A sensitive ELISPOT assay to detect low-frequency human T lymphocytes

M. McCutcheon, N. Wehner, A. Wensky, M. Kushner, S. Doan, L. Hsiao, Peter Calabresi, T. Ha, T. V. Tran, K. M. Tate, J. Winkelhake, E. G. Spack

Research output: Contribution to journalArticle

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 x 105 PBMC with the standard assay compared to 19 SFC per 2 x 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 lymphocytes cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.

Original languageEnglish (US)
Pages (from-to)149-166
Number of pages18
JournalJournal of Immunological Methods
Volume210
Issue number2
DOIs
Publication statusPublished - Dec 29 1997
Externally publishedYes

    Fingerprint

Keywords

  • Autoantigen
  • Cytokine
  • ELISPOT
  • Human

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

McCutcheon, M., Wehner, N., Wensky, A., Kushner, M., Doan, S., Hsiao, L., ... Spack, E. G. (1997). A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. Journal of Immunological Methods, 210(2), 149-166. https://doi.org/10.1016/S0022-1759(97)00182-8