Antigen - Antibody Interactions

1. Antigen -Antibody Interactions Hugh B. Fackrell

2. Antigen-Antibody Interactions • Assigned Reading • Content Outline • Performance Ojectives • Key terms • Key Concepts • Short Answer Questions

3. Assigned Reading • Chapter: 6 pp 144-164 • Janis Kuby’s Immunology 3rd Ed

4. Content Outline • Radioimmunoassay (RIA) • Enzyme Linked Immunosorbent Assay (ELISA) • Western Blots • Immunofluorescence • Immunoelectron Microscopy

5. Strength of Antigen-Antibody Interactions • affinity • avidity

6. Immunoadsorbent Assays • Enzyme Linked Immuno Sorbent Assay • Fluorescent Immuno Sorbent Assay • Radio Immuno Assay

7. Enzyme Linked Immunosorbent Assay (ELISA) • indirect ELISA • sandwich ELISA • Competitive ELISA

12. Indirect ELISA

13. Competitive ELISA

14. Sandwich ELISA

15. ELISA: Advantages • Specific & Sensitive- Wide Application • Equipment cheap & available • Reagents “Cheap”, long shelf life • Assays may be rapid • Simultaneous assays; variety of labels • Potential for automation • no radiation hazards

16. ELISA:Disadvantages • Number of separation methods limited • Expertise required to label and purify conjugates • Susceptible to interference from non specific factors

17. ELISA: features of labeled enzymes • Stable when conjugated • High substrate turnover number • High Extinction coefficient of product • Enzyme & substrate not present in test samples

18. ELISA: Enzyme Choices • Horse Radish Peroxidase • Alkaline Phosphatase • Glucose Oxidase • Urease

19. RIA: Advantages • Measurement simple, not affected by composition of sample matrix • Sensitivity & precision not dependent on the measurement of the magnitude of the signal • Large variety of radiolabelled compounds • labels do not affect reaction kinetics • Mathematically documented

20. RIA: Disadvantages • Labeled reagents have short shelf life • Potential health hazards • Disposal of radioactive wastes • Equipment is expensive • Variability between batches of labels • Dependence on duration of count time may limit sensitivity of assays

21. Western Blot • Electrophoresis proteins to separate • Molecular weight, charge, pI etc • 2D electrophoresis possible • Immobilize separated proteins • Electrophoresis onto nitrocellulose • Develop as an ELISA • Product MUST be INSOLUBLE chromogen

22. oligomer monomer Western Blot with MABS • Same antigen was exposed to 6 different MABS • Staphylococcal alpha toxin • Each MAB reacted with a monomer and a oligomer form of the toxin Maria Sawicki 1996

23. Immunofluorescent Methods • Fluoresecence Immuno Assay • Fluorescence Quenching • Fluorescence Enhancement • Fluorescence Polarization

24. Characteristics of Fluorescent Molecules • Many loosely bound electrons • Resonance of double bonds • Hybridization

25. Plasma cell function

26. Antigen localization in Spleen

27. Flow Cytometry

29. The End

30. Performance Objectives Key terms, concepts short answers

31. Key Terms • agglutination, direct agglutination reaction, indirect agglutination reaction • antibody affinity, antiserum, association constant (K), average affinity, • average intrinsic association constant(Ka), avidity, ELISA, equilibrium constant, • equilibrium dialysis, fluorescein, fluorochromes, hemagglutination,

32. passive hemagglutination, passive hemagglutination inhibition, • reverse passive hemagglutination, immune precipitation, immunoelectrophoresis • immunofluorescence, Indirect fluorecent antibody test, ring test,

33. Ouchterlony methods, plasma, primary antigen-antibody interactions, Radioimmunoassay(RIA • Rhodamine, secondary antigen-antibody interactions, serology, • serum, titer, zone phenomena (antibody excess, antigen excess, equivalence)

34. Key Concepts • Explain a primary antigen-antibody interaction and include at least three important characteristics. • Describe the forces that encourage primary antigen-Antibody interactions • Assess the reasons for using the different gel preciptitin reactions

35. Distinguish betweeen antibody affinity and avidity. • Describe the strength of the primary antigen-antibody interactions using equilibrium dialysis. Include the terms K and Ka • Compare and contrast RIA and ELISA • Describe direct and indirect fluorescent antibody methods. • Explain zone phenomena.

36. Describe a secondary antigen-antibody interaction in terms of lattice formation and antigen:antibody ratios. • Construct a table to compare the various procedures used to determine the presence of soluble antigen or antibody in a fluid and in a gel. • Distinguish between agglutination and preciptin reactions and give the advantages and disadvantages of each.

37. Short Answer Questions

38. Cross reactivity of antibodies creates problems for their application in serology. Explain. • Differentiate between a primary and a secondary antigen-antibody reaction. • What are three important characteristics that distinguish the two reactions?

39. What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why? • How is equilibrium dialysis used to measure PRIMARY antigen-antibody reactions? • Differentiate between avidity and affinity.

40. Discuss the term lattice formation. • What are the pros and cons of RIA? • Describe two types of immunofluorescence tests. • What is the advantages of the indirect procedure over the direct procedure? • What are some commonly used fluors? • What colour does each fluor emit? • What makes precipitin reactions visible?

41. What two factors are important in the development of precipitin reactions? • Three patterns can be observed in the Ouchterlony test. DRAW and LABEL diagrams to illustrate these patterns. What does each pattern show? • What is the major advantage of immunoelectrophoresis over immunodiffusion? • What are the disadvantages?

42. How does agglutination differ from precipitation? • Why are agglutinatin tests more sensitive that precipitin tests? • Differentiate between direct and indirect agglutination reactions? • What is a major advantage of indirect agglutination reaction over direct reactions?