Enumeration of IFN-gamma-producing human lymphocytes by spot-ELISA. A method to detect lymphokine-producing lymphocytes at the single-cell level

Abstract

We present a method to detect and enumerate individual interferon (IFN)-producing human lymphocytes. The assay is based on the ELISA-plaque assay developed by Sedgwick and Holt (J. Exp. Med. (1983) 157, 2178; J. Immunol. Methods (1986) 87, 37). Mitogen-stimulated T cells are seeded in anti-IFN-gamma-coated wells. After a 16 h incubation period, the cells are removed. Subsequently a rabbit anti-IFN-gamma-antiserum followed by goat anti-rabbit antiserum conjugated to alkaline phosphatase are used to detect the IFN-gamma spots. Application of the spot-ELISA in combination with the conventional ELISA reveals the amount of IFN-gamma produced per cell. The spot-ELISA is a highly sensitive, easy to perform and rapid assay. Provided specific antisera are available, this method is suitable to detect production of other lymphokines at the single-cell level. To our knowledge, this is the first report of a single, well-defined T cell product measurement by the spot-ELISA.