A Sensitive ELISPOT Assay to Detect Low-Frequency Human T Lymphocytes

J Immunol Methods. 1997 Dec 29;210(2):149-66. doi: 10.1016/s0022-1759(97)00182-8.

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 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.

MeSH terms

  • Amino Acid Sequence
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Humans
  • Interleukin-2 / pharmacology
  • Lymphokines / analysis
  • Molecular Sequence Data
  • Sensitivity and Specificity
  • T-Lymphocytes / immunology*

Substances

  • Interleukin-2
  • Lymphokines