Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: Evidence for intramolecular epitope spreading

H. Vlase, M. Nakashima, P. N. Graves, Yaron Tomer, J. C. Morris, T. F. Davies

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

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 sara 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 call 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.

Original languageEnglish (US)
Pages (from-to)4415-4423
Number of pages9
JournalEndocrinology
Volume136
Issue number10
StatePublished - 1995
Externally publishedYes

Fingerprint

Thyrotropin Receptors
Epitopes
Inbred CBA Mouse
Peptides
Antibodies
B-Lymphocyte Epitopes
Amino Acids
Immune Sera
Swine
Rabbits
Epitope Mapping
Protein Sorting Signals
Immunoenzyme Techniques
Immunization
B-Lymphocytes
Western Blotting
Antigens
Serum

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice : Evidence for intramolecular epitope spreading. / Vlase, H.; Nakashima, M.; Graves, P. N.; Tomer, Yaron; Morris, J. C.; Davies, T. F.

In: Endocrinology, Vol. 136, No. 10, 1995, p. 4415-4423.

Research output: Contribution to journalArticle

Vlase, H. ; Nakashima, M. ; Graves, P. N. ; Tomer, Yaron ; Morris, J. C. ; Davies, T. F. / Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice : Evidence for intramolecular epitope spreading. In: Endocrinology. 1995 ; Vol. 136, No. 10. pp. 4415-4423.
@article{c6c1d2c812d34fbab26c04048d3023f4,
title = "Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: Evidence for intramolecular epitope spreading",
abstract = "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 sara 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 call 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.",
author = "H. Vlase and M. Nakashima and Graves, {P. N.} and Yaron Tomer and Morris, {J. C.} and Davies, {T. F.}",
year = "1995",
language = "English (US)",
volume = "136",
pages = "4415--4423",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "10",

}

TY - JOUR

T1 - Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice

T2 - Evidence for intramolecular epitope spreading

AU - Vlase, H.

AU - Nakashima, M.

AU - Graves, P. N.

AU - Tomer, Yaron

AU - Morris, J. C.

AU - Davies, T. F.

PY - 1995

Y1 - 1995

N2 - 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 sara 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 call 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.

AB - 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 sara 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 call 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.

UR - http://www.scopus.com/inward/record.url?scp=0029093756&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029093756&partnerID=8YFLogxK

M3 - Article

C2 - 7664661

AN - SCOPUS:0029093756

VL - 136

SP - 4415

EP - 4423

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 10

ER -