Combining ELISPOT and ELISA to Measure Amounts of Cytokines Secreted by a Single Cell

Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of �cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, “blank,” ELISPOT plate. After finishing the incubations, the amount of IFN gamma was measured by ELISA in culture media collected from both plates. The “blank” plate served to measure a total amount of secreted IFN gamma, whereas the ELISPOT plate served to measure the amount of unbound (UB) IFN gamma. Subtracting the amount of unbound IFN gamma from its total amount and dividing it by the number of spots in the ELISPOT plate allows for the calculation of the average amount of IFN gamma in a spot formed by a single cell.