Immunology Lecture Robert J. Boackle, Ph.D. Antigen-Antibody Reactions

1. Immunology Lecture Robert J. Boackle, Ph.D. Antigen-Antibody Reactions Specific Objectives: THE STUDENT SHOULD BE ABLE TO 1. Discuss immunoglobulin variability (ie. the variable region) 2. Describe bonds between the variable region and the antigenic determinant 3. Define antibody affinity and antibody avidity 4. Describe a precipitin curve and discuss lattice formation involving proteins verses carbohydrate antigens and be able to define "zone of equivalence". 5. Understand immunodiffusion in agar gels. (identity, nonidentity and partial identity) 6. Have a conceptual understanding of immunoelectrophoresis, Fluorescent antibody techniques and ELISA (enzyme-linked immunoassay) 7. Define "agglutination" and understand the functional differences between monomeric Ab (ie. IgG) and polymeric Ab (ie. IgM and S-IgA)

2. Definitions: 1. The "antibody affinity" of an antibody-antigen reaction is related to the strength of attractiveness between an antibody (Fab region) and its antigenic determinant. 2. The "antibody avidity" is the total strength of binding of the Fab regions of the population of antibodies evoked to an antigen, and involves the reaction with all the antigenic determinates. Thus it is the total strength of the binding of antibodies to antigens. 3. Immune Complex = Antigen-Antibody Complex [the size depends on the ratio of antigen to antibody]. Also the student should be prepared to answer and discuss the following: 1. List and describe the possible bonds between the immunoglobulin variable region and an antigenic determinant. Then draw and explain a precipitin curve and "lattice formation" involving protein antigens and polyclonal Ab. 2. What is meant by "hypervariable regions" on immunoglobulins? How do B cell clones differ in regard to the hypervariable regions of the immunoglobulins on their surface? At the level of the gene, explain what is believed to account for these clonal diversities. 3. Can two different classes of immunoglobulins have identical variable regions? In your answer include a discussion of the switch mechanism.

3. ANTIGENIC DETERMINANTS INTERACT WITH SPECIFIC ANTIBODY

4. IgG has a Valence of 2 TWO Identical ANTIGEN BINDING SITES CH2 CH3 CH2 CH3

5. Movement at the Hinge Region IgG Surface of an Antigen i.e. bacterial cell surface CH2 CH3 CH2 CH3

6. Non-Covalent Interactions Ball in glove fit VL Antigenic Determinant VH

7. Charge-Charge Interactions Hydrophobic Interactions - And good fit ! VL + - + VH - Gene rearrangements and Mutational Hot Spots

8. VL + - Antibody Affinity Y + VH -

9. Antigenic determinant 1 Antigenic determinant 2 PROTEIN ANTIGEN Antigenic determinant 3 Antigenic determinant 4

10. MUST HAVE POLYCLONALANTIBODY and at least two different antigenic determinants TO CROSS-LINK PROTEIN ANTIGENS Y Y Y Immune Complexes

11. Excess Antibody Y Y Y Y Y Y Y Y Y Y ANTIBODY EXCESS NO CROSSLINKS NO Precipitate Y Y

12. Excess Antigen = Not enough Cross-links to cause a Precipitation Y

13. More cross-links, and higher individual affinities = higher AVIDITY of the Immune Complexes Y Y Y Y Y Y

14. ZONE of Equivalence No Soluble Ag or Ab ANTIGEN EXCESS ANTIBODY EXCESS Amount of Precipitate Ab CONC

15. Repeating Antigenic Determinants e.g. PEPTIDOGYCAN

16. CHO ANTIGENS may cross-link with MONOCLONAL Ab Y Y Y Y

17. Immune Complex DOUBLE DIFFUSION Antigen Antibody

18. Immune Complexes Zone of Equivalence Antibody Antigen Antigen

19. Rabbit Serum as antigens 1:4 1:20 Goat anti-rabbit serum (Antibodies to rabbit serum)

20. Non-Identity Antigen #1 Antigen #2

21. No Shared Antigenic Determinants Antigen #1 Antigen #2

22. OUCHTERLONY ANALYSIS Diffusion of Antigens and Polyclonal Antibodies Non-Identity Antigen 1 (Molecule #1) Antigen 2 (Molecule #2) Antibodies to both antigens The same Animal was injected with antigen 1 and with antigen 2

23. This animal was only injected with Antigen #4 OUCHTERLONY ANALYSIS Partial - Identity Antigen 3 is a part of antigen 4 Antigen 4 Remember that Protein Antigens have different antigenic determinants Also remember that this antibody is a multi-clonal antibody such as an anti-serum to an antigenic preparation Antibody

24. OUCHTERLONY ANALYSIS Partial - Identity Antigen 3 Antigen 4 Antibody

25. Partial - Identity Antibodies to determinants c and d are only on Antigen 3 and they pass by antigen 4 Antigen 3 Antigen 4 Antibodies polyclonal antibody

26. OUCHTERLONY ANALYSIS Identity These two Antigens are the Same Molecule Antigen 6 is Antigen 5 Antigen 5 No spikes were formed because: Antigenic determinants on Antigen 5 captured all the antibodies to Antigen 6 and antigenic determinants on Antigen 6 captured all the antibodies to Antigen 5 Antibody

27. Fluorescence UV Light Antigens on Cells or on Tissue Sections

28. Double layer Sandwich UV Light Fluorescence Antigens

29. ELISA Substrate that turns from clear to green Enzyme Linked Immuno-Sorbant Assay Peroxidase Enzyme is permanently attached to the Antibody Probe Ag Ag Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate

30. ELISA Enzyme Linked Immuno-Sorbant Assay Peroxidase Enzyme is permanently attached to the Antibody Probe Substrate that turns from clear to green Ag Ag Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate

31. Capture ELISA -- using pre-immobilized mouse monoclonal Ab to capture the Specific Antigen and a second Probe monoclonal Antibody against a different antigenic determinant Ag Ag

32. Agglutination Y Y Y IgM >>IgG