ELISPOT
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ELISPOT is an immunological assay based on ELISA (Enzyme-Linked Immunosorbent Assay). Basically, the difference between the two is that in ELISA, the substance containing the "unknown" is stuck at the bottom of the well, whereas in ELISPOT the substance with the "unknown" is placed in the well after the bottom of the well has been coated with cytokine-specific antibody. In both cases, the wells are typically contained within a generic microtiter plate.
The ELISPOT method is most often used to determine the amount (i.e. the concentration) of activated antigen-specific cytotoxic T-cells in a given sample of splenocytes harvested from immunized animals, usually mice.
Elispots rely on the principle that T cells secrete cytokines following activation. In this assay, a given number (around 10e6) splenocytes or peripheral blood lymphocytes are plated in a 96-well nitrocellulose plate with antigen. The T cells settle to the bottom of the plate and, if they are specific for the given antigen, they will become activated. Because the plates are pre-coated with antibodies to the cytokine of interest, cytokines secreted by activated T cells will be “captured” locally. Typically, CD4 responses are measured by Interleukin-4 capture, while CD8 responses are measured by Ifn-γ (Interferon-gamma) capture.
Following incubation, the T cells can be washed away and a secondary antibody to the same cytokine will be added. This secondary antibody is usually biotinylated and can be visualized by adding streptavidin-alkaline phosphitase reagent. This reagent catalyses the conversion of a substrate to a deep purple stain, causing purple spots to appear wherever an activated T cell was. By counting these spots, we can ascertain what fraction of T cells can be activated by a given antigen.