In the ELISPOT assay, a well is coated with antibody against the antigen of interest, a cytokine in this example, and then a suspension of a cell population thought to contain some members synthesizing and secretfrig the cytokine are layered onto the bottom of well and incubated.
Most of the cytokine molecules secreted by a particular cell react with nearby well bound antibodies.
Atlet the incubation period, the well is washed and an enzyme-labeled anti-cytokibe antibody is added.
After washing away unbourd antibody, a chromogenic substrate that forms an insoluble colored product is added.
The colored product(purple) precipliates and forms a spot only on the areas of the well where cytokine secreting cells had been deposited.
By couriting he number of colored spots.
it is possible to determine how many cytokine secreting cells were present in the added cell suspension.