Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor

L. Chiovato, P. Vitti, G. Bendinelli, F. Santini, E. Fiore, A. Capaccioli, M. Tonacchera, C. Mammoli, M. Ludgate, A. Pinchera

Research output: Contribution to journalArticlepeer-review

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°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. 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

Original languageEnglish
Pages (from-to)809-816
Number of pages8
JournalJournal of Endocrinological Investigation
Volume17
Issue number10
DOIs
Publication statusPublished - 1994

Keywords

  • autoimmune thyroiditis
  • CHO-R cells
  • FRTL-5 cells
  • hypothyroidism
  • TSH blocking antibody
  • TSH-receptor antibody

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

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