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

We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (trunc ated at amino acid residue 289 with a B-histidine tail) to investigate prop erties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 ( similar to 20 -40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Gra ves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, t o partially purified TSHR-289. Screening of a TSHR complementary DNA fragme nt 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 conf ormational. 3BD10 preferentially recognized native, as opposed to reduced a nd denatured, TSHR-289, but did not interact with the TSH holoreceptor on t he cell surface, Moreover, mAb 3BD10 could extract from culture medium TSHR -289 nonreactive with autoantibodies, but not the lesser amount (similar to 25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Althoug h the active form of TSHR-289 in culture medium was stable at ambient tempe rature, 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 exq uisite conformational requirements of TSHR autoantibodies. Even under "nati ve" conditions, only a minority of molecules in highly potent TSHR-289 prep arations neutralize patients' autoantibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than pr eviously thought. Finally, reciprocally exclusive binding to TSHR-289 by hu man autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.