Quantitative liquid-phase chemiluminescence ELISA: Detection of subtle epitope differences in HuIFN-beta

We have developed a new liquid-phase, chemiluminescence-enhanced, inhibitio n ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutr alizing monoclonal antibodies (mAb) against recombinant human IFN-beta ser (rHuIFN-beta ser). In this assay, the initial antigen-antibody reaction occ urs in solution under more physiologic conditions than in a standard solid- phase ELISA, Subsequently, the reaction mixture is applied to a membrane th at is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagent s are added, and the membrane is photographically recorded. Competitive inh ibition of binding of a second, labeled mAb by the first mAb decreases the signal detected, Two well-characterized mAb A1 and A7, have been shown to r ecognize distinct epitopes on rHuIFN-beta ser and to neutralize its antivir al and antiproliferative activity (Proc, Natl. Acad, Sci, USA 88, 4040-4044 , 1991), In conventional solid-phase ELISA, mAb Al does not inhibit the bin ding of A7 to rHuIFN-beta ser, but we observed partial inhibition in the ne w liquid-phase assay. In contrast, A7 did not inhibit the binding of Al, co nsistent with the solid-phase ELISA results. This observation suggests th;d t in the LP-CEI-ELISA, Al and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, an d quantifiable, appears also to be able to detect subtle, conformational di fferences of epitopes not evident in a standard solid-phase ELISA.