A SENSITIVE ELISPOT ASSAY TO DETECT LOW-FREQUENCY HUMAN T-LYMPHOCYTES

We extended the sensitivity of the ELISPOT assay by including an antig en-driven proliferation step prior to a final restimulation with antig en and irradiated antigen presenting cells (APCs). This improved sensi tivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridi zation. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-t o-noise ratio for counting cytokine spots, and use of video computer i maging software improved objective quantitation. Analysis of antigen-r eactive peripheral blood mononuclear cells (PBMC) from multiple sclero sis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic prot ein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 X 10(5) PBMC with the standard assay compared to 19 SFC per 2 X 10(5) PBMC with the modified assay). In addition, t he modified ELISPOT assay could be performed with frozen PBMC, which p ermitted greater flexibility in sample processing, multiple use of a s ingle sample as an internal standard, and simultaneous analysis of sam ples collected at different time points. This modified ELISPOT assay h as many applications, including analysis of cytokine profiles in rare T cell populations. identification of antigen-responsive individuals a s PBMC donors for T lymphocyte cloning or for therapeutic intervention , and assessment of vaccine or therapeutic efficacy as a surrogate cli nical marker. (C) 1997 Elsevier Science B.V.