A competitive inhibition ELISA for the quantification of human interferon-gamma.
A competitive enzyme immunoassay has been developed for the measurement of human interferon-gamma (IFN-gamma) in cell culture supernatants. The assay is based on the dose-dependent inhibitory effect of liquid phase IFN-gamma on the binding of a specific monoclonal antibody to recombinant 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 readings were taken and results for test samples were extrapolated from standard rIFN-gamma 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-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, 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-gamma, it is considerably more economical in its use of monoclonal anti-IFN-gamma, requiring < 50 ng of a single antibody per 96 well plate. It also uses relatively small volumes of test samples (50 microliters/well) which is particularly advantageous where limited amounts of cell culture supernatant are available for cytokine assays.
Wilson
A B
AB
Molecular Immunopathology Unit, Medical Research Council Centre, Cambridge, UK.
McHugh
S M
SM
Deighton
J
J
Ewan
P W
PW
Lachmann
P J
PJ
D000485:Allergens / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology; D039741:Antigens, Dermatophagoides; D001667:Binding, Competitive; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007371:Interferon-gamma / Q000032:analysis*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008925:Mites / Q000276:immunology; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000485:Allergens; D000911:Antibodies, Monoclonal; D000941:Antigens; D039741:Antigens, Dermatophagoides; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007371:Interferon-gamma