Chap. 6 – Antigen-Antibody interactions Characterized as



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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 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



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