Enzyme-linked bio-immunoassay for IFN-gamma by HLA-DR induction

A novel enzyme-linked bio-immunoassay (bio-ELISA) has been developed to detect interferon-gamma (IFN-gamma) induced HLA-DR antigen on the surface of human tumor cells. Cells are cultured at 37 degrees C in 96-well microtiter plates in the presence of IFN-gamma for 2 days. After fixation with reagent alcohol the HLA-DR antigen is detected using a monoclonal antibody, followed by goat anti-mouse IgG-HRP conjugate. Four human cell lines were evaluated and all expressed HLA-DR in response to IFN-gamma in a dose-related fashion. Based on sensitivity, reproducibility and absence of antiproliferative effect by IFN-gamma, the COLO 205 cells (colon adenocarcinoma) were determined to be optimal. The bioassay is sensitive to 0.3 ng/ml IFN-gamma with a range to 10 ng/ml. The specificity of HLA-DR induction by IFN-gamma was demonstrated using an isotype specific monoclonal antibody as well as IFN-gamma neutralizing monoclonal and polyclonal antibodies. The effect of other cytokines on HLA-DR induction with COLO 205 cells was also investigated in this bioassay and only IFN-beta and interleukin-1 (IL-1) showed slight induction of HLA-DR. IFN-alpha had no effect at the concentration tested. Evaluation of assay parameters including reproducibility, sensitivity, simplicity, speed, cost and ability to standardize support the conclusion that this bioassay is a substantial improvement over the routinely used viral inhibition assay as a measure of IFN-gamma biological activity. The bio-ELISA technique also has potential applications for the quantitation of other cellular surface antigens induced by cytokines.