A Bioassay for Thyroid Stimulating Immunoglobulins of Patients with Graves' Disease Using Porcine Thyroid Monolayer Cells

抄録

A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2×10⁶ cells/1.5ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1ml of serum-free, 0.5mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay.
Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30min. Responses of cAMP were in a dose-related manner up to 10mU/m1 of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228±51.8%, mean±SD; 127-393%, range) than normal subjects (103±13.3%, mean±SD, n=24;80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI.
These results indicate that this bioassay is clinically useful for detecting TSI.