This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jaume, J. C. Articles by McLachlan, S. M. Search for Related Content PubMed PubMed Citation Articles by Jaume, J. C. Articles by McLachlan, S. M.

Thyrotropin Receptor Autoantibodies in Serum Are Present at Much Lower Levels Than Thyroid Peroxidase Autoantibodies: Analysis by Flow Cytometry¹

Thyroid Molecular Biology Unit, Veterans Administration Medical Center, and the University of California, San Francisco, California 94121

Address all correspondence and requests for reprints to: Juan Carlos Jaume, M.D., Veterans Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.

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 (250 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.

This article has been cited by other articles:

				Y. Mizutori, C.-R. Chen, F. Latrofa, S. M. McLachlan, and B. Rapoport Evidence that Shed Thyrotropin Receptor A Subunits Drive Affinity Maturation of Autoantibodies Causing Graves' Disease J. Clin. Endocrinol. Metab., March 1, 2009; 94(3): 927 - 935. [Abstract] [Full Text] [PDF]

				J. A. Gilbert, A. G. Gianoukakis, S. Salehi, J. Moorhead, P. V. Rao, M. Z. Khan, A. M. McGregor, T. J. Smith, and J. P. Banga Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways. J. Immunol., April 15, 2006; 176(8): 5084 - 5092. [Abstract] [Full Text] [PDF]

				S. M. McLachlan, Y. Nagayama, and B. Rapoport Insight into Graves' Hyperthyroidism from Animal Models Endocr. Rev., October 1, 2005; 26(6): 800 - 832. [Abstract] [Full Text] [PDF]

				S. Costagliola, M. Bonomi, N. G. Morgenthaler, J. Van Durme, V. Panneels, S. Refetoff, and G. Vassart Delineation of the Discontinuous-Conformational Epitope of a Monoclonal Antibody Displaying Full in Vitro and in Vivo Thyrotropin Activity Mol. Endocrinol., December 1, 2004; 18(12): 3020 - 3034. [Abstract] [Full Text] [PDF]

				T. Ando, R. Latif, S. Daniel, K. Eguchi, and T. F. Davies Dissecting Linear and Conformational Epitopes on the Native Thyrotropin Receptor Endocrinology, November 1, 2004; 145(11): 5185 - 5193. [Abstract] [Full Text] [PDF]

				F. Latrofa, G. D. Chazenbalk, P. Pichurin, C.-R. Chen, S. M. McLachlan, and B. Rapoport Affinity-Enrichment of Thyrotropin Receptor Autoantibodies from Graves' Patients and Normal Individuals Provides Insight into Their Properties and Possible Origin from Natural Antibodies J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4734 - 4745. [Abstract] [Full Text] [PDF]

				C.-R. Chen, P. Pichurin, G. D. Chazenbalk, H. Aliesky, Y. Nagayama, S. M. McLachlan, and B. Rapoport Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves' Disease Endocrinology, January 1, 2004; 145(1): 228 - 233. [Abstract] [Full Text] [PDF]

				J. Van Sande, M. J. Costa, C. Massart, S. Swillens, S. Costagliola, J. Orgiazzi, and J. E. Dumont Kinetics of Thyrotropin-Stimulating Hormone (TSH) and Thyroid-Stimulating Antibody Binding and Action on the TSH Receptor in Intact TSH Receptor-Expressing CHO Cells J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5366 - 5374. [Abstract] [Full Text] [PDF]

				L. Schwarz-Lauer, P. N. Pichurin, C.-R. Chen, Y. Nagayama, C. Paras, J. C. Morris, B. Rapoport, and S. M. McLachlan The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors Endocrinology, May 1, 2003; 144(5): 1718 - 1725. [Abstract] [Full Text] [PDF]

				C.-R. Chen, G. D. Chazenbalk, S. M. McLachlan, and B. Rapoport Targeted Restoration of Cleavage in a Noncleaving Thyrotropin Receptor Demonstrates that Cleavage Is Insufficient to Enhance Ligand-Independent Activity Endocrinology, April 1, 2003; 144(4): 1324 - 1330. [Abstract] [Full Text] [PDF]

				T. Ando, M. Imaizumi, P. Graves, P. Unger, and T. F. Davies Induction of Thyroid-Stimulating Hormone Receptor Autoimmunity in Hamsters Endocrinology, February 1, 2003; 144(2): 671 - 680. [Abstract] [Full Text] [PDF]

				R. Metcalfe, N. Jordan, P. Watson, S. Gullu, M. Wiltshire, M. Crisp, C. Evans, A. Weetman, and M. Ludgate Demonstration of Immunoglobulin G, A, and E Autoantibodies to the Human Thyrotropin Receptor Using Flow Cytometry J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1754 - 1761. [Abstract] [Full Text] [PDF]

				P. Pichurin, O. Pichurina, G. D. Chazenbalk, C. Paras, C.-R. Chen, B. Rapoport, and S. M. McLachlan Immune Deviation Away from Th1 in Interferon-{gamma} Knockout Mice Does Not Enhance TSH Receptor Antibody Production after Naked DNA Vaccination Endocrinology, April 1, 2002; 143(4): 1182 - 1189. [Abstract] [Full Text] [PDF]

				G. D. Chazenbalk, S. M. McLachlan, P. Pichurin, X.-M. Yan, and B. Rapoport A Prion-Like Shift between Two Conformational Forms of a Recombinant Thyrotropin Receptor A-Subunit Module: Purification and Stabilization Using Chemical Chaperones of the Form Reactive with Graves' Autoantibodies J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1287 - 1293. [Abstract] [Full Text]

				G. D. Chazenbalk, Y. Wang, J. Guo, J. S. Hutchison, D. Segal, J. C. Jaume, S. M. McLachlan, and B. Rapoport A Mouse Monoclonal Antibody to a Thyrotropin Receptor Ectodomain Variant Provides Insight into the Exquisite Antigenic Conformational Requirement, Epitopes and in Vivo Concentration of Human Autoantibodies J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 702 - 710. [Abstract] [Full Text]

				S. Costagliola, N. G. Morgenthaler, R. Hoermann, K. Badenhoop, J. Struck, D. Freitag, S. Poertl, W. Weglöhner, J. M. Hollidt, B. Quadbeck, et al. Second Generation Assay for Thyrotropin Receptor Antibodies Has Superior Diagnostic Sensitivity for Graves' Disease J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 90 - 97. [Abstract] [Full Text]

				B. Rapoport, G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies Endocr. Rev., December 1, 1998; 19(6): 673 - 716. [Abstract] [Full Text]

				S. Kikuoka, N. Shimojo, K.-I. Yamaguchi, Y. Watanabe, A. Hoshioka, A. Hirai, Y. Saito, K. Tahara, L. D. Kohn, N. Maruyama, et al. The Formation of Thyrotropin Receptor (TSHR) Antibodies in a Graves' Animal Model Requires the N-Terminal Segment of the TSHR Extracellular Domain Endocrinology, April 1, 1998; 139(4): 1891 - 1898. [Abstract] [Full Text] [PDF]

				S. Costagliola, P. Rodien, M.-C. Many, M. Ludgate, and G. Vassart Genetic Immunization Against the Human Thyrotropin Receptor Causes Thyroiditis and Allows Production of Monoclonal Antibodies Recognizing the Native Receptor J. Immunol., February 1, 1998; 160(3): 1458 - 1465. [Abstract] [Full Text] [PDF]

				G. D. Chazenbalk, J. C. Jaume, S. M. McLachlan, and B. Rapoport Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera J. Biol. Chem., July 25, 1997; 272(30): 18959 - 18965. [Abstract] [Full Text] [PDF]

				A. Kakinuma, G. D. Chazenbalk, J. C. Jaume, B. Rapoport, and S. M. McLachlan The Human Thyrotropin (TSH) Receptor in a TSH Binding Inhibition Assay for TSH Receptor Autoantibodies J. Clin. Endocrinol. Metab., July 1, 1997; 82(7): 2129 - 2134. [Abstract] [Full Text] [PDF]

				C.-R. Chen, K. Tanaka, G. D. Chazenbalk, S. M. McLachlan, and B. Rapoport A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain J. Biol. Chem., April 27, 2001; 276(18): 14767 - 14772. [Abstract] [Full Text] [PDF]