Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: Antibodies can bind to discrete conformational or linear epitopes and block TSH binding

Gattadahalli Seetharamaiah, N. M. Wagle, J. C. Morris, B. S. Prabhakar

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Abstract

Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG(2a), and IgG(2b). The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20%, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39% and 43%, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR.

Original languageEnglish (US)
Pages (from-to)2817-2824
Number of pages8
JournalEndocrinology
Volume136
Issue number7
StatePublished - 1995
Externally publishedYes

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Thyrotropin Receptors
Epitopes
Monoclonal Antibodies
Immunoglobulin G
Amino Acids
Peptides
Competitive Binding
Antibodies
Immunoglobulin Isotypes
Hybridomas
thyrotropin-binding inhibitory immunoglobulin
Thyroid Gland

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{f261c0c492284ca3b3e4e4aab92edac8,
title = "Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: Antibodies can bind to discrete conformational or linear epitopes and block TSH binding",
abstract = "Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG(2a), and IgG(2b). The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20{\%}, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39{\%} and 43{\%}, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR.",
author = "Gattadahalli Seetharamaiah and Wagle, {N. M.} and Morris, {J. C.} and Prabhakar, {B. S.}",
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TY - JOUR

T1 - Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor

T2 - Antibodies can bind to discrete conformational or linear epitopes and block TSH binding

AU - Seetharamaiah, Gattadahalli

AU - Wagle, N. M.

AU - Morris, J. C.

AU - Prabhakar, B. S.

PY - 1995

Y1 - 1995

N2 - Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG(2a), and IgG(2b). The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20%, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39% and 43%, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR.

AB - Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG(2a), and IgG(2b). The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20%, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39% and 43%, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR.

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