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Chap. 6 Antigen-Antibody interactions
Characterized as:
Non-covalent interaction (similar to lock and key fit of
enzyme-substrate)
Does not lead to irreversible alteration of Ag or Ab
This exact and specific interaction has led to many
immunological assays used to:
detect Ag orAb
diagnose disease
measure magnitude ofhumoral IR
identify molecules ofbio and med interest
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Ag-Ab interactions
Bonds:
Hydrogen
Ionic
Hydrophobic interactions
Van der Waals forces
Each bond is weak; many arestrong
To hold they must be close requiring high amts of
complementarity!
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Measuring affinity of Ab to Ag
Assoc between CDR and monovalent Ag can be expressed
as:
Ag + Ab Ag-Ab;
k1 = forward (assoc) rate constant whereby k1/k-1 = Kak-1 = reverse (dissoc) rate constant the assoc/equilibrium
constantKa = [Ag-Ab] value of Ka depends on k1;
[Ag] [Ab] for small haptens, k1 is high
for large protein Ags, k1 is lower
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Ka determined by
equilibrium dialysis
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Cross-reactivity
Sometimes, Ab can cross-react with unrelated Ag.
(can occur if Ags share an identical/similar epitope)
Often seen with polysaccharide Ags
e.g. ABO Blood groups glycoproteins-persons lacking one or both of the blood (AB) Ags will
have serum Abs vs.the missing Ags
-these Abs produced from cross-reactive MO Ags!!
-provides basis forblood typing tests-necessitates compatible blood types during transfusions, etc.
OtherMO cross-reactions: 1)Streptococcus pyogenes
2) Vaccinia virus
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Immunologic tests:
1. Precipitation Rxns:
-Abs and Ags in aqueous solns form a lattice => Precipitin
Lattice formation requires: 1) polyvalent Abs
2) Ag must be bivalent, polyvalent
Precipitation rxns, once popular, have been replaced by faster, more sensitive tests
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Immunologic tests:
Precipitation rxns in gels
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Immunologic tests:
2. Immunoelectrophoresis: Incorp electrophoresisw/ double diffusion
An Ag mixture is 1st separated by charge
Then, troughs are cut to direction of elec fieldand antisera is added to trough
Ags and Abs diffuse towards each other toproduce precipitin bands
Used to detect: a)presence/absence of specificproteins or Ig classes
b) immunodeficiency or immunoproliferative disorder
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Immunologic tests:
Immunoelectrophoresis:
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Immunologic tests:
3) Agglutination reactions simple,
inexpensive, but sensitive!
Several types exist:
a) Hemagglutination of RBCs
b) Bacterial Agglutination
c) Passive Agglutination
d) Agglutination Inhibition
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Immunologic tests:
4) Radioimmunoassay (RIA) very sensitive test; usedfor measuring hormones, serum proteins, drugs, etc.
at low [C]s ( 0.001ug/ml)
measures competitive binding of radiolabelled Ag +
unlabelled (test) Ag to high affinity Ab
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Immunologic tests:
5. EL
ISA tests: dep on enzyme conugated to2
Abreacting with a specific substrate to produce a color
rxn. Most sensitive of tests for Ag/Ab!!
Variations of ELISAs:
Allows for qualitative or quantitative testing.Each one can be used for qualitative detection of Ag or Ab
Also, a std curvebased on known [C]s of Ag/Ab can be
prepped and an unknown [C} can be ascertained
a. Indirect ELISA
b. Sandwich ELISA
c. Competitive ELISA
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Immunologic tests: Types of ELISAs
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Immunologic tests:
6) Western Blot
Used to id specific
proteins in mixtures
Proteins are separated on
SDS-PAGE Proteins then transferred
to membrane
Membrane flooded w/
radio-labelled or enz-linked poly/monoclonal
Abs specific for protein
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Immunologic tests: 7) Immunoprecipitation
Provides a quick and
sensitive test for finding
proteins/Ags
Especially in low [C]s
Binds Ab to synthetic
bead support
centrifuged
Or2 Ab w/ bead or
magnetic bead -> collect
by magnetism
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Immunologic tests: 8) Immunofluorescence
Provides a quick method for the id of
pathogens and lymphocytes
Abs are conjugated with a fluorescent dye(fluorescein, rhodamine, phycoerythrin)
If Abs bind to specific Ags, they can be illum
w/ UV light and emit bright colors
There are currently 2 methods employed:
Direct staining
Indirect staining
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Direct and indirect
Immunofluorescence