Development of a dual color enzyme-linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells.

The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies and cytokines. For simultaneous differentiation of individual cells producing interleukin-2 (IL-2) and/or interleukin-4 (IL-4) at a single cell level, a dual color ELISPOT assay has been developed. In the present system, the red spots corresponding to IL-2-secreting cells (T helper type 1, Th1 cells) were developed with a horseradish peroxidase and the amino ethyl carbazole (AEC)/H2O2. The light blue spots corresponding to IL-4-secreting cells (T helper type 2, Th2 cells) were developed with an alkaline phosphatase and the Vector blue. The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (T helper type 0, Th0 cells) were developed with both substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in crude spleen cell preparation or purified CD4 fraction. This procedure provides a useful tool for quantitatively analyzing micro-levels of dynamic immune responses.