Principles of immunodetection

1. Principles of
immunodetection

2. Aims and Objectives
•Basis of antigen-antibody interaction
•Conceptualise the different techniques
based on this interaction
•Examples of clinical application
•Research problems requiring
immunoanalyses

3. Role 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

4. 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

5. 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

6. 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)

7. Antigen-antibody binding

8. Structure of an antibody

9. 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

10. Generation of an antibody:
antigen processing

11. B cell activation

12. Antibody and VDJ recombination

13. 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

14. 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

15. Antigen-antibody interaction:
concentration dependence
Concentration of unknown samples are determined from a standard curve

16. •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.)
Sigmoidal dose response curve
10%
90%

17. •Antibody antigen interaction
–RIA, ELISA
–Ligand receptor interaction
–Growth factors
–Hormones
•Activity of chemotherapeutics
•Enzymatic inhibitors
Doses response curves

18. Cross reactivity

19. One and two sites competition

20. Detection principles
•Radiolabelled isotopes
–
125
I,
14
C,
32
P,
35
S
•Enzymes
–Peroxydase
•Chromophores
–Fluorogenic probes, fluorescent proteins

21. Peroxydase reaction

22. RIA: radio immuno assay

23. RIA interference

24. Elisa: Enzyme-linked immunosorbent assay

25. Sandwich Elisa

26. Western blotting

27. Two dimensional electrophoresis
pH
Molecular weight kDa

1st dimension 2nd dimension

28. Immunoprecipitation
Western Blotting

29. Immunohistochemistry

30. 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)

31. 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)

32. •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

33. DNA damage inducible cascades

34. Phosphospecific detections

35. Cytoskelet
on
Translati
on
ERK
5
ELK
1/T
CF
MEF2
A-C
ATF2 NFAT4
, NFAT
c1
MAX CHOP/
GADD1
53
Transcription Factors
c-jun
SAPK
s
Inhibits
nuclear
transloca
tion
Effector
Kinases
MAPKAP-
K2/3
PRAK MSK1/2 MNK1/2 RSKs
p38
s
HSP25/27
CREB, Histone
H3, HMG14
eIF4E
Chromatin
Remodelli
ng
ASK
1
Tpl-2 MEK
K2
MEK
K3
MEKK
1
RAF
1
SEK
1
MK
K7
a
MK
K3
a
MK
K6
ME
K5
MEK
1/2
ERK1/
2
MAP3Ks
MAPKs
MEKs
a

Inhibited by
CSAIDS
(Cytokine-
Suppressive
Anti-
Inflammatory
Drugs)
eg SB203580
Synergize in
SAPK
activation
p53
PP2B/
Calcineurin
MK
P1
CDC2
5B
CDC2 WIP
1
Pac
1
Pac
1
MK
P5
MK
P4
MK
P2
MK
P3
M3/
6
(Hematopoi
etic only)
Inhibited by
PD98059
(MEK2)
c-Abl
Rac1
dsDNA
breaks
Inflammator
y cytokines
ATM
MEK
K4
TAK
1
TAO
s
MLK
s
UV,
MMS
Pyk2 Ly
n
SHPT
P1
Cdc42
Hs
Kinases and signal transduction

36. FRET:
Fluorescence resonance energy transfer

37. 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

38. cDNA librairies

39. Expression librairies

40. Phage display

41. 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

42. 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)

43. Concentration of serum peptides
•Blood levels of:
–Hormones
–Antibodies
–Enzymes
–Metabolites

44. •Identification of signaling pathways
–Protein modifications
–Signaling partners
•Activity of drugs (lead compounds)
•Lack of specific molecules
–Specific ligands (side effects)
–New antibodies


Research problems requiring
immunoanalyses

45. The problems of chemotherapy
Chemotherapy/
radiotherapy
Sensors
Transducers
Cytoplasmic/Nuclear effectors
Chromatin
Structure
Transcription
DNA repair
Cell cycle
checkpoints
Apoptosis
Drug resistance arising
from sensor/transducer
defects
Drug resistance arising
from effector defects
DNA Damage
Drug resistance arising
from altered drug
delivery to target