Idiotypes on monoclonal antibodies to bovine insulin. I. Two public idiotypes on anti-bovine insulin hybridomas define idiotypically distinct families of hybridomas.

Abstract

We used isoelectric focusing (IEF) to show that individual mice responding to bovine insulin (BI) present complex spectrotypes that are not conserved between individuals. Competition of 125I-BI-reactive bands with cold BI or cold pork insulin (PI), showed that the majority of the antibody response is sensitive to the two amino acid difference between BI and PI. This difference resides in the three amino acid intrachain disulfide-bonded A-loop. To begin dissection of the antibody response to BI, we prepared a panel of 30 monoclonal antibodies against insulin. Anti-idiotypic sera were produced in guinea pigs against seven of these hybridoma proteins (HP), and 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. Both the G.P. anti-5.10C6 and G.P. anti-5.2B8 were found to define distinct groups of idiotypically related HP. The 5.10C6 idiotype group contains three closely related members, 5.10C6, 5.5F10, and 5.4C5, which are shown to bear the same L chain by IEF. The 5.2B8 idiotype group has six members that are less closely related, defined by inhibition solid phase RIA. The two most closely related members of this family, 5.2B8 and 5.3C6, possess co-focusing L chains. Within each idiotype group, however, members were found that bind to distinct determinants on insulin. Four of the members of the 5.2B8 idiotype group appear to bind overlapping determinants on insulin that define a topographic region that includes the three amino acid A-loop. Two other HP that have been shown to bind the A-loop do not bear determinants that cross-react with the G.P. anti-5.2B8 antiserum.