Thyrotropin Receptor Autoantibodies in Serum Are Present at Much Lower Levels Than Thyroid Peroxidase Autoantibodies: Analysis by Flow Cytometry

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Abstract
Using Chinese hamster ovary (CHO) cells that express high num- bers of TSH receptor (TSHR) on their surface, we studied the feasi- bility of detecting directly by flow cytometry the binding of autoan- tibodies in patients' sera to the native TSHR. After using a serum (BBl) with high potency in the TSH binding inhibition (TBI) assay to establish the protocol, we studied an additional 38 sera: 10 without TBI activity (1- 4.2% inhibition), 10 with moderately high TBI values (17.3-39.4% inhibition), 10 with high TBI levels (52-95.1% inhibi- tion), 4 from normal individuals without autoimmune thyroid disease, and 4 from patients with systemic lupus erythematosus. We observed that a number of sera, including some without thyroid autoantibodies, contain antibodies against unknown antigens on CHO cells. Pread- sorption with untransfected CHO cells before addition to the TSHR- 10,000 cells eliminated or greatly reduced this nonspecific back- ground. None of the sera from normal individuals, subjects with negative TBI values, or patients with systemic autoimmunity gen- erated a positive signal on flow cytometry with TSHR-10,000 cells relative to the signal on untransfected cells. Remarkably, only 4 of 21 TBI-positive sera (including BBl) unequivocally recognized the TSHR on flow cytometry. In contrast, when thyroid peroxidase (TPO) au- toantibodies in the same sera were studied using CHO cells overex- pressing TPO on their surface, all 20 sera with TPO autoantibodies clearly elicited positive net fluorescence relative to untransfected cells. Study of the potent serum, BBl, revealed similar fluorescence (;250 U) for TPO autoantibodies and TSHR autoantibodies at dilu- tions of 1:1000 and 1:10, respectively. Thus, by flow cytometry, the titer of TPO autoantibodies in the BBl serum is about 100-fold higher than that for TSHR autoantibodies in the same serum. In conclusion, the present data provide the strongest support for the idea that TSHR autoantibodies in the sera of patients with au- toimmune thyroid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves' disease. (J Clin Endocrinol Metab 82: 500 -507, 1997)

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