ELISPOT assay to measure antigen-specific murine CD8(+) T cell responses

The enzyme-linked immunospot technique (ELISPOT) relies on the visualization of cytokine secretion by individual T cells following in vitro stimulation with antigen. This assay has been developed and standardized for the quantitative detection of antigen-specific CD8(+) T cells in mice subjected to different immunization protocols [J. Immunol. Methods 181 (1995) 45]. We have identified important variables that affect the efficacy of the ELISPOT assay and in this protocol we describe this methodology in detail. As a model, we used the production of interferon-gamma by CD8(+) T cells from peripheral blood, spleen and liver of mice immunized with malaria sporozoites expressing the H-2K(d)-restricted SYVPSAEQI. This protocol has also been used successfully to detect Th1 and Th2 epitope specific CD4(+) T cells.