TriSpot Human IFNγ/IL-2 ELISPOT Kit allows for the detection of a third, distinct color attributable to the secretion of two distinct analytes from a single cell, in addition to the detection of each of the two distinct analytes independently. IFNγ secretion produces blue spots, IL-2 spots are red and dual secretion is purple. The kit contains the components and protocol to develop 2 ELISPOT plates. The antibody pairs, antibody concentration and volume, plates, and detection strategies, were all evaluated to provide the optimal conditions for a TriSpot ELISPOT. Plate coating and blocking conditions have been selected to provide well-to-well and within-well consistency of spot forming cells (SFC), high signal-to-noise ratios and low background. All components undergo quality control testing to reduce lot-to-lot variability.