(a) Stimulating TSHR autoantibodies recognize the wild-type TSHR less well than the TSHR ectodomain with a GPI anchor. Sera (diluted 1:50) from hyperthyroid Graves patients, all with high levels of TSHR autoantibodies, were tested by flow cytometry on cells expressing the wild-type TSH holoreceptor (TSHR-10,000) and the TSHR ectodomain with a GPI anchor (see Methods). Net mean fluorescence values for the Graves sera (and for control sera from normal individuals) are plotted after subtraction of background fluorescence obtained with untransfected CHO cells. Statistical significance of differences between individual Graves serum recognition of the TSHR-10,000 and the ECD-GPI cells was determined by the paired t test; P < 0.001. (b) Sera from rare hypothyroid patients with TBAb’s show no difference in their recognition of the wild-type TSHR and the GPI-anchored TSHR ectodomain. Sera (diluted 1:50) were tested by flow cytometry on cells expressing the wild-type TSH holoreceptor (TSHR-10,000) and the TSHR ectodomain with a GPI anchor (see Methods). Net mean fluorescence values for the sera (and for control sera from normal individuals) are indicated after subtraction of background fluorescence obtained with untransfected CHO cells. Note the difference scales for the Y axes in a and b, reflecting the higher titers of TBAb’s versus TSAb’s.