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, IgG2a, and IgG2b. 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.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555-1019, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Seetharamaiah
authors.forename: N M
authors.initials: NM
authors.lastname: Wagle
authors.forename: J C
authors.initials: JC
authors.lastname: Morris
authors.forename: B S
authors.initials: BS
authors.lastname: Prabhakar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism*; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D013972:Thyrotropin / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin