1. Antigen-Antibody Reactions 2011 Features Examples in vivo in vitro John F Kearney 410 Shelby Building 4-6557 jfk@uab.edu

2. Learning Objectives To understand – The features of the antigen-antibody interaction Reversible Affinity Avidity – What makes a good antigen – Examples of in vivo antigen-antibody interactions – Examples of in vitro antigen-antibody interactions

3. Structure of an IgG Antibody

4. Features of the Antigen-Antibody Interaction A reversible bimolecular association involving various non-covalent interactions Is similar to an enzyme-substrate interactions, but not lead to an irreversible chemical alteration However catalytic antibodies have been described

5. Non-covalent binding

6. - Four types of non-covalent forces operates over a very short distance ( generally 1 angstrom ) Nature of Ag/Ab Reactions

7. Specificity The ability of an individual antibody combining site to react with only one antigenic determinant.

11. 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 Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions K.R. Deepthi

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

13. Calculation of Affinity Ag + Ab  Ag-Ab K eq = [Ag-Ab] [Ag] x [Ab] Applying the Law of Mass Action:

14. Avidity The overall strength of binding between an Ag with many determinants and multivalent Abs K eq = 10 4 Affinity 10 6 Avidity 10

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

16. Precipitation reactions in fluids yield a precipitin curve. FIGURE 6-4 ( Lattices or large aggregates ) ( no precipitate is formed if an Ag contains only a single copy of each epitope ) Precipitation Reactions

18. Antigen-Antibody Binding Epitope

19. 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

20. Antigen-Antibody Binding

21. Terminology AntigenOriginal - Substance able to generate antibody More general - Substance that can be recognized by antibody or T cells ImmunogenSubstance able to generate antibody or T cell responses

22. What Makes a Good Antigen? For T cell responses antigens should be degradable

23. Terminology AntigenOriginal - Substance able to generate antibody More general – able to bind antibody ImmunogenSubstance able to generate antibody or T cell responses HaptenNon-immunogenic substance Usually low molecular weight Can induce antibody formation when coupled to a larger “carrier” molecule

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

25. Examples of Haptens Cortisol Estradiol Testosterone Thyroid hormonesPenicillin

26. Antigen-Antibody Interactions in vivo

27. Direct Inhibition © 1998 Gold Standard Multimedia Inc. Toxin

28. Inhibit attachment of bacteria to cells

29. Pseudo colored EM image of Helicobacter pylori attached to a gastric epithelial cell

30. Would antibodies be effective at preventing H. pylori attachment? 1.Yes 2.No

31. Antibodies Enhance Phagocytosis

32. Opsonization © 1998 Gold Standard Multimedia Inc. Fc Receptor

33. Complement Lysis Complement activation can be initiated by antigen-antibody complexes © 1998 Gold Standard Multimedia Inc.

34. Antigen-Antibody Reactions In Vivo Neutralization of viruses and toxins Opsonization of pathogens Complement lysis of bacteria Prevention of bacterial adherence The Bad Autoimmunity - myasthenia gravis Transfusion reactions The Ugly Allergy The Good

35. Antigen-Antibody Interactions in vitro Techniques

36. Hemagglutination Used to identify blood group antigens or antibodies to them

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

38. FIGURE 6-7 Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs). Agglutination Reactions +++ (control) -visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads. -routinely performed to type RBCs for blood transfusion.

39. Hemagglutination Used to identify blood group antigens or antibodies to them

40. ELISA Enzyme Linked Immunosorbent Assay HIV Ag Human Serum Enz-Goat Anti-Human IgG Colorless → Yellow HIV Testing

41. 1. ELISA, determine if patient sera is + or – for antibodies to HIV 2. Western Blot, determine which viral antigens the patient has made antibodies to.

43. Immunofluorescence mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope. FIGURE 6-14 Fluorochromes -Fluorescein (490→517nm) -Rhodamine (515→546nm) -Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.

44. Bacillus anthracisB. thuringiensis B. subtilis

45. Examples of markers to dissect heterogeneity of immune cells

46. Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample

47. Hematoxlin eosin staining

48. Staining of spleen with antibodies to distinguish cell types Marginal zone macrophages Metallophilic macrophages IgM +B cells CD4/CD8 T ells

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

53. 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

54. 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: For example, ELISA and Western blot>>Agglutination

55. Affinity : 1.is due to covalent bonds between antigen and antibody. 2.is always the same as avidity. 3.is an expensive imported automobile. 4.is a measure of the strength of antigen- antibody binding. 5.Answers 2 and 4 are correct.

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

57. Evolution of Flow Instruments WA Bonner, HR Hulett, RG Sweet and LA Herzenberg, Fluorescence Activated Cell Sorting, Review of Scientific Instruments 43, 1972 Slide Kindly Supplied by Compucyte