A sensitive assay utilizing enzyme-linked immunosorbent assay methodology has been developed for the quantitation of single cells secreting interferon (IFN)-gamma or tumor necrosis factor (TNF). Cloned T cells or cells from lymphoid organs were stimulated with antigen, concanavalin A, or phorbol myristate acetate and ionomycin in microwells coated with antibodies specific for IFN-gamma. Discrete "spots" overlying areas where cells secrete IFN-gamma were then developed by incubation with a second antibody to IFN-gamma, followed by an enzyme-labeled antibody conjugate and substrate. Similarly, using TNF-specific antibody reagents, TNF-secreting cells were detected and quantitated in cell populations obtained from normal lymphoid tissues, bone marrow and peripheral blood, following activation with phorbol myristate acetate and ionomycin. Provided specific antibodies are available, this method has the potential to measure the frequency of cells secreting any cytokine.
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