Abstract
We have developed a new liquid-phase, chemiluminescence-enhanced, inhibition ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutralizing monoclonal antibodies (mAb) against recombinant human IFN- beta ser (rHuIFN- beta ser). In this assay, the initial antigen-antibody reaction occurs in solution under more physiologic conditions than in a standard solid-phase ELISA. Subsequently, the reaction mixture is applied to a membrane that is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagents are added, and the membrane is photographically recorded. Competitive inhibition 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 recognize distinct epitopes on rHuIFN- beta ser and to neutralize its antiviral and antiproliferative activity ( Proc. Natl. Acad. Sci. USA 88, 4040-4044, 1991). In conventional solid-phase ELISA, mAb A1 does not inhibit the binding of A7 to rHuIFN- beta ser, but we observed partial inhibition in the new liquid-phase assay. In contrast, A7 did not inhibit the binding of A1, consistent with the solid-phase ELISA results. This observation suggests that in the LP-CEI-ELISA, A1 and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, and quantifiable, appears also to be able to detect subtle, conformational differences of epitopes not evident in a standard solid-phase ELISA.
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