1 / 27

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.

daryl-lara
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related