QUANTIFICATION OF ANTIGEN-SPECIFIC CD8(-CELLS USING AN ELISPOT ASSAY() T)

An ELISPOT assay to detect and determine the number of antigen specifi c CD8(+) T cells was standardized using cloned murine CD8(+) T cells s pecific for the epitope SWPSAEQI of a rodent malaria antigen. This ass ay is based on the detection of IFN-gamma secretion by single cells af ter their stimulation with antigen. The interferon secretion is visual ized as spots revealed by using enzyme labeled anti-IFN-gamma monoclon al antibodies. Using known numbers of cloned murine CD8(+) T cells it was determined that the assay detects 80-95% of these CD8(+) T cells. The optimal culture conditions for the stimulation of the CD8(+) T cel ls were determined and the antigen concentration, number of antigen pr esenting cells and supplement of growth factors required to perform th e assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen s pecific CD8(+) T cells present in mixed lymphocyte populations. This m ethod is more sensitive than the chromium-51 release assay, and much s impler than the conventional precursor frequency analysis, providing t he number of antigen specific CD8(+) T cells in 36-48 h.