A COMPETITIVE-INHIBITION ELISA FOR THE QUANTIFICATION OF HUMAN INTERFERON-GAMMA

A competitive enzyme immunoassay has been developed for the measuremen t of human interferon-gamma (IFN-gamma) in cell culture supernatants. The assay is based on the dose-dependent inhibitory effect of liquid p hase IFN-gamma on the binding of a specific monoclonal antibody to rec ombinant IFN-gamma (rIFN-gamma) immobilized on microtitre plate wells. The extent of monoclonal anti-IFN-gamma inhibition was determined by the uptake of alkaline phosphatase-conjugated goat anti-mouse IgG and the subsequent development of enzyme substrate colour. Absorbance read ings were taken and results for test samples were extrapolated from st andard rIFN-gamma inhibition curves constructed as logit-log plots. As say performance was assessed using three different monoclonal antibodi es (clones 20G7, H-22 and GZ-4). Optimum sensitivity was achieved with the antibodies of higher affinity, 20G7 and H-22, which gave reliable quantification of IFN-gamma over a wide range of concentrations from 0.4 ng/ml (3.4 IU/ml), or less, to 250 ng/ml approximately 2000 IU/ml) . The inhibition assay incorporates the advantages of specificity, rep roducibility and convenience of performance which are the hallmarks of monoclonal antibody-based ELISAs. However, compared to the sandwich E LISAs previously described for human IFN-gamma, it is considerably mor e economical in its use of monoclonal anti-IFN-gamma, requiring < 50 n g of a single antibody per 96 well plate. It also uses relatively smal l volumes of test samples (50 mul/well) which is particularly advantag eous where limited amounts of cell culture supernatant are available f or cytokine assays.