principles of immunodetection 2

7/31/2019 Principles of Immunodetection 2

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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 analyticaltechniques based on this interaction

• Examples of clinical application

• Research problems requiringimmunoanalyses

• 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

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 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

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 in

units of sec. or min.

Y Total binding Usually expressed in

units of cpm, mol/mg,

sites/cellSpan Difference

between binding

at time zero and

plateau

Specific binding

(same units as Y)

Plateau Binding that

doesn't dissociate

Nonspecific binding

(same units as Y).

K Dissociation rateconstant

Expressed In units of inverse time (inverse

of 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 logarithmof 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

M o l e c u l a r w e i g h t k D a

1st dimension 2nd dimension

Stable

pH gradient



Immunoprecipitation

Proteomics

Western 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



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 yetpowerful 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

Fluoresent

proteins

CFP

GFP

YFP

RFP

a-FP Ab Direct visualisation

TAGS

His

Myc

Flag

Strep

GST

Affinity a-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



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

Sensors

Transducers

Cytoplasmic/Nuclear effectors

ChromatinStructureTranscription

DNA repairCell cyclecheckpoints

Apoptosis

Drug resistance arisingfrom sensor/transducer

defects

Drug resistance arisingfrom effector defects

DNA Damage

Drug resistance arisingfrom altered drugdelivery 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 islimiting the progression of your

research!



Expression librairies

principles of immunodetection 2

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