Four-Color T And B Cell Elispot For Simultaneous Detection of Analytes

ELISPOT assays are a key research tool for enumerating antigen-specific T and B cells in PBMC. As both T and B cells occur in major classes, immune monitoring has to be concerned with identifying these as well. So far, ELISPOT assays have been primarily done single or double color. We report the development of four color T and B cell ELISPOT assays. Cells secreting any of the four analytes can therefore be identified unambiguously and without overlap in an automated fashion, without the need for compensation. Cells co-expressing analytes can be identified by superimposing the individual detection planes. Studying B cells and T cells experimentally has permitted us to verify the accuracy of co-expression measurements. Each B cell secretes only one type of Ig class/subclass. T cells, in contrast, frequently co-express cytokines. Serial dilution experiments showed that for T cells the numbers of co-expressers linearly decreased with the numbers of cells plated. For B cells, no co-expressers were found.