A sensitive ELISPOT assay to detect low-frequency human T lymphocytes |
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Authors: | M McCutcheon N Wehner A Wensky M Kushner S Doan L Hsiao P Calabresi T Ha TV Tran KM Tate J Winkelhake EG Spack |
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Institution: | a Anergen Incorporated, 301 Penobscot Drive, Redwood City, CA 94063, USA b National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA |
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Abstract: | We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity 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 hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a >10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2×105 PBMC with the standard assay compared to 19 SFC per 2×105 PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocyte cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker. |
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Keywords: | ELISPOT Human Cytokine Autoantigen |
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