Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum

Purification of TSH receptor autoantibodies (TRAb) from the serum of patients with Graves’ disease (GD) might help to elucidate the nature of these disease causing autoantibodies. We describe here for the first time the successful affinity purification of human TRAb. Affinity purification was performed in a four step procedure with human recombinant TSH receptor (TSH-R) expressed in K562 cells. Purification from six different serum pools from patients with GD and two individual sera (one with only thyroid stimulating antibodies (TSAb) one with only thyroid blocking antibodies (TBAb)) resulted in a purity of 39.2±3.8 IU/mg TRAb or 25.7±2.1 μg IgG/IU (about 3.5–13.7 μg TRAb/ml serum). The average enrichment based on the respective original serum was 3420-fold (range 1200–10,000). The kDa of the purified TRAb were in the range of 0.7–2.6×10 −10 M. All purified TRAb (except from the TBAb serum which showed blocking activity) showed a more than 1000-fold stronger stimulation in the TSAb bioassay based on the IgG content than the original serum, and similar stimulation based on international units (IU/l) TRAb. When labelled purified TRAb were used in a competitive assay as tracer instead of bovine TSH, their binding to the human recombinant TSH-R on tubes was displaced by 99 of 100 GD sera (selected for TBII activity). Correlation to the standard TSH tracer was r=0.92. Interestingly, the use of TRAb tracer derived from a patient with TSAb and a patient with TBAb gave virtually identical results ( r=0.93) with these patients, suggesting similar if not identical binding sites for both TRAb subtypes. In conclusion, this is the first report on the purification of human TRAb from the serum of patients with GD. The purified TRAb are of low concentration with high affinity, strong TBII and TSAb activity. Further characterisation may allow new insights in TRAb epitope localisation, the pathology of GD and the differences between TSAb and TBAb. Also, their use as tracer in a competitive assay is the first report on a completely homogenous assay with high sensitivity for TSH-R autoantibodies.