Transcript Titre - Faculty of Medicine, McGill University

Principles of
immunodetection
by
Martin Loignon Ph.D.
Lady Davis Institute for Cancer Research
Jewish General Hospital
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
• Type 1 diabetes
– Graft rejection
Lupus
Croh’n disease
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
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%)
• IC50 (inhibitory conc.)
90%
10%
Doses response curves
• Antibody antigen interaction
– RIA, ELISA
– Ligand receptor interaction
– Growth factors
– Hormones
• Activity of chemotherapeutics
• Enzymatic inhibitors
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
2nd dimension
Molecular weight kDa
1st dimension
pH
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)
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
dsDNA
breaks
Kinases and signal transduction
UV, Inflammator
MMS y cytokines
Tpl-2
ATM
Cdc42
Hs
SHPT
P1
c-Abl Pyk2
MEKK
1
TAK
1
MEK
K4
MAP3Ks
MLK
s
RAF
1
TAO
s
SEK
MK
1
K7
Synergize in
SAPK
activation
SAPK
s
MK
K3
a
MK
K6
a
ATF2
Inhibited by
PD98059
(MEK2)
ME
K5
Inhibited by
CSAIDS
(CytokineSuppressive
AntiInflammatory
Drugs)
M3/
6
MK
P1
p38
s
Pac
1
ERK
5
a

MK
P5
c-jun
MEK
K3
Rac1
ASK
1
Ly
n
MEK
K2
NFAT4
, NFAT
c1
MAX CHOP/
GADD1
53
MEF2
A-C
p53
ELK
1/T
CF
eg SB203580
CDC2
5B
MAPKAPK2/3
MEKs
MEK
1/2
MK
P2
MK
P4
PRAK
MK
P3
MAPKs
ERK1/
Pac
(Hematopoi
2
etic 1only)
MSK1/2
MNK1/2
Effector
Kinases
Transcription Factors
HSP25/27
WIP
PP2B/ CDC2
1 Calcineurin
Inhibits
nuclear
transloca
tion
RSKs
eIF4E
CREB, Histone
H3, HMG14
Cytoskelet
on
Translati
on
Chromatin
Remodelli
ng
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
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
Research problems 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
The problems of chemotherapy
Chemotherapy/
radiotherapy
DNA Damage
Drug resistance arising
from altered drug
delivery to target
Sensors
Drug resistance arising
from sensor/transducer
defects
Transducers
Cytoplasmic/Nuclear effectors
Chromatin
Structure
Transcription
DNA repair
Cell cycle
checkpoints
Drug resistance arising
from effector defects
Apoptosis