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Principles of Immunology Antigen-Antibody Interactions 4/25/06

Principles of Immunology Antigen-Antibody Interactions 4/25/06. Word/Terms List. Agglutinin EIA Equivalence zone FIA Immunodiffusion Immunoelectrophoresis RIA Titer. Low Affinity. High Affinity. Ab. Ab. Ag. Ag. Affinity.

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Principles of Immunology Antigen-Antibody Interactions 4/25/06

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  1. Principles of ImmunologyAntigen-Antibody Interactions4/25/06

  2. Word/Terms List • Agglutinin • EIA • Equivalence zone • FIA • Immunodiffusion • Immunoelectrophoresis • RIA • Titer

  3. Low Affinity High Affinity Ab Ab Ag Ag Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site Affinity =  attractive and repulsive forces

  4. Specificity • The ability of an individual antibody combining site to react with only one antigenic determinant. • The ability of a population of antibody molecules to react with only one antigen.

  5. Cross reactions Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag

  6. Ab excess Ag excess Equivalence – Lattice formation Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag

  7. Tests Based on Ag/Ab Reactions • All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes • All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

  8. Agglutination Tests Lattice Formation

  9. Qualitative agglutination test • Ag or Ab Y +  Y Y Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

  10. 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8 Agglutination/Hemagglutination • Quantitative agglutination test • Titer • Prozone

  11. 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Applications • Blood typing • Bacterial infections • Fourfold rise in titer • Practical considerations • Easy • Semi-quantitative

  12. Y Y +  Y Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of antibodies to soluble antigens

  13. Prior to Test Y Y +  Y Y Test Y + +  Y Patient’s sample Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

  14. Agglutination/Hemagglutination Inhibition • Applications • Measurement of soluble Ag • Practical considerations • Same as agglutination test • Definition

  15. Precipitation Tests Lattice Formation

  16. Ab in gel Ag Ag Ag Ag Diameter2 Ag Concentration Radial Immunodiffusion • Method • Ab in gel • Ag in a well • Interpretation • Diameter of ring is proportional to the concentration • Quantitative • Ig levels

  17. - + Ag Ag Ab Ag Ab Immunoelectrophoresis • Method • Ags are separated by electrophoresis • Interpretation • Precipitin arc represent individual antigens

  18. Immunoelectrophoresis • Method • Interpretation • Qualitative • Relative concentration

  19. Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (EIA) Lattice formation not required

  20. Prior to Test Y Y +  Labeled Ag Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method • Determine amount of Ab needed to bind to a known amount of labeled Ag • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

  21. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ab detection • Immobilize Ag • Incubate with sample • Add labeled anti-Ig • Amount of labeled Ab bound is proportional to amount of Ab in the sample

  22. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ag detection • Immobilize Ab • Incubate with sample • Add labeled antibody • Amount of labeled Ab bound is proportional to the amount of Ag in the sample

  23. Tests for Cell Associated Antigens Lattice formation not required

  24. Fluorochrome Labeled Ab Y Ag Tissue Section Immunofluorescence • Direct • Ab to tissue Ag is labeled with fluorochrome

  25. Fluorochrome Labeled Anti-Ig Unlabeled Ab Y Y Ag Tissue Section Immunofluorescence • Indirect • Ab to tissue Ag is unlabeled • Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi-Quantitative

  26. Assays Based on Complement Lattice formation not required

  27. No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y Complement Fixation • Ag mixed with test serum to be assayed for Ab • Standard amount of complement is added • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag

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