A competitive inhibition ELISA for the quantification of human interferon-γ

A competitive enzyme immunoassay has been developed for the measurement of human interferon-γ (IFN-γ) in cell culture supernatants. The assay is based on the dose-dependent inhobitory effect of liquid phase IFN-γ on the binding of a specific monoclonal antibody to recombinant IFN-γ (rIFN-γ) immobilized on microtitre plate wells. The extent of monoclonal anti-IFN-γ inhibition was determined by the uptake of alkaline phosphatase-conjugated goat anti-mouse IgG and the subsequent development of enzyme substrate colour. Absorbance readings were taken and results for test samples were extrapolated from standard rIFN-γ inhibition curves constructed as logit-log plots. Assay performance was assessed using three different monoclonal antibodies (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-γ over a wide range of concentrations from 0.4 ng/ml (3.4 IU/ml), or less, to 250 ng/ml ∼ 2000 IU/ml). The inhibition assay incorporates the advantages of specificity, reproducibility and convenience of performance which are the hallmarks of monoclonal antibody-based ELISAs. However, compared to the sandwich ELISAs previously described for human IFN-γ, it is considerably more economical in its use of monoclonal anti-IFN-γ, requiring < 50 ng of a single antibody per 96 well plate. It also uses relatively small volumes of test samples (50 μl/well) which is particularly advantageous where limited amounts of cell culture supernatant are available for cytokine assays.