A Mouse Monoclonal Antibody to a Thyrotropin Receptor Ectodomain Variant Provides Insight into the Exquisite Antigenic Conformational Requirement, Epitopes and in Vivo Concentration of Human Autoantibodies 1

Abstract

We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (truncated at amino acid residue 289 with a 6-histidine tail) to investigate properties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (20-40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Graves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, to partially purified TSHR-289. Screening of a TSHR complementary DNA fragment expression library localized the 3BD10 epitope to 27 amino acids at the N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conformational. 3BD10 preferentially recognized native, as opposed to reduced and denatured, TSHR-289, but did not interact with the TSH holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract from culture medium TSHR-289 nonreactive with autoantibodies, but not the lesser amount (25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Although the active form of TSHR-289 in culture medium was stable at ambient temperature , stability was reduced at 37 C, explaining the mixture of active and inactive molecules in medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoanti-bodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients' auto-antibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally , reciprocally exclusive binding to TSHR-289 by human autoan-tibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition. (J Clin Endocrinol Metab 84: 702-710, 1999)