Molecular and Cellular Endocrinology, 13 (1990) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED R13-R18 Elsevier Scientific Publishers Ireland, Ltd. R13 MOLCEL 02390 Rapid Paper Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSH-R autoantibodies M. Ludgate ‘, J. Perret ‘, M. Parmentier ‘, C. Gerard ‘, F. Libert ‘, J.E. Dumont ’ and G. Vassart 2 ’ I.RI.B.H.N. and 2 Service de Gh%iques, UL.B, Campus Erasme, Brwels 1070, Belgium (Received 13 August 1990; accepted 24 August 1990) Key work Thyrotropiu receptor; Autoimmunity; Graves’ disease; CHO cells We report on two assays for autoantibodies to the TSH-R which have been developed using materials from mammalian cells transfected with the cDNA for the human TSH-R. In the first, a particulate fraction has been prepared from COS cells, transiently expressing the human TSH-R and used in a radioreceptor assay in conjunction with bovine ‘*‘I-TSH. Immunoglobulins (IgGs) from patients with Graves’ disease (n = 11) and idiopathic myxoedema (n = 2) have been used as competitors of ‘251-TSH binding to the COS TSH-R membranes and the results have been compared with those obtained with a commercially available kit for measuring TSH-R autoantibodies, which uses solubilised porcine TSH-R. Both assays showed similar performance, being particularly sensitive to antibodies from patients with idiopathic myxoedema. In the second assay system we have used a CHO cloned cell line (JP26) stably transfected with the human TSH-R. A selection of IgG preparations from patients with Graves’ disease and of six normal controls was used to test the ability of this cell line to detect thyroid stimulating immunoglobulins (TSAb) by increasing its CAMP production. The assay was performed under two conditions: in standard (isotonic) medium or in hypotonic medium. Freshly thawed human thyrocytes incubated in hypotonic medium served as a reference method. Only five patients scored positive when tested in the JP26 cell line under isotonic conditions. When the assay was performed in a hypotonic medium, a significant positive correlation was observed between the results given by JP26 cells and human thyrocytes. We conclude that the recombinant human TSH-R, as expressed in non-thyroid cells, has potential in the measurement of TSH-R autoantibodies and in understanding their mechanism of action. zyxwvutsrqponmlkjihgfed Introduction Patients with hyperthyroid Graves’ disease pro- duce antibodies which mimic the action of TSH (Dorrington and Munro, 1966; Manley et al., 1974), leading to chronic stimulation of adenylate cyclase (TSAb). The autoantibodies in some pa- tients with idiopathic myxoedema are also able to bind to the TSH-R (Grgiazzi et al., 1976; Mat- Address for correspondence: M. Ludgate, I.R.I.B.H.N., ULB, H6pita.l Campus Erasme, B&iment C, 808 route de Lennik, B-1070 Brussels, Belgium. suura et al., 1980), but do not result in an increase in intracellular cyclic AMP (TBAb). Many assays have been developed to measure TSH-R autoanti- 0303-7207/90/$03.50 8 1990 Ekevier Scientific Publishers Ireland, Ltd.