Stimulating and Blocking Thyroid-Stimulating Hormone (TSH) Receptor Autoantibodies from Patients with Graves’ Disease and Autoimmune Hypothyroidism Have Very Similar Concentration, TSH Receptor Affinity, and Binding Sites

Abstract

The distinct biological properties of TSH receptor (TSH-R) autoantibodies (TRAbs) from patients with Graves' disease (GD) are yet unexplained on the molecular level. Here we compare serum concentration, affinity to the TSH-R, and binding sites on the TSH-R of stimulating (TSAb) and blocking (TBAb) TRAbs. Four-step affinity purification using human recombinant TSH-R was performed with 22 TRAb-positive sera from GD patients (11 with only TSAb and 11 with only TBAb) and five control sera. Antibody concentration, TSH binding inhibition (TBII), and TSAb/TBAb activity of the purified TRAb were assessed. Labeled purified TRAbs were used for displacement studies with TRAb and an additional 30 patients and 10 control sera. TRAbs could be purified to 80-93% purity with recovery of the TBII and TSAb and TBAb activity. No TRAbs could be purified from healthy individuals. The mean +/- SD concentration of TRAb was 17.3 +/- 5.4 microg/IU for the TSAb sera (range, 9.6-25.9) and 18.2 +/- 8.5 microg/IU for the TBAb sera (range, 4.6-29.2), respectively (P = 0.79). Affinity was in the picomolar range for both TRAb subtypes with mean +/- sd dissociation constant of 167 +/- 109 pM (60-410 pM) for TSAb and 253 +/- 132 pM (80-410 pM) for TBAb (P = 0.12). Purified and labeled TSAb and TBAb showed a very similar binding pattern to the TSH-R in displacement studies with unlabeled TSAb/TBAb or unpurified patients sera, indicating binding sites on the TSH-R in close proximity to each other. TSAbs and TBAbs in the serum of patients with GD have similar characteristics. They are of low concentration with high affinity and have also similar binding epitopes on the TSH-R.