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 or Ab
• diagnose disease
• measure magnitude of humoral IR
• identify molecules of bio and med interest

Ag-Ab interactions

Bonds:

• Hydrogen
• Ionic
• Hydrophobic interactions
• Van der Waals forces

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Each bond is weak; many are strong

To “hold” they must be close 🡪 requiring high amts of

complementarity!

Measuring affinity of Ab to Ag

Assoc between CDR and monovalent Ag can be expressed as:

Ag + Ab ⇆ Ag-Ab;

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k₁ = forward (assoc) rate constant whereby k₁/k_-1 = K_a

k_-1 = reverse (dissoc) rate constant the assoc/equilibrium constant

K_a = [Ag-Ab] value of K_a depends on k_1;

[Ag] [Ab] for small haptens, k₁ is high

for large protein Ag’s, k₁ is lower

K_a determined by� equilibrium dialysis

Cross-reactivity

Sometimes, Ab can “cross-react” with unrelated Ag….

(can occur if Ag’s share an identical/similar epitope)

Often seen with polysaccharide Ag’s

e.g. ABO Blood groups – glycoproteins

-persons lacking one or both of the blood (AB) Ag’s will have serum Ab’s vs.the missing Ag’s

-these Ab’s produced from cross-reactive MO Ag’s!!

-provides basis for blood typing tests

-necessitates compatible blood types during transfusions, etc.

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Other MO cross-reactions: 1) Streptococcus pyogenes

2) Vaccinia virus

Immunologic tests:

1. Precipitation Rxns:

-Ab’s and Ag’s in aqueous soln’s form a lattice => Precipitin Lattice formation requires: 1) polyvalent Ab’s

2) Ag must be bivalent, polyvalent

Precipitation rxns, once popular, have been replaced by faster, more sensitive tests

Immunologic tests:

Precipitation rxns in gels

Immunologic tests:

2. Immunoelectrophoresis: Incorp electrophoresis w/ double diffusion

• An Ag mixture is 1^st separated by charge
• Then, “troughs” are cut ∥to direction of elec field

and antisera is added to trough

• Ag’s and Ab’s diffuse towards each other to produce precipitin bands
• Used to detect: a)presence/absence of specific proteins or Ig classes

b) immunodeficiency or immunoproliferative disorder

Immunologic tests:

Immunoelectrophoresis:

Immunologic tests:

3) Agglutination reactions – simple, inexpensive, but sensitive!

Several types exist:

a) Hemagglutination of RBC’s

b) Bacterial Agglutination

c) Passive Agglutination

d) Agglutination Inhibition

Immunologic tests:

4) Radioimmunoassay (RIA)– very sensitive test; used for 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

Immunologic tests:

5. ELISA tests: dep on enzyme conugated to 2 Ab reacting with a specific substrate to produce a color rxn. Most sensitive of tests for Ag/Ab!!

Variations of ELISA’s:

Allows for qualitative or quantitative testing.

Each one can be used for qualitative detection of Ag or Ab

Also, a std curve based on known [C]’s of Ag/Ab can be prepped and an unknown [C} can be ascertained

1. Indirect ELISA
2. Sandwich ELISA
3. Competitive ELISA

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Immunologic tests: Types of ELISA’s…

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 Ab’s specific for protein

Immunologic tests: 7) Immunoprecipitation

• Provides a quick and sensitive test for finding proteins/Ag’s
• Especially in low [C]’s
• Binds Ab to synthetic bead support 🡪 centrifuged
• Or 2° Ab w/ bead or magnetic bead -> collect by magnetism

Immunologic tests: 8) Immunofluorescence

• Provides a quick method for the id of pathogens and lymphocytes
• Ab’s are conjugated with a fluorescent dye (fluorescein, rhodamine, phycoerythrin)
• If Ab’s bind to specific Ag’s, 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