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 human 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 groups A and B were boosted once, and mice from group C received three antigen boosts. All individual mice developed highly specific hTSHR-ecd antibodies (hTSHR-ecd-Ab), confirmed by Western blot analyses. The B cell epitopes recognized by these murine hTSHR-ecd-Ab were mapped by enzyme-linked immunoassays using 26 synthetic overlapping peptides spanning 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 recognized peptide 1 (aa 22-41), the hyperimmunized CBA/J mice (group C) demonstrated recognition of additional peptides (numbers 21-26) clustered toward the carboxyl-terminus of the hTSHR-ecd (aa 322-415). Furthermore, group C serum blocked the binding of [125I]bTSH to native porcine 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 repeatedly with hTSHR-ecd and found the same recognition pattern of peptide 1 and additional peptides clustered near the carboxyl-terminus of the hTSHR-ecd (aa 322-341 and 367-415). These rabbit antisera also inhibited the binding of [125I]bTSH to native porcine TSHR. These data provide a comprehensive B cell epitope-mapping study of induced hTSHR-ecd-Ab and demonstrate intramolecular spreading of the epitopes recognized. Although the N-terminal region was highly antigenic, repeated immunization induced hTSHR-ecd-Ab targeted to a region critical for TSH binding.
Vlase
H
H
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Nakashima
M
M
Graves
P N
PN
Tomer
Y
Y
Morris
J C
JC
Davies
T F
TF
D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D011817:Rabbits; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins