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 Activates Additional features
transfert complement

No Yes First Ab in development and response

IgM

No No B-cell receptor
IgD

Yes Yes Involved in opsonization and ADCC.

IgG Four subclasses; IgG1, IgG2, IgG3,
IgG4

No No Involved in allergic responses

IgE

No No Two subclasses; IgA1, IgA2. Also found

IgA as dimer (sIgA) in secretions.
Commercial production of antibodies:
polyclonal vs monoclonal
• Host animals ca be used to raise antibodies • Slected clones from a polyclonal each recognizing
against a given antigen 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 Comment

X Time Usually expressed in

units of sec. or min.
Y Total binding Usually expressed in
units of cpm, mol/mg,
sites/cell
Span Difference Specific binding
between binding (same units as Y)
at time zero and
plateau

Plateau Binding that Nonspecific binding

doesn't dissociate (same units as Y).

K Dissociation rate Expressed In units of

constant inverse time (inverse
of units of X-axis)

T1/2 Half-life 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 90%
• X is the logarithm of agonist
concentration and Y is the
response
• Log EC50 is the logarithm
10%
of the EC50 (effective
concentration, 50% of
maximal response)
• IC50 (inhibitory conc.)
 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
Molecular weight kDa
pH gradient

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 post-
translational 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 UV, Inflammator
Kinases and signal transduction
breaks MMS y cytokines
Tpl-2 MEK MEK
K2 K3
ATM Cdc42 Rac1
SHPT
Hs
P1
ASK TAK MEK
1 1 K4
c-Abl Pyk2 Ly
n MAP3Ks
RAF
MEKK MLK TAO
1
1 s s
Inhibited by
PD98059
SEK MK MK MK ME
(MEK2)
MEK
MEKs
1 K7 K3 K6 K5 Inhibited by 1/2
Synergize in a a CSAIDS MK
SAPK M3/ (Cytokine- P3
MK
activation 6 Suppressive
Anti- P2 MAPKs
Pac ERK1/ Pac
SAPK MK p38 ERK Inflammatory (Hematopoi
1 2 etic 1only)
s P1 s 5 Drugs) MK
P4
MK a eg SB203580
P5 

MAPKAP- PRAK MSK1/2 MNK1/2 RSKs

c-jun ATF2 NFAT4 MAX CHOP/ MEF2 p53 ELK K2/3
CDC2
, NFAT GADD1 A-C 1/T
c1 53 CF
5B Effector
Transcription Factors Kinases
HSP25/27 eIF4E
WIP PP2B/ CDC2 CREB, Histone
1 Calcineurin H3, HMG14
Inhibits
nuclear Chromatin
transloca Cytoskelet Remodelli
on Translati
tion ng
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
TAGS Gene of interest proteins
GST CFP

His GFP

Myc YFP

Strep RFP

Flag

a-FP Ab Direct visualisation

Affinity a-Tag Ab
 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 Drug resistance arising
from altered drug
DNA Damage delivery to target

Sensors
Drug resistance arising
from sensor/transducer
defects
Transducers

Drug resistance arising

Cytoplasmic/Nuclear effectors from effector defects

DNA repair
Chromatin
Cell cycle Apoptosis
Structure
checkpoints
Transcription
 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