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

Abstract

Using Chinese hamster ovary (CHO) cells that express high numbers of TSH receptor (TSHR) on their surface, we studied the feasibility of detecting directly by flow cytometry the binding of autoantibodies 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% inhibition), 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. Preadsorption with untransfected CHO cells before addition to the TSHR-10,000 cells eliminated or greatly reduced this nonspecific background. None of the sera from normal individuals, subjects with negative TBI values, or patients with systemic autoimmunity generated 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) autoantibodies in the same sera were studied using CHO cells overexpressing 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 (ϳ250 U) for TPO autoantibodies and TSHR autoantibodies at dilutions 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 autoimmune 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)

Purification and Characterization of a Soluble Bioactive Amino-Terminal Extracellular Domain of the Human Thyrotropin Receptor

“…Titration experiments demonstrated that neutralization of 50% of the cAMP-stimulating activity of 50 µL of serum was achieved by 5 pmol (5 nM final concentration) of the ectodomain. This result was consistent with previous flow cytometry experiments (30,45) demonstrating low concentrations of TSAb in Graves' sera, as compared with blocking antibodies (TSBAb). It was also compatible with data obtained with the ECD261 (28).…”

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

“…This quantitative information will be useful as a future benchmark by which to judge the potency of purified TSHR of bacterial and insect cell origin. A corollary of the minute amount of antigen necessary for TSHR autoantibody neutralization is that the autoantibody concentration in patients' sera is very low, consistent with previous flow cytometry data (40). The large difference between TSHR and thyroid peroxidase autoantibody concentrations is of potential significance in understanding the pathogenesis of Graves' disease (56).…”

Production of bioactive amino‐terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor

“…Direct detection of the interaction between autoantibodies and the TSH receptor has been achieved in rare instances, and only with a handful of potent antisera [33, 34]. It was concluded from these observations that TSAbs must be present in very low concentrations in the serum of patients with Graves' disease [33, 34]. Incubation of intact GT14 cells with a panel of sera from patients with Graves' disease or autoimmune hypothyroidism yielded clearly positive signals in flow cytometry for the majority of them (Fig.…”

“…This observation suggests that blocking antibodies have a higher titer than stimulating antibodies. Whether the positive results obtained with the GPI‐ECD are explained by the higher amounts of antigens at the surface of GT14 cells, as compared to those expressing the holoreceptor [33, 34], or to a structural peculiarity of the GPI‐ECD antigen remains to be determined. The GPI‐TSHr at the surface of GT14 cells constitutes a novel reagent, with promising characteristics for the development of new direct assays of autoantibodies against the TSHr.…”