Antigen-Antibody Interactions

"Predigested food" should be inscribed over every hall of learning as a warning to all
who do not wish to lose their own personalities and their original sense of judgement
Emma Goldman

Who Am I? As an endocrinologist I developed the technique of radioimmunoassay for which I received the Noble prize in medicine in 1977

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Precipitation reactions
Immobilization Assays

Precipitin Reactions:graphics presentation of classic immunologic precipitin reactions

Immobilization Assays: graphics presenation of newer asssays such as ELISA, RIA and Western Blots

Molecular Probes

Founded in 1975, Molecular Probes, Inc. is a biotechnology company specializing in the area of fluorescence technology. They produce a wide range of fluorescent probes and chemicals for research use in cell biology, molecular biology, immunology, microbiology, biochemistry and neuroscience. A fluorescent microscopic image from Molecular Probes (link below) illustrates how antibodies can be used to localize antigens in a cell.

URL --> http://www.probes.com/

A fluorescent microscopic image URL --> http://www.probes.com/cgi-bin/photo.cgi?file=g000453.gif&id=000001

 

Fluorescence-activated cell sorter (FACS)

Introduction to the Flow Cytometry Facility at the University of Massachusetts, Amherst.

URL --> http://www.bio.umass.edu/mcbfacs/intro.htm

ASSIGNED READING

Kuby's Immunology 4th Ed Chapter 6 pp 149-172
Kuby's Immunology 3rd Ed Chapter 6: pp 144-164

OUTLINE/SUMMARY

1. Strength of Antigen-Antibody Interactions
   • affinity
   • avidity
2. Cross Reactivity
3. Precipitin Reactions
   • reactions in fluids
   • reaction in gels
   • radial immunodiffusion
   • Ouchterlony
   • immunoelectrophoresis
4. Agglutination Reactions
   • hemagglutination
   • bacterial agglutination
   • passive agglutination
   • agglutination inhibition
5. Radioimmunoassay (RIA)
6. Enzyme Linked Immunosorbent Assay (ELISA)
   • indirect ELISA
   • sandwich ELISA
   • Competitive ELISA
7. Western Blots
8. Immunofluorescence
9. Immunoelectron Microscopy

PERFORMANCE OBJECTIVES

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Explain a primary antigen-antibody interaction and include at least three important charactistics
2. Describe the forces that encourage primary antigen-Antibody interactions
3. Distinguish betweeen antibody affinity and avidiy.
4. Describe the strength of the primary antigen-Antibody interactions using equilibrium dialysis. Include the terms K and Ka
5. Compare and contrast RIA and ELISA
6. Describe direct and indirect fluorescent antibody methods.
7. Describe a secondary antigen-antibody interaction in terms of lattice formation and antigen:antibody ratios.
8. Explain zone phenomena.
9. Construct a table to compare the various procedures used to determine the presence of solbule antigen or antibody in a fluid and in a gel.
10. Assess the reasons for using the different gel preciptitin reactions
11. Distinguish between agglutination and preciptin reactions and give the advantages and disadvantages of each.

SHORT ANSWER QUESTIONS

1. Cross reactivity of antibodies creates problems for their application in serology. Explain.
2. Differentiate between a primary and a secondary antigen-antibody reaction. What are three important characteristics that distinguish the two reactions?
3. What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why?
4. How is equilibrium dialysis used to measure PRIMARY antigen-antibody reactions?
5. Differentiate between avidity and affinity.
6. Discuss the term lattice formation.
7. What are the pros and cons of RIA?
8. Describe two types of immunofluorescence tests.
9. What is the advantages of the indirect procedure over the direct procedure?
10. What are some commonly used fluors?
11. What colour does each fluor emit?
12. What makes precipitin reactions visible?
13. What two factors are important in the development of precipitin reactions?
14. Three patterns can be observed in the Ouchterlony test.
15. DRAW and LABEL diagrams to illustrate these patterns.
16. What does each pattern show?
17. What is the major advantage of immunoelectrophoresis over immunodiffusion?
18. What are the disadvantages?
19. How does agglutination differ from precipitation?
20. Why are agglutinatin tests more sensitive that precipitin tests?
21. Differentiate between direct and indirect agglutination reactions?
22. What is a major advantage of indirect agglutination reaction over direct reactions?