Abstract

ELISpot is a highly sensitive method in immunology to enumerate cells producing a given cytokine. Cells are stimulated in a microtiter plate pre-coated with a specific anti-analyte antibody. In response to the stimulation, cells release cytokines that are bound to the anti-analyte antibody. After a washing step, which removes the cells from the wells, the location of the cytokine-releasing cell is visualized by an enzymelabeled detection antibody and its corresponding chromogenic substrate. The end result is a set of colored spots, each of which represents an area where a cell secreting the cytokine had been located. Here we describe the standard ELISpot protocol and a variation denominated dendritic cell (DC)ELISpot for the detection of IFN gamma-secreting cells upon stimulation with oligopeptides and protein antigens, respectively.