ELISPOT

From cells, alternate forms of the different plaque-forming cell assays can be utilized. An ELISPOT assay, puts cells over antigen, which are attached to a plastic surface, trap the cells' secreted product, which can then be detected by using an antibody coupled to an enzyme that cleaves a substrate into a colored product.

Hemolytic plaque assays, as Jerne's can also be utilized (differ by use of agar instead of chambers) and reverse PFC assays that use target RBCs with conjugated anti-Ig antibody. Moreover, we can take a representative sample of cells and separate them individually by flow cytometry into individual wells, which can then be stimulated by addition of antigen. Any precipitate formed in a well, can depcit that the cell is a plasma cell specific to the antigen (extrapolate to get magnitude of antibody response). The use of indicator cells, filled with Chromium 51, which would be released upon complement activation could indicate the level of an antibody response. In addition, immunodiffusion, the detection of antibody (or antigen) by the formation of an antigen:antibody precipitate in a clear agar gel can also be used. Using the reverse transcriptase polymerase chain reaction, to view the level of a transcript found in lysed cells, as for the transcript for the secreted antibody, we can get a sense of the level of antibody produced.

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. Kissinger R, and Myl A.D. 1984. Improvements to the Plaque Assay for Antibody Secreting Cells. J. of Immun. Meth. 66:377-382

3. Wilson S., Munson A., and Meade J. 1999. Assessment of the Functional Integrity of the Humoral Immune Response: The Plaque-Forming Cell Assay and the Enzyme-Linked Immunosorbent Assay. Methods 19:2-7

4. Cunningham, A.J. 1965. Nature (London) 207, 1106

5. Cunningham A.J. and Szenberg A. 1968. Immuniligy 14: 599

6. Jerne N. and Nordin A. 1963. Science 140: 405

7. Delves P. and Roitt I. 1999. Encyclopedia of Immunology, Academic Press Inc., 2nd ed., San Diego, USA page 238.

8. Cruse, J and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965