Assay principles

More about the ELISA Spot assay:

The ELISPOT assay can be done with freshly isolated PBMC or with PBMC frozen by certain specifications. Specifically designed 96-well plates are coated with a cytokine-specific monoclonal antibody (e.g., IFN-). PBMC (or purified T cell subsets) are pipetted into the coated wells, and are cultured with the test antigen(s). Control wells contain either irrelevant antigen, or media alone. T cells that are specific for the test antigen when activated secrete molecules that are captured by the membrane-bound antibody (Figure 3).

Induction of maximal cytokine production typically requires 24 h (IFN-, IL-2 and IL-3, IL-10 and TNF or 48 h for IL-4 and IL-5). After the activation culture, the cells are discarded (or transferred for further characterization/propagation), and a labeled cytokine-specific secondary antibody is added. Subsequently the plate bound secondary antibody is visualized via an enzymatic reaction. When ELISPOT assays are optimized (as is the case for ImmunoSpot® assays) each color precipitation (“spot”) represents the footprint of a single cell’s cytokine secretion. Spot number denotes the accurate frequency of the antigen specific T cells among the plated cells, spot size and morphology providing additional information on the magnitude and kinetics of the cells’ secretory activity.