Characterization of epitope regions of thyrotropin β-subunit recognized by the monoclonal antibodies mAb279 and mAb299:: a chimeric peptide approach

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSH beta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSH beta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop LI [bTSH beta(14-20)] and loop L3 [bTSH beta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSH beta[Cys(16)-Cys(67)]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSH beta [I-125-bTSH beta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSH beta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of I-125-bTSH beta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSH beta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSH beta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.