Quantification of antigen specific CD8+ T cells using an ELISPOT assay

Yasushi Miyahira, Kenichiro Murata, Dolores Rodriguez, Juan R. Rodriguez, Mariano Esteban, Mauricio M. Rodrigues, Fidel Zavala

Research output: Contribution to journalArticlepeer-review

320 Scopus citations


An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-γ secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-γ monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Original languageEnglish (US)
Pages (from-to)45-54
Number of pages10
JournalJournal of Immunological Methods
Issue number1
StatePublished - Apr 12 1995
Externally publishedYes


  • CD8 T cell
  • IFN-γ
  • Malaria
  • Recombinant vaccinia virus
  • Synthetic peptide

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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