Chap. 6 – Antigen-Antibody interactions Characterized as

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

• Each bond is weak; many are strong

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

• 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

• 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:

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:

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

Direct and indirect Immunofluorescence