Characterization of murine monoclonal antibodies to human interferon-gamma (IFN-gamma) and their application for sandwich enzyme-linked immunosorbent assay (ELISA)

The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-gamma (IFN-gamma), but not human IFN-alpha and IFN-beta. They react weakly with heat-treated IFN-gamma. The three antibodies recognize different epitopes of the IFN-gamma molecule, as evaluated by antibody-binding inhibition experiments. We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA). The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer. When we measured IFN-gamma in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained. This may suggest that human IFN-gamma exists in oligomeric form. Recombinant human IFN-gamma expressed in E. coli is detectable at a concentration of 1 ng/ml in this sandwich ELISA. This assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule as well as measurement of IFN-gamma in human sera and tissue culture fluids.