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Demonstration of Immunoglobulin G, A, and E Autoantibodies to the Human Thyrotropin Receptor Using Flow Cytometry

Department of Medicine, Clinical Sciences Center, Northern General Hospital (R.M., P.W., A.W.), Sheffield, United Kingdom S5 7AU; and Departments of Medicine (N.J., S.G., M.C., M.L.) and Pathology (M.W.), University of Wales College of Medicine, and Department of Medical Biochemistry, University Hospital of Wales National Health Service Trust (N.J., C.E.), Heath Park, Cardiff, United Kingdom CF14 4XN

Address all correspondence and requests for reprints to: Dr. M. Ludgate, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom CF14 4XN. E-mail: . ludgate{at}cf.ac.uk

Abstract

Human TSH receptor (TSHR) autoantibodies with biological activity result in thyroid dysfunction, but antibodies that simply bind do not. We have applied flow cytometry to the measurements of IgG, IgA, and IgE immunoreactivity to the TSHR in patients with Graves’ disease (GD) and thyroid eye disease (TED) and in normal controls.

CHO cells stably expressing the extracellular domain of the TSHR with a glycophosphatidylinositol anchor were produced and found to express approximately 4 times as many receptors, but of similar affinity, as JP09 in TSH binding studies. Substantial increases in median fluorescence and peak channel fluorescence were obtained by flow cytometry using TSHR monoclonal antibodies on the glycophosphatidylinositol cells.

IgG autoantibodies were demonstrated in 55 of 65 untreated GD patients, 3 of 25 normal subjects, and 4 of 8 atypical TED sera (negative for TSHR autoantibodies with biological activity) by flow cytometry and correlated poorly with thyroid-stimulating antibodies. IgA antibodies were present in 1 of 12 normal, 1 of 7 treated GD with TED, and 3 of 8 atypical TED sera. IgE binding was observed in 1 of 12 normal, 2 of 8 treated GD without TED, 1 of 6 treated GD with TED, and 0 of 8 atypical TED sera.

In conclusion, we have demonstrated autoantibodies that bind directly to the TSHR in the majority of GD patients and in 50% of patients with atypical TED and a small number of normal controls lacking TSHR antibodies that affect function. Although predominantly IgG , TSHR autoantibodies of the IgA and IgE isotypes are also detectable.

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