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 Each bond is weak; many are strong complementarity!
Measuring affinity of Ab to Ag
Ka determined by equilibrium dialysis
Cross-reactivity
Immunologic tests:
Immunologic tests:
Immunologic tests: 2. Immunoelectrophoresis: Incorp electrophoresis w/ double diffusion An Ag mixture is 1st separated by charge 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
Immunologic tests:
Immunologic tests: 3) Agglutination reactions – simple, inexpensive, but sensitive! Several types exist: a) Hemagglutination of RBC’s b) Bacterial 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:
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 Binds Ab to synthetic bead support centrifuged Or 2° Ab w/ bead or magnetic bead -> collect by magnetism
Immunologic tests: 8) Immunofluorescence - 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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