A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFN gamma-producing T cells

We describe an ELISPOT technique for the detection of antigen specific IFN gamma-producing T cells. The technique is performed on spleen cells plated directly ex vivo into ELISPOT trays without an in vitro pre-culture step. T hus, the assay is likely to reflect the in vivo activity of the cells. We h ave found that very high cell densities (at least 10(6) cells/well) are req uired for optimal detection of spot forming cells, and only at a high densi ty of cells is the number of spots detected linearly related to the number of primed cells plated. If lower numbers of antigen primed cells are used, then unprimed spleen cells from syngeneic mice can be added to the well to increase the cell density. Under these conditions, we find that the number of spots is linearly proportional to the number of primed cells plated, eve n if these are well below a million cells/well. Experiments with MHC congen ic mice indicate that the high density of spleen cells required to obtain o ptimal spot formation reflects a requirement for an MHC restricted function , probably efficient antigen presentation to T cells. The formation of IFN gamma spots is antigen dependent and abrogated by depleting the antigen pri med cells of T cells. We conclude that this linear assay can be used to eff iciently detect ex vivo antigen-specific IFN gamma-producing T cells. (C) 1 999 Elsevier Science B.V. All rights reserved.