Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA

Abstract

Two stable hybridoma cell lines secreting specific antibodies against human gamma interferon (HuIFN-gamma) were established. Both monoclonal antibodies (designated as MD-1 and MD-2) belong to the IgG1/kappa subclass and neutralize the antiviral activity of natural and recombinant DNA derived HuIFN-gamma (nHuIFN-gamma and rHuIFN-gamma respectively), although MD-1 is far more effective than MD-2. MD-1 and MD-2 recognize different epitopes and do not compete with each other in binding to HuIFN-gamma as concluded from competition assays. In a 'Western' blot, both antibodies reacted with the 20 kDa and 25 kDa polypeptides present in nHuIFN-gamma preparations. A sandwich enzyme immunoassay using microtiter plates coated with unlabeled MD-2 was developed. Biotinylated MD-1 was used as the second antibody. Bound MD-1 was detected by an avidin/alkaline phosphatase enzyme reaction. This immunoassay is highly specific and as sensitive as a bioassay. A radioimmunoassay using MD-2 coated on polystyrene balls and 125I-labeled MD-1 as the second antibody showed a sensitivity comparable to that of the enzyme immunoassay.