Detection of autoantibodies to the TSH receptor (TSH-R) in Graves' disease has found widespread use in clinical routine and is performed mostly by commercial RRAs measuring TSH binding inhibitory activity. We report in this study on a second generation TSH binding inhibitory assay using the human recombinant TSH-R with two major improvements: 1) superior diagnostic sensitivity for Graves' disease, and 2) for the first time, nonradioactive and radioactive coated tube (CT) technology. Full-length human recombinant TSH-R was expressed in the K562 leukemia cell line and grown in suspension at a high density. A murine monoclonal antibody was selected for binding to the native TSH-R without interfering with autoantibodies or TSH and was coated to polystyrene tubes. After detergent extraction, TSH-R was affinity immobilized on antibody-coated tubes. The binding of TSH to the TSH-R could be demonstrated by the addition of 125I- or acridinium ester-labeled bovine TSH, and this binding could be inhibited by sera from patients with Graves' disease up to 95%. Subsequently, these novel assays, a CT RRA and a CT luminescence receptor assay, were compared to the conventional RRA based on porcine antigen in a blinded clinical multicenter trial. Sera from 328 patients with Graves' disease (86 untreated, 116 treated, and 126 in remission) and 520 controls (comprised of healthy blood donors and patients with autoimmune diseases or goiter) were tested in all 3 assays. Receiver-operating characteristic plot analysis resulted in a specificity of 99.6% with a sensitivity of 98.8% for both CT assays, compared to 99.6% specificity and 80.2% sensitivity for the conventional RRA (P < 0.001). In all 3 groups of patients with Graves' disease, the 2 CT assays were significantly more sensitive for the disease than the conventional assay, without loss of specificity in the control groups. This increase in sensitivity and the nonradioactive or radioactive CT format constitute a significant improvement over the currently available assays.

In the serum of patients with malignant endocrine gastroenteropancreatic (GEP) tumours, both alpha-subunit (alpha-SU), common to all glycoprotein hormones, as well as free beta-subunit of human chorionic gonadotropin (hCG-beta) have been reported to be elevated in a substantial fraction. Both have been discussed as markers of malignancy in these neoplasms. In the present study we evaluated the diagnostic significance of alpha-SU and hCG-beta as serum markers in patients with malignant endocrine gastroenteropancreatic tumours. The study group consisted of 52 patients with endocrine GEP-malignancies (24 nonfunctioning, 23 carcinoid syndromes, four gastrinoma, one glucagonoma), located in the small intestine (n = 29), pancreas (n = 17), colon or rectum (n = 3), retroperitoneum (n = 2) and stomach (n = 1). alpha-SU and hCG-beta immunoreactivity was also assessed in the serum of patients with benign GEP-tumors (five insulinoma, and three gastrinoma). Concentrations of alpha-SU and hCG-beta were determined using two highly sensitive and specific immunoradiometric assays employing two monoclonal antibodies each. In 19 of 52 patients (37%), either alpha-SU (n = 9), hCG-beta (n = 7) or both subunits (n = 3) were elevated. In the subgroup of 24 patients with nonfunctioning GEP-tumours, increased concentrations of either alpha-SU (n = 6) or hCG-beta(n = 3) or both subunits (n = 1) were found in 10 of 24 patients (42%). In four of 23 patients with carcinoid syndrome (17%), either alpha-SU (n = 2), hCG-beta(n = 1) or both subunits (n = 1) were above the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

We present the results of a pre-evaluation of the thyroid function test free thyroxine, free triiodothyronine and third generation TSH using the Elecsys electrochemiluminescence immunoassay system. A collaborative field study between the development center of the manufacturer and a clinical chemistry laboratory addressed the reliability and comparability of the new Elecsys assays to established methods under clinical laboratory conditions using samples from routine in vitro thyroid testing. Preliminary (reference) formulations of the reagents and several electrochemiluminescent pilot models were used for assay measurements, either in the company's research center or in the clinical setting. The new thyroid assays were compared with the respective Enzymun-Test assays, performed on the ES300 automated immunoassay analyzer. A WHO standard was used for standardization of TSH, whereas an equilibrium dialysis method was applied for free triiodothyronine. The free thyroxine assay was standardized against the Enzymun-Test free thyroxine assay, which had previously been calibrated against equilibrium dialysis. The aim of this field study was to support the optimization of the technology used for Elecsys in an early stage of development and thereby prepare the ground for the adaptation of the immunoassays to the final Elecsys 2010 random access analyzer. A subsequent multicenter evaluation demonstrated that the requirements of routine thyroid testing in terms of reliability were fulfilled by the system.

Toxic thyroid nodules have been shown to be of clonal origin. In a portion of them, point mutations affecting either the gene of the TSH receptor (TSHr) or the alpha-subunit of stimulating G-protein, consecutively leading to enhanced cAMP levels, which may enhance growth or functional activity of the thyrocyte or both, were recently found. To complement these studies, we evaluated hormone response (i.e. T3 release) in vitro from tissues derived from toxic thyroid nodules as compared directly to the surrounding paranodular tissues as well as tissues derived from euthyroid goiter and from patients with Graves' disease. Experiments were conducted in the presence and absence of bTSH or Graves' immunoglobulines. Tissues obtained during surgery were incubated over 5 h, followed by equilibrium dialysis for 24 h, and determination of free T3 in an aliquot by RIA. Basal T3 release in nodular tissues (n = 10) was significantly higher (median: 7.3 ng/l) compared to paranodular tissues (3.2 ng/l; P < 0.01), tissues derived from euthyroid goiter (1.3 ng/l; n = 12; P < 0.001) and thyroid tissues derived from patients with Graves' disease (2.5 ng/l; n = 6; P < 0.001). Upon stimulation with bTSH (1 IU/l), median T3 concentrations markedly increased to 11.5 ng/l (P < 0.05), 7.3 ng/l (P < 0.05), 4.2 ng/l (P < 0.01) and 3.2 ng/l (P = N.S.), respectively. Stimulation over basal values was 1.6-fold in nodular tissues, 2.3-fold in paranodular tissues, 3.2-fold in euthyroid goiter and 1.3-fold in Graves' disease. In toxic thyroid nodules basal hormone-releasing activities were stimulated by fifteen out of twenty (75%) Graves' sera tested. For comparison, stimulation in other tissues occurred in 45% (paranodular), 80% (euthyroid goiter) and 35% (Graves' disease), respectively. In conclusion, tissue derived from toxic thyroid nodules exhibits enhanced basal hormone release as compared to both, the surrounding paranodular tissues and tissues from euthyroid goiter in vitro, which may reflect constitutional activation of TSHr, alpha-subunit of stimulating G-protein or other so far unknown intermediate by point mutations affecting the respective genes. Hyperactivities in toxic thyroid nodules may be even further enhanced by external stimulators such as TSH or TSH receptor antibodies. The first stimulator may have clinical relevance in patients with toxic thyroid nodules and not yet suppressed TSH; the latter could play a role in the rare Marine Lenhart syndrome.

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