TSH-binding inhibitor immunoglobulins (TBII) have been detected in patients with Graves' disease and Hashimoto's thyroiditis by using the radioreceptor assay of TSH. In untreated Graves' patients, TBII levels correlated well with thyroidal 99mTc uptake at 30 min and the grade of epithelial hyperplasia of thyroid follicles. There were many Graves' patients whose sera contained high TBII levels but no detectable bioassayable thyroid-stimulating activity (LATS), and in these patients, close correlation was observed between serum levels of TBII and bioassayable LATS-protector activity. TBII were detectable in 2 (10%) of 20 patients with Hashimoto's thyroiditis, both of whom were clinically hypothyroid. The serum or IgG fraction from one of them, however, did not contain any significant LATS, LATS-protector, or human thyroid adenylate cyclase-stimulating activity and caused inhibition of adenylate cyclase stimulation by TSH. In that patient, TBII may be acting to block TSH binding to TSH receptors, thus causing TSH unresponsiveness and hypothyroidism.

A simple, sensitive, and practical assay for thyroid-stimulating autoantibodies (TSAb) was developed in which cryopreserved porcine thyroid cells were incubated with crude immunoglobulin fractions sedimented from serum with polyethylene glycol. In the assay, 1.4- to 2.0-fold and 6- to 12-fold increases in cAMP released into Hank's medium without NaCl were found at 1 and 10 microU/ml bovine TSH, respectively. TSAb were detected in 41 (97.6%) of 42 patients with untreated hyperthyroid Graves' disease, 29 (55.8%) of 52 patients with hyperthyroid Graves' disease who were euthyroid while taking antithyroid drugs, 22 (78.6%) of 28 patients with euthyroid Graves' disease, and none of the patients with simple goiter, adenomatous goiter, thyroid adenoma, or thyroid cancer tested. TSAb activities measured using porcine thyroid cells significantly correlated with those measured using human thyroid adenoma cells (r = 0.908; n = 46; P less than 0.001). Thyroid-stimulating activity was also detected in 11 (28.9%) of 38 patients with Hashimoto's thyroiditis. However, the activity was considered to be due to TSH in the patients' sera, because it was completely abolished by pretreatment with anti-TSH antibodies. Serum TSH concentrations lower than 50 microU/ml did not affect the assay result. In Graves' disease after cessation of antithyroid drugs, 85.7% (12 of 14) of TSAb-positive patients relapsed, while 77.8% (14 of 18) of TSAb-negative patients remained in remission. Thus, the assessment of TSAb was useful as an index to predict prognosis.

A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.