Analysis of Autoantibody Reactivity in Patients with Graves' Disease Using Recombinant Extracellular Domain of the Human Thyrotropin Receptor and Synthetic Peptides

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Abstract
Graves' disease is characterized by hyperthyroidism leading to enhanced production of thyroid hormones. Hyperthyroidism is primarily mediated by the binding of autoantibodies to the thyrotropin receptor (TSHr). In the past, either thyroid cells or thyroid membranes were used as a source of TSHr to detect anti-TSHr antibodies. Recently, we expressed the extracellular domain of the human TSHr (ETSHr) using the baculovirus expression system. In this study, we used ETSHr protein in an ELISA to detect anti-TSHr antibodies. Our data show that this assay can be used to analyze and quantitate isotype specific antibodies against the TSHr. To map immunogenic epitopes on the TSHr, we tested patients sera against synthetic peptides derived from two highly immunogenic regions (amino acid, AA 12-46 and 316-397) of the receptor. Although sera from patients with Graves' disease reacted with several peptides, they showed particularly strong reactivity against peptides from a relatively narrow region (i.e. AA 352-394) of the TSHr. The present study demonstrates the usefulness of the recombinant ETSHr to detect and characterize anti-TSHr antibodies in a simple and sensitive ELISA, and has lead to the identification of some of the immunoreactive epitopes on the TSHr.

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