This assay is used to detect and quantify the number of T cells that secrete a particular cytokine (e.g Interferon-γ) upon recognition of a specific antigen. T cells are cultured with antigen-presenting cells in wells coated with an antibody recognizing a particular cytokine. The coating antibody captures the secreted cytokine and a second cytokine-specific antibody that is coupled to a chromogenic substrate is used for detection. Results appear as spots, with each spot corresponding to one cytokine-secreting cell. The number of spots allows the calculation of the frequency of cytokine-secreting cells for a specific antigen. However this assay does not determine the amount of cytokine secreted.

This assay is highly sensitive (1 cell in 100,000) and can be performed directly ex-vivo using relatively few T cells (compared with ICS).