Bispecific antibody against two different epitopes in the same antigen

Abstract

For the enhancement of monoclonal antibody affinity, a bispecific antibody against two different epitopes in the same antigen has been prepared by chemical recombination. After purification of humain chorionic gonadotropin and immunization, splenocytes of immunized mice and myeloma cells were fused using standard PEG fusion technique. Among 288 clones, 8 clones were selected on the basis of subunit specificity, and finally 4 clones(R111, B71, B91, and R40) were selected based upon epitope specificity. The relative positions of epitopes recognized by these clones were investigated by competitive radioimmuno assay, two-site sandwich radioimmuno assay, and additivity assay. After analyzing these three assay data, we selected a pair of antibody (antibody R111 on $\alpha$ -subunit and B91 on $\beta$ -subunit) as a proper pair for the preparation of bispecific antibody. Bispecific antibody between antibody R111 and B91 was prepared by chemical recombination using 5, 5``-dithiobis(2-nitrobenzoic acid), purified by Sephadex G-150, and characterized. By the results of SDS-PAGE, dual antigen binding radioimmuno assay, enzyme linked immunosorbant assay, and western blott analysis, we confirmend that this antibody has bispecificity against two different epitopes, one is on $\alpha$ -subunit and the other is on $\beta$ ,-subunit. Using Scatchard plot and the other graphical analysis, we confirmed that the affinity of bispecific antibody is as much as 17-fold higher than that of monoclonal antibody with higher affinity and bispecific antibody: antigen reaciton is equi-molar interaction. The characterization of binding mechanism between bispecific antibody and its antigen and application for the development of new immuno assay systems remain to be studied.