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
IgM
IgD
IgG
IgE
IgA
Structure
Placenta
transfert
Activates
Additional features
complement
No
Yes
First Ab in development and response
No
No
B-cell receptor
Yes
Yes
Involved in opsonization and ADCC.
Four subclasses; IgG1, IgG2, IgG3,
IgG4
No
No
Involved in allergic responses
No
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
Variable Meaning
Comment
X
Time
Y
Total binding
Span
Difference
between binding
at time zero and
plateau
Usually expressed in
units of sec. or min.
Usually expressed in
units of cpm, mol/mg,
sites/cell
Specific binding
(same units as Y)
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
• 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.)
90%
10%
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
Stable
pH gradient
2nd dimension
Molecular weight kDa
1st dimension
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 posttranslational 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
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
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
TAGS
Gene of interest
GST
Fluoresent
proteins
CFP
His
GFP
Myc
YFP
Strep
RFP
Flag
Affinity
a-Tag Ab
a-FP Ab Direct visualisation
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
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
Physiological roles of antibodies
• Protect against
– Viral infections
– Bacterial infections
– Foreign bodies
• Antigens
• Deleterious in
– Autoimmune diseases
• Reumathoid arthritis
• Type 1 diabetes
Lupus
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