ELISPOT
The Enzyme-linked immunospot (ELISPOT) assay was developed by Czerkinsky in 1983. The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. It is a sensitive method for monitoring immune responses in humans and animals and a superior tool for accurate quantification of cytokine-secreting T cells in single cell suspensions of lymphoid tissue, CNS tissue, bone marrow or preparations of peripheral blood mononuclear cells (PBMC).
Brief description of the ELISPOT procedure
Cells are incubated for a defined length of time in the wells of the ELISPOT plate precoated with a high-affinity monoclonal antibody to which the cytokine, produced during incubation, will bind. Subsequently, cells are lysed and debris is washed away. Areas in which the cytokine has been captured by the coating antibody are detected with a combination of biotinylated anti-cytokine detector antibodies and enzyme-labeled streptavidin or anti-biotin antibodies. The last step in the assay is the addition of a substrate yielding a colored zone ('spot'), which reveals the site of cytokine secretion.