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Antigen-Antibody Reactions Lou Justement lbjust@uab.edu

Antigen-Antibody Reactions Lou Justement lbjust@uab.edu. Features Antibody-based assays. Antigen-antibody Interaction. Heavy Chain. Lysozyme. Antigenic Determinant Epitope. Light Chain. Features of the Antigen-Antibody Interaction. Reversible Non-covalent Interactions. ;. ;.

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Antigen-Antibody Reactions Lou Justement lbjust@uab.edu

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  1. Antigen-Antibody ReactionsLou Justement lbjust@uab.edu • Features • Antibody-based assays

  2. Antigen-antibody Interaction Heavy Chain Lysozyme Antigenic Determinant Epitope Light Chain

  3. Features of the Antigen-Antibody Interaction • Reversible • Non-covalent Interactions

  4. ; ; Non-covalent binding

  5. Structure of IgG Antibody

  6. Antigen-antibody Interactions

  7. Protein antigens may have multiple epitopes Tetanus toxin

  8. Antigen-antibody Interaction Heavy Chain Lysozyme Antigenic Determinant Epitope Light Chain

  9. Features of the Antigen-Antibody Interaction • Reversible • Non-covalent Interactions • Affinity • Measure of the strength of the binding • Ease of association or dissociation • Avidity • Increase in affinity due to multivalent binding • The summation of multiple affinities

  10. Affinity and Avidity

  11. Terminology Antigen Original - Substance able to generate antibody More general - Substance that can be recognized by antibody or T cells Immunogen Substance able to generate an immune response

  12. What Makes a Good Immunogen?

  13. Terminology Antigen Original - Substance able to generate antibody More general - Substance able to bind antibody Immunogen Substance able to generate antibody Hapten Non-immunogenic substance Usually low molecular weight Induces antibody formation when coupled to a larger “carrier” molecule Can bind antibody

  14. Hapten - DNP Protein Carrier - Bovine Serum Albumin Immunize with Antibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA

  15. Testosterone Estradiol Cortisol Penicillin Thyroid hormones Examples of Haptens

  16. Antibody-based assays

  17. Enzyme-Linked Immunosorbent Assay (ELISA) Colorless > Yellow Human Serum HIV Ag AP-Goat Anti- Human IgG AP = alkaline phosphatase

  18. ELI SA Enzyme-Linked Immunosorbent Assay (ELISA) 1. Antigen HIV Protein 2. Test antibody Human serum 3. Developing Ab Enzyme-Goat anti-Human IgG 4. Substrate Colorless > Blue © 1998 Gold Standard Multimedia Inc.

  19. - + Western Blot

  20. Western Blot for HIV Antibodies + - Patient sera

  21. Pregnancy Testing Assay for human chorionic gonadotrophin 1. Bioassay (Rabbit Test) 2. Agglutination inhibition

  22. Agglutination Inhibition Assay for HCG

  23. 3. ELISA HCG Test Control HCG Pregnancy Testing Assay for human chorionic gonadotrophin 1. Bioassay (Rabbit Test) 2. Agglutination inhibition

  24. Tissue Immunofluorescence/Immunohistochemistry

  25. Anti-B Anti-A Immunofluorescence Analysis of Single Cells

  26. CD8 CD4

  27. Summary of Antigen-Antibody Interactions Specificity depends on the variable regions of the immunoglobulin heavy and light chains In vivo After antibody binds antigen, the outcome depends on the constant region of the heavy chain. Effector functions are determined by antibody isotype.

  28. Summary of Antigen-Antibody Interactions In vitro All assays depend on exquisite specificity of the antibody. Assays differ in the means used to detect the bound antibody or bound antigen Sensitivity: Agglutination<immunofluorescence<ELISA/RIA

  29. Hemagglutination Used to identify blood group antigens or antibodies to them Variations: Chemically couple haptens to RBC Use other particles - bacteria, antigen-coated latex beads

  30. Radioimmunoassay (RIA)

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