The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies. This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbant assay (ELISA) technique. A monoclonal antibody specific for a cytokine has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37° C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted cytokine. After washing away cells and any unbound substances, a biotinylated polyclonal antibody specific for the cytokine is added to the wells. Following a wash to remove any unbound biotinylated antibody, alk aline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. Blue-black colored precipitate forms at the sites of cytokine localization and appears as spots, with each individual spot representing an individual cytokine-secreting cell. The spots can be counted with automated ELISpot reader systems or manually, using a stereomicroscope.
|Typical images of human IFN-gamma ELISpot assay (Catalog # EL285) wells following development. Distinct spots are readily apparent in wells containing human PBMCs that were stimulated and cultured overnight with calcium ionomycin and PMA (left panel), whereas no spots are detected in wells containing non-stimulated cells (right panel).|