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 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)
Chiovato
L
L
Istituto di Endocrinologia, University of Pisa, Italy.
Vitti
P
P
Bendinelli
G
G
Santini
F
F
Fiore
E
E
Capaccioli
A
A
Tonacchera
M
M
Mammoli
C
C
Ludgate
M
M
Pinchera
A
A
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
D000906:Antibodies; D011989:Receptors, Thyrotropin; D013972:Thyrotropin