A New in Vitro Assay for Human Thyroid Stimulator Using Cultured Thyroid Cells: Effect of Sodium Chloride on Adenosine 3′,5′-Monophosphate Increase*

Abstract

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.

Predictive value of maternal second-generation thyroid-binding inhibitory immunoglobulin assay for neonatal autoimmune hyperthyroidism

“…Individual cases are shown in Table 2. The median age of the women at the beginning of pregnancy was 31 years (range, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]. Twenty mothers of 21 neonates (one twin pregnancy) had a past history of GD.…”

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

“…Initial dose-response studies were conducted in NaCl-free, sucrose-containing medium routinely used for its increased sensitivity for detecting TSAbs (36). As shown in Fig.…”

“…1A) and therefore also behaved as full agonists for the TSHR. We also performed dose-response studies under physiological salt concentrations, although these assay conditions demonstrate reduced sensitivity compared with the use of salt-free, sucrosecontaining isotonic HBSS buffer (36). The dose-response for TSH induced cAMP production was not altered significantly in the NaCl-containing buffer, with 40U/ml giving maximal stimulation (not shown).…”

Evaluation of a 1 step TRAb Assay for the Detection of High-affinity Components to hTSHR: Evidences Indicating Superiority of the Assay in the Lower TRAb Range

“…Autoantibodies including TRAb are essentially polyclonal, and therefore, heterogeneous TRAb components should exist in individual patient serum [4]. Recent progress in TRAb assay methods, such as the 2 nd generation thyrotropin binding inhibitor immunoglobulin (TBII) assays and the high-sensitivity thyroid stimulating antibody (TSAb) assay, have made it possible to detect TRAb in nearly all Graves' patient sera with very high specificity [5][6][7][8][9]. Clinical diagnosis of Graves' disease became much easier and more relevant.…”