DETECTION OF ANTIBODIES BLOCKING THYROTROPIN EFFECT USING CHINESE-HAMSTER OVARY CELLS TRANSFECTED WITH THE CLONED HUMAN TSH RECEPTOR

Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto's thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5-2 mg/ml), TSH alone (0.2-625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37 degrees C in 5% CO2 -95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by ISH, the results obtained ranging between -30% and +18% (mean+/-SD=-3+/-14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (>25%) were considered positive for blocking antibodies. TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and in 16/47 (34.0%) patients with AT. When the same IgGs were tested in FRTL-5 cells, TSHBAb were detected in 1/8 (12.5%) patients with HT-SH, in 5/30 (16.6%) with HT-H and in 15/47 (31.9%) with AT. TSHBAb results in CHO-R cells showed a good correlation with those in FRTL-5 cells (r=0.74, p<0.0001), but 3/24 IgGs were positive for TSHBAb in CHO-R cells and negative in FRTL-5 cells. Using the radioreceptor assay, TSH-binding inhibiting antibodies were detected in 17/24 (70.8%) sera that contained TSHBAb when tested in the CHO-R cell system. Thyroid stimulating antibody (TSAb) and TSHBAb, that coexisted in 5 IgGs, were simultaneously detected using CHO-R cells. These IgGs belonged to patients in whom spontaneous hypothyroidism developed after hyperthyroidism, or viceversa. In conclusion a new in vitro assay for the detection of TSHBAb was developed using CHO-R cells. The sensitivity of this assay is slightly greater than that obtained in FRTL-5 cells and definitely greater than that of the radioreceptor assay. CHO-R cells have the advantages of expressing the human TSH receptor and of requiring less cumbersome procedures for cell culture than FRTL-5 cells.