ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is 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 analyte.
After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the first analyte and an HRP-conjugated polyclonal antibody specific for the second analyte is added to the wells.
Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added.
Unbound enzyme is subsequently removed by washing and the first substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual analyte-secreting cell. After incubation with BCIP/NBT chromogen, the AEC substrate is added. Red spots develop at the site of secretion of the second analyte. The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope.