Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

Figure 1

Schematic representation of ELISPOT assays. (a) The standard ELISPOT is used to measure antigen-specific effector T cells. The membrane’s plate is coated with an antibody (Ab) specific for the cytokine of interest (capture Ab). Peripheral blood mononuclear cells (PBMCs) are added with or without antigen and incubated overnight. The cytokine released by the cells binds to the capture Ab. After washing, an anti-cytokine biotinylated detection Ab is added followed by streptavidin conjugated with an enzyme, and finally an enzyme substrate is added which produces colored spots. (b) The cultured ELISPOT assay is used to measure antigen-specific memory T cells. PBMCs are cultured with or without antigen for 10 days, with the addition of interleukin-2 (IL-2) at days 3 and 7, allowing the expansion of antigen-specific T cells. Then, cells are restimulated with the same antigen used during the 10-day period in the ELISPOT assay. (c) Spots are counted using an automated ELISPOT reader. Representative images of interferon-γ (IFN-γ) ELISPOT wells are shown. PBMCs from an Epstein-Barr virus (EBV) seropositive healthy subject were evaluated in response to medium alone (negative control), phytohemagglutinin (PHA, positive control) and a peptide pool (15 amino acids in length with an 11 amino acid overlap) spanning full-length EBV latent protein EBNA1.