A sensitive ELISPOT assay to detect low-frequency human T lymphocytes
| Autor: | M Kushner, M McCutcheon, T Ha, T.V Tran, K.M Tate, P Calabresi, L Hsiao, J Winkelhake, S Doan, N Wehner, A Wensky, E.G Spack |
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| Rok vydání: | 1997 |
| Předmět: |
T-Lymphocytes
medicine.medical_treatment T cell Molecular Sequence Data Immunology Enzyme-Linked Immunosorbent Assay Sensitivity and Specificity Peripheral blood mononuclear cell Antigen medicine Humans Immunology and Allergy Amino Acid Sequence Antigen-presenting cell Lymphokines biology Chemistry ELISPOT T lymphocyte Molecular biology Myelin basic protein Cytokine medicine.anatomical_structure biology.protein Interleukin-2 |
| Zdroj: | Journal of Immunological Methods. 210:149-166 |
| ISSN: | 0022-1759 |
| Popis: | 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 x 10(5) PBMC with the standard assay compared to 19 SFC per 2 x 10(5) 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. |
| Databáze: | OpenAIRE |
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