DEFINING THE MAJOR ANTIBODY EPITOPES ON THE HUMAN THYROTROPIN RECEPTOR IN IMMUNIZED MICE - EVIDENCE FOR INTRAMOLECULAR EPITOPE SPREADING

To evaluate the B cell response to the extracellular domain of the hum an TSH receptor (hTSHR-ecd), we used recombinant hTSHR-ecd to immunize BALB/c mice (group A) and CBA/J mice (groups B and C). Mice from grou ps A and B were boosted once, and mice from group C received three ant igen boosts. All individual mice developed highly specific hTSHR-ecd a ntibodies (hTSHR-ecd-Ab), confirmed by Western blot analyses. The B ce ll epitopes recognized by these murine hTSHR-ecd-Ab were mapped by enz yme-linked immunoassays using 26 synthetic overlapping peptides spanni ng the entire mature hTSHR-ecd [amino acids (aa) 22-415], i.e. without the signal sequence. Although all BALB/c and CBA/J mice antisera reco gnized peptide 1 (aa 22-41), the hyperimmunized CBA/J mice (group C) d emonstrated recognition of additional peptides (numbers 21-26) cluster ed toward the carboxyl-terminus of the hTSHR-ecd (aa 322-415). Further more, group C serum blocked the binding of [I-125]bTSH to native porci ne TSHR, whereas sera from groups A and B were inactive. We were also able to map the B cell epitopes of antisera from rabbits immunized rep eatedly with hTSHR-ecd and found the same recognition pattern of pepti de 1 and additional peptides clustered near the carboxyl-terminus of t he hTSHR-ecd (aa 322-341 and 367-415). These rabbit antisera also inhi bited the binding of [I-125]bTSH to native porcine TSHR. These data pr ovide a comprehensive B cell. epitope-mapping study of induced hTSHR-e cd-Ab and demonstrate intramolecular spreading of the epitopes recogni zed. Although the N-terminal region was highly antigenic, repeated imm unization induced hTSHR-ecd-Ab targeted to a region critical for TSH b inding.