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Flow Cytometric Analyses of Antibody Binding to Chinese Hamster Ovary Cells Expressing Human Thyrotropin Receptor¹

Departments of Microbiology and Immunology (S.A.P., G.S.S., B.S.P.) and Pediatrics (J.S.D.), University of Texas Medical Branch, Galveston, Texas 77555-1019; and the Cell Regulation Section (K.T., L.D.K.), Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Bellur S. Prabhakar, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019. E-mail: bprabhak{at}mspo6.utmb.edu

To develop a method that can be used to directly detect binding of antibodies to TSH receptor (TSHr), we employed Chinese hamster ovary (CHO) cells permanently transfected with a human TSHr complementary DNA (CHOR). These cells showed increased cAMP production when treated with either human TSH or thyroid-stimulating antibodies and decreased TSH-mediated cAMP production when treated with stimulation-blocking antibodies. We employed flow cytometry and rabbit antibodies against the extracellular domain of the TSHr (ETSHr) to test whether these cells can be used to directly detect and quantitate the binding of anti-TSHr antibodies. Rabbit anti-ETSHr bound specifically to CHOR cells, and the binding could be blocked with purified ETSHr. To test the feasibility of using these cells for epitope mapping, we tested the binding of rabbit antibodies raised against several synthetic TSHr peptides. Rabbit antipeptide 92 (amino acids 12–30) and 91 (amino acids 32–46) showed little or no binding to the CHOR cells. In contrast, antibodies raised against peptides 93 (amino acids 316–330), 95 (aa 325–345), 3A (aa 357–372), 367 (aa 367–386), and 1B (aa 362–376) showed significant binding to the CHOR cells. The specificity of binding of antipeptide antibodies was demonstrated by a complete inhibition of binding by corresponding peptides. When TSH-binding inhibitory Ig-positive sera from 15 patients with hyperthyroidism were tested, 8 of them showed specific binding to the CHOR cells compared to their relative binding to normal CHO cells; sera from all normal individuals tested did not exhibit specific binding to CHOR cells. These studies showed the usefulness of CHOR cells and flow cytometry in epitope mapping using sera with known specificities and the potential usefulness of the technique to detect anti-TSHr antibodies in patient sera.

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