1 5/28/2016 Antigen -Antibody Interactions Precipitin Reactions Hugh B. Fackrell

2 5/28/2016 Antigen-Antibody Interactions ä Assigned Reading ä Content Outline ä Performance Ojectives ä Key terms ä Key Concepts ä Short Answer Questions

3 5/28/2016 Assigned Reading ä Chapter: 6 pp ä Janis Kuby’s Immunology 3rd Ed

4 5/28/2016 Content Outline ä Strength of Antigen-Antibody Interactions ä Cross Reactivity ä Precipitin Reactions ä Agglutination Reactions

5 5/28/2016 Strength of Antigen-Antibody Interactions ä affinity ä avidity

6 5/28/2016 Cross reactivity

7 5/28/2016 Mammoth

8 5/28/2016 Precipitin Reactions ä reactions in fluids ä reaction in gels ä radial immunodiffusion ä Ouchterlony ä immunoelectrophoresis

9 5/28/2016 Ring Test

10 5/28/2016 Ring Test: Controls

11 5/28/2016 C reactive Protein ring test

12 5/28/2016 QPT

13 5/28/2016 QPT: Zones

14 5/28/2016 QPT:# Epitopes

15 5/28/2016

16 5/28/2016 QPT: Simple system

17 5/28/2016 QPT: Multiple Interactions

18 5/28/2016 QPT: Multiple Reactions

19 5/28/2016 Radial Immuno Diffusion

20 5/28/2016 Double Immuno Diffusion

21 5/28/2016 Immunodiffusion: Single reactions

22 5/28/2016

23 5/28/2016

24 5/28/2016 Immunoelectrophoresis

25 5/28/2016 Counter Immuno Electrophoresis Fig 12-7 Barrett 1988

26 5/28/2016 IEF: Concept

27 5/28/2016 IEF: IgG IgM IgA

28 5/28/2016 IEF: Partial Identity

29 5/28/2016 IEF: Human Serum

30 5/28/2016 Rocket Immunoelectrophoresis

31 5/28/2016 Rocket Immunoelectrophoresis 2

32 5/28/2016 Rocket Immunoelectrophoresis 3

33 5/28/2016 Agglutination Reactions ä hemagglutination ä bacterial agglutination ä passive agglutination ä agglutination inhibition

34 5/28/2016

35 5/28/2016 Hemagglutination

36 5/28/2016 The End

37 5/28/2016 Performance Objectives Key terms, concepts short answers

38 5/28/2016 Key Terms ä agglutination, direct agglutination reaction, indirect agglutination reaction ä antibody affinity, antiserum, association constant (K), average affinity, ä average intrinsic association constant(Ka), avidity, ELISA, equilibrium constant, ä equilibrium dialysis, fluorescein, fluorochromes, hemagglutination,

39 5/28/2016 ä passive hemagglutination, passive hemagglutination inhibition, ä reverse passive hemagglutination, immune precipitation, immunoelectrophoresis ä immunofluorescence, Indirect fluorecent antibody test, ring test,

40 5/28/2016 ä Ouchterlony methods, plasma, primary antigen-antibody interactions, Radioimmunoassay(RIA ä Rhodamine, secondary antigen-antibody interactions, serology, ä serum, titer, zone phenomena (antibody excess, antigen excess, equivalence)

41 5/28/2016 Key Concepts ä Explain a primary antigen-antibody interaction and include at least three important characteristics. ä Describe the forces that encourage primary antigen-Antibody interactions ä Assess the reasons for using the different gel preciptitin reactions

42 5/28/2016 ä Distinguish betweeen antibody affinity and avidity. ä Describe the strength of the primary antigen-antibody interactions using equilibrium dialysis. Include the terms K and Ka ä Compare and contrast RIA and ELISA ä Describe direct and indirect fluorescent antibody methods. ä Explain zone phenomena.

43 5/28/2016 ä Describe a secondary antigen-antibody interaction in terms of lattice formation and antigen:antibody ratios. ä 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. ä Distinguish between agglutination and preciptin reactions and give the advantages and disadvantages of each.

44 5/28/2016 Short Answer Questions

45 5/28/2016 ä Cross reactivity of antibodies creates problems for their application in serology. Explain. ä Differentiate between a primary and a secondary antigen-antibody reaction. ä What are three important characteristics that distinguish the two reactions?

46 5/28/2016 ä What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why? ä How is equilibrium dialysis used to measure PRIMARY antigen-antibody reactions? ä Differentiate between avidity and affinity.

47 5/28/2016 ä Discuss the term lattice formation. ä What are the pros and cons of RIA? ä Describe two types of immunofluorescence tests. ä What is the advantages of the indirect procedure over the direct procedure? ä What are some commonly used fluors? ä What colour does each fluor emit? ä What makes precipitin reactions visible?

48 5/28/2016 ä What two factors are important in the development of precipitin reactions? ä Three patterns can be observed in the Ouchterlony test. DRAW and LABEL diagrams to illustrate these patterns. What does each pattern show? ä What is the major advantage of immunoelectrophoresis over immunodiffusion? ä What are the disadvantages?

49 5/28/2016 ä How does agglutination differ from precipitation? ä Why are agglutinatin tests more sensitive that precipitin tests? ä Differentiate between direct and indirect agglutination reactions? ä What is a major advantage of indirect agglutination reaction over direct reactions?

50 5/28/2016 Ouchterlony

51 5/28/2016 Ouchterlony:Partial Identity