OBJECTIVE: 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. METHODS AND PATIENTS: 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. RESULTS: 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. CONCLUSION: 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.