title: Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor.
abstract: 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 TSH, 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. TSHABAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and 16/47 (34.0%) patients with AT.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Istituto di Endocrinologia, University of Pisa, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Chiovato
authors.forename: P
authors.initials: P
authors.lastname: Vitti
authors.forename: G
authors.initials: G
authors.lastname: Bendinelli
authors.forename: F
authors.initials: F
authors.lastname: Santini
authors.forename: E
authors.initials: E
authors.lastname: Fiore
authors.forename: A
authors.initials: A
authors.lastname: Capaccioli
authors.forename: M
authors.initials: M
authors.lastname: Tonacchera
authors.forename: C
authors.initials: C
authors.lastname: Mammoli
authors.forename: M
authors.initials: M
authors.lastname: Ludgate
authors.forename: A
authors.initials: A
authors.lastname: Pinchera
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001667:Binding, Competitive; D016466:CHO Cells / Q000276:immunology* / Q000502:physiology; D002460:Cell Line; D002648:Child; D002999:Clone Cells / Q000502:physiology; D006224:Cricetinae; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics*; D013959:Thyroid Diseases / Q000175:diagnosis; D013961:Thyroid Gland / Q000166:cytology; D013972:Thyrotropin / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000276:immunology*; D014162:Transfection
chemical_list: D000906:Antibodies; D011989:Receptors, Thyrotropin; D013972:Thyrotropin