ORF49-06 - New Insights into the Graves' Disease Reactome - Characterization of Hinge Region TSH Receptor Antibodies in the Serum of Patients with Graves' Disease

Objective: The TSH receptor antibody (TSHR-Ab) reactome found in patients with Graves’ disease (GD) includes a variety of autoantibodies with differing biological action. We have previously shown that one group of such antibodies, often referred to as “Neutral” or “Hinge region” antibodies have the potential to induce thyroid cell apoptosis and potentially limit the degree of hyperthyroidism. This study aimed to define the prevalence of such hinge-region TSHR Abs in the serum of patients with GD using a newly established cell-based detection platform.

Methods: Serum samples were collected from a consecutive series of 42 patients with GD and 8 healthy controls. The presence of TSHR binding antibodies was assessed by flow cytometry, and stimulating TSHR Abs (TSAb) were measured using our luciferase-based Glo cell bioassay. We generated a novel CHO-TSHR-Hinge cell line expressing the human TSHR hinge region on the cell surface via a GPI-linker and used flow cytometry to detect hinge-region antibodies. A series of ELISAs using ten overlapping 20-mer peptides, which covered the entire human TSHR hinge region (amino acids 280–410) was used to characterize hinge region antibodies. Positivity was determined using the cut-off criteria of ±2 SD above mean of the control group.

Results: Among 42 patients with GD, 30 (71.4%) still showed TSHR binding antibodies by flow cytometry, and 26 (61.90%) had stimulating TSHR antibody detected by TSHR Glo assay. Thirteen patients (30.95%) demonstrated detectable hinge region TSHR antibodies without reactivity to CHO-only cells. Peptide ELISAs identified 11 of these 13 patients with significant responses to one or more hinge peptides. Specificity of such antibodies was confirmed using unlabeled peptide competition.

Conclusion: A new TSHR hinge-region assay detected TSHR antibodies in 30% of GD patients which indicated that the immune repertoire in GD is broad and may play an important role in the disease process. These data provide further evidence that hinge region TSHR antibodies may help shape the clinical picture secondary to their pro-apoptotic potential.