immunodetection

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 Placentatransfert

Activatescomplement

Additional features

IgMNo Yes First Ab in development and response

IgDNo No B-cell receptor

IgGYes Yes Involved in opsonization and ADCC.

Four subclasses; IgG1, IgG2, IgG3,IgG4

IgENo No Involved in allergic responses

IgANo 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 curveSTD 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

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

Variable Meaning Comment

X Time Usually expressed inunits of sec. or min.

Y Total binding Usually expressed inunits of cpm, mol/mg,sites/cell

Span Differencebetween bindingat time zero andplateau

Specific binding(same units as Y)

Plateau Binding thatdoesn't dissociate

Nonspecific binding(same units as Y).

K Dissociation rateconstant

Expressed In units ofinverse time (inverseof units of X-axis)

T1/2 Half-life 0.69302/k

• 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.)

Sigmoidal dose response curve

10%

90%

• Ligand receptor interaction– Growth factors

– Hormones

• Antibody antigen interaction – RIA, ELISA

• Activity of chemotherapeutics

• Enzymatic activators/inhibitors

Doses response curves

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

pH

Mol

ecu

lar

wei

ght

kD

a

1st dimension 2nd dimension

Stable

pH gradient

Immunoprecipitation

ProteomicsWestern Blotting

Immunohistochemistry

• 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

• Phospho Ser, Thr, Tyr

• Sequence specific (-Ser18 p53)

Antibodies against other post- translational modifications

• Ubiquitination

• Sumoylation

• Acetylation

• Methylation

• Geranylation

• Etc...

• Specific DNA damage (CPD, 6-4PP)

• Sugars

• Lipids

• Vitamins (vit D)

• Iodine

Antibodies against non-protein antigens

• Identification of signaling pathways– Protein modifications

– Signaling partners

• Activity of drugs (lead compounds)

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

– New antibodies

Research requiring immunoanalyses

Cytoskeleton Translati

on

ERK5

ELK1/

TCF

MEF2A-C

ATF2 NFAT4, NFAT

c1

MAX CHOP/ GADD1

53

Transcription Factors

c-jun

SAPKs

Inhibits nuclear

translocation

Effector Kinases

MAPKAP-K2/3

PRAK MSK1/2 MNK1/2 RSKs

p38s

HSP25/27CREB, Histone

H3, HMG14

eIF4E

Chromatin Remodelli

ng

ASK1

Tpl-2 MEKK2

MEKK3

MEKK1

RAF1

SEK1

MKK7

MKK3

MKK6

MEK5

MEK1/2

ERK1/2

MAP3Ks

MAPKs

MEKs

Inhibited by CSAIDS

(Cytokine-Suppressive

Anti-Inflammatory

Drugs)

eg SB203580

Synergize in SAPK

activation

p53

PP2B/Calcineurin

MKP1

CDC25B

CDC2WIP1

Pac1

Pac1

MKP5

MKP4

MKP2

MKP3M3/

6

(Hematopoietic only)

Inhibited by PD98059 (MEK2)

c-Abl

Rac1

dsDNA breaks

Inflammatory cytokines

ATM

MEKK4

TAK1

TAOs

MLKs

UV, MMS

Pyk2 Lyn

SHPTP1

Cdc42Hs

Kinases and signal transduction

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

Gene of interest

Fluoresentproteins

CFP

GFP

YFP

RFP

-FP Ab Direct visualisation

TAGS

His

Myc

Flag

Strep

GST

Affinity -Tag Ab

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

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, -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- – 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

Sensors

Transducers

Cytoplasmic/Nuclear effectors

Chromatin StructureTranscription

DNA repairCell cycle checkpoints

Apoptosis

Drug resistance arisingfrom sensor/transducer

defects

Drug resistance arisingfrom effector defects

DNA Damage

Drug resistance arisingfrom altered drug delivery to target

Physiological roles 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 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