Enzyme-linked immunosorbent assays for kinins using high-affinity monoclonal kinin antibodies

Splenocytes from mice immunized either with bradykinin conjugated with carbodiimide to keyhole limpet hemocyanin or ovalbumin were fused using polyethylene glycol with the mouse myeloma cell line SP2/o. Nine monoclonal antibodies reactive with kinins were obtained from two fusions. All of the antibodies were of the IgG1k isotype, except for one, which was an IgG2ak. Based on their reactivities with biologically active kinins and biologically inactive degradation products, the antibodies were separated into three groups. The first group, which had the highest affinities for bradykinin, displayed about equal reactivities for bradykinin and des-Arg9-bradykinin, but little reactivities for the kinin fragments, des-Arg1-bradykinin and des-Phe8-Arg9-bradykinin, or for lysyl-bradykinin and methionyl-lysyl-bradykinin. The second group was similar to the first except that it showed about a 2.5- to 3.5-fold greater reactivity for des-Arg9-bradykinin than for bradykinin. The third group, which had the lowest affinities for bradykinin [50% inhibition of antibody binding to an enzyme-linked immunosorbent assay (ELISA) plate occurring with bradykinin concentrations ranging from about 8 to 39 nM], showed little reactivities with des-Arg1-bradykinin, des-Arg9-bradykinin and des-Phe8-Arg9-bradykinin, but 50-100% cross-reactivities with lysyl-bradykinin and methionyl-lysyl-bradykinin. The useful ranges for bradykinin detection (ng/well, 50 microL assay volume) using the highest affinity antibody in each group in ELISAs were: 0.01 to 0.5, 0.03 to 3, and 0.1 to 3 for groups 1, 2, and 3, respectively.