Common and Unique Idiotypes in the Murine Immune Response to Bovine Insulin.

Abstract

The purpose of this research is to develop the murine immune response to bovine insulin as a model system for the examination of antibody diversity generated in response to a protein antigen. These studies provide information not only on the heterogeneity among antibodies made in response to a complex antigen, but, also demonstrate shared structural features among monoclonal antibodies which bind to different epitopes on the same antigen. It has been shown utilizing the technique of isoelectric focusing (IEF), that individual mice responding to bovine insulin present spectrotypes of limited heterogeneity that are not conserved between individuals. Competition of ('125)I-bovine insulin reactive b and s with cold bovine or cold porcine insulin show that the majority of the response is more reactive with bovine insulin than porcine insulin. Thus, most of the serum antibodies made against bovine insulin are sensitive to the two amino acid difference between bovine and porcine insulin which resides in the intrachain disulfide bonded A-loop. To begin dissection of the antibody response to bovine insulin a panel of 25 monoclonal antibodies (HP) was prepared against bovine insulin. Anti-idiotypic sera were produced in guinea pigs against three of these HP; two, anti-5.10C6 and anti-5.2B8, were found to define public idiotypes. The level of the 5.10C6 idiotype is controlled by genes linked to the Igh locus. In situ localization of b and s in immune sera which cross-reacted with the anti-5.10C6 or anti-5.2B8 antisera indicated that these idiotypes were present on heterogeneous populations of antibodies. Both the anti-5.10C6 and anti-5.2B8 sera were found to define distinct groups of idiotypically related HP's. The 5.10C6 idiotype group contains three closely related members, 5.10C6, 5.5F10, and 5.4C5, which were shown to bear the same L-chain by IEF. The 5.2B8 idiotype group has seven members which were less closely related as defined by inhibition SRIA. The two most closely related members of this family, 5.2B8 and 5.3C6, possessed co-focusing L-chains. However, within each idiotype group, members were found which bound to distinct determinants on insulin. Four of the members of the 5.2B8 idiotype group appeared to bind overlapping, but distinct determinants on insulin which define a topographical region that includes the three amino acid A-loop.