The Enzyme-linked Immunospot (ELISPOT) assay is a method that allows the determination of T or B cell responses by allowing enumeration of cells secreting a specific product (cytokine or antibody). As an example, for a T cell ELISPOT, a PVDF plate is coated with antibody towards a particular cytokine. Cells are then incubated with antigen or mitogen, secreting cytokine that is bound by the antibody on the plate. The cells are washed away, and a biotinylated detection antibody specific for the cytokine is added. Enzyme-conjugated streptavidin and substrate is then added, revealing a spot on the membrane for each cytokine-secreting cell. The ELISPOT assay is 10 times more sensitive than flow cytometry whe trying to enumerate antigen-specific cells.
For a generalized ELISPOT protocol, click here.