Neutralization by purified TSHR A subunits of TSHR autoantibodies from Graves patients with hyperthyroidism. Flow cytometry was performed using sera from five Graves patients whose signal upon flow cytometry gave the largest differential between recognition of the wild-type TSHR and GPI-anchored ectodomain (Figure 4a). Sera were preincubated in either buffer alone or in buffer containing a large excess of TSHR-289 (0.1 μg/0.1 ml of serum diluted 1:50) before addition to CHO cells expressing ECD-GPI. TSHR-289 is a secreted, soluble, heavily glycosylated protein that comprises amino acid residues 22–289 of the TSHR, essentially the A subunit (residues 1–21 being the signal peptide). Net fluorescence values for the sera are indicated after subtraction of background fluorescence obtained with untransfected CHO cells.