Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin

Abstract

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG 1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr 1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH 2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg 1-bradykinin and des-Arg 9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.