Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial

J Immunol Methods. 2000 Oct 20;244(1-2):81-9. doi: 10.1016/s0022-1759(00)00257-x.


The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.

Publication types

  • Comparative Study
  • Evaluation Study
  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral / immunology*
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism
  • Chromium Radioisotopes
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Epitopes, T-Lymphocyte / immunology
  • HIV Reverse Transcriptase / immunology
  • HLA-A2 Antigen / immunology
  • Humans
  • Immunodominant Epitopes / immunology
  • Indicator Dilution Techniques
  • Interferon-gamma / metabolism
  • Leukocytes, Mononuclear / immunology
  • Lymphocyte Activation / immunology
  • Peptide Fragments / immunology
  • T-Lymphocytes, Cytotoxic / immunology
  • Viral Matrix Proteins / immunology


  • Antigens, Viral
  • Chromium Radioisotopes
  • Epitopes, T-Lymphocyte
  • HLA-A2 Antigen
  • Immunodominant Epitopes
  • Peptide Fragments
  • Viral Matrix Proteins
  • influenza virus membrane protein (58-66)
  • Interferon-gamma
  • HIV Reverse Transcriptase