A stable, reproducible radioreceptorassay for thyrotropin binding inhibiting immunoglobulins (TBII)

A radioreceptorassay for thyrotropin binding inhibiting immunoglobulins (TBII) is presented. A stable reproducible method was established based on the use of thyroid glands obtained by autopsy for the preparation of a highly purified plasma membrane from human thyroid homogenate. Lower detection limit for TSH was below 5 miu/l. The TBII activity was measured in a crude IgG precipitate from serum and the results were calculated as an index value compared to normal controls, the mean of which was defined as 1.00. The binding of [125I] TSH to this membrane preparation was stable for more than 6 months. Interassay variations of the TBII index in three ranges were 1.30 +/- 0.19, 0.37 +/- 0.08 and 0.04 +/- 0.07 (mean +/- SD) over a period of 6 months. The intra-assay variations of the TBII index in three ranges were 1.30 +/- 0.08, 0.46 +/- 0.03 and 0.09 +/- 0.03 (mean +/- SD). Seventy-six of 116 patients with untreated Graves' disease had index values below the reference interval and TBII was also detected in 4 of 14 patients with multinodular goitre. The value of the TBII index in patients with untreated Graves' disease was significantly correlated to S-T3, the 4 h [131I] uptake and the 20 min [99mTc] uptake. No significant correlation between the TBII index and long-acting thyroid stimulator (LATS) measured in a bioassay was found. However, the LATS-positive patients also had positive TBII index.     

The role of precision in determining the performance of a thyrotropin assay in diagnosing hyperthyroidism

We examined the relationship between analytical sensitivity, precision at the lower limit of the reference interval, and diagnostic performance in hyperthyroidism for one radioimmunoassay and five immunometric assay kits for thyrotropin. The analytical sensitivity of these kits extended from 0.05 to 1.56 milli-int. units/L. Diagnostic efficiencies of the immunometric assays, in discriminating between euthyroidism and hyperthyroidism, ranged between 93% and 98%. There was a highly significant correlation (r = 0.99, P less than 0.001) between analytical sensitivity and diagnostic efficiency. The between-assay coefficients of variations, at the lower limit of the reference interval, ranged from 26% to 87%. There was no correlation (r = 0.36) between precision, at this concentration, and diagnostic efficiency. We conclude that analytical sensitivity and not precision is the major determinant in controlling the diagnostic performance of a thyrotropin assay in hyperthyroidism.     

Clinical value of immunoradiometric assay of thyrotropin for patients with nonthyroidal illness and taking various drugs

Using a two-site immunoradiometric assay, we measured concentrations of thyrotropin (TSH) in serum of 134 clinically euthyroid subjects, 93 patients with nonthyroidal illness, and 80 patients who were being treated with various drugs. Abnormal concentrations of TSH, free thyroxin, and free triiodothyronine, respectively, were recorded in serum of three (3.2%), 19 (20.4%), and 37 (39.8%) of the patients with nonthyroidal illness and in three (3.8%), five (6.3%), and 10 (12.5%) of the patients taking drugs. TSH could be detected in all patients' serum samples. We conclude that, for most patients without thyroid disease, a basal (i.e., unstimulated) measurement of their TSH concentration in serum will indicate their thyroid status more reliably than will assay of free thyroxin or free triiodothyronine.     

Clinical application of a sensitive non-isotopic immunometric assay of thyrotropin

We have evaluated an immunometric assay of thyrotropin (TSH) based on enhanced chemiluminescence signal; its detection limit is 0.06 milli-int. unit/L. Values in 101 clinically euthyroid subjects with normal thyroid hormone concentrations ranged from 0.39 to 6.83 milli-int. units/L. TSH in 15 hypothyroid patients ranged from 10.3 to greater than 200 milli-int. units/L, whereas in 31 hyperthyroid subjects with increased concentrations of free thyroxin and free triiodothyronine, TSH was undetectable serum of all but one subject. Of 32 clinically and biochemically euthyroid patients with goiter, two had undetectable serum TSH and six had values below the normal range. In 19 clinically euthyroid patients from an intensive-care unit, TSH was undetectable in two and below the normal range in another two. This immunometric chemiluminescence assay distinguishes thyrotoxic from euthyroid subjects, but caution is required in interpreting TSH values alone in subjects with goiter or nonthyroidal illness.     

Diagnostic value of thyrotropin concentrations in serum as measured by a sensitive immunoradiometric assay

A new, highly sensitive immunoradiometric thyrotropin (TSH) assay involving solid-phase-coupled monoclonal antibodies (Boots-Celltech Sucrosep IRMA-TSH) has been evaluated in a wide variety of patients with thyroidal and nonthyroidal illnesses and the results compared with those obtained by conventional diagnostic TSH RIAs. The sensitivity of the present assay ranged from 0.036 to 0.1 milli-int. unit/L (mean 0.056). TSH, measurable in serum of each of 128 euthyroid patients, ranged from 0.1 to 6.3 milli-int. units/L (mean 1.7, SD 1.1). Similar concentrations were found in 15 healthy pregnant women. TSH was undetectable in 27 hyperthyroid patients, of whom six were tested with thyroliberin stimulation and failed to respond. The mean TSH concentration measured in 62 seriously ill hospital patients of 2.7 (SD 2.5) milli-int. units/L was significantly higher (p less than 0.05) than in the euthyroid patients. Basal values and peak TSH responses to thyroliberin testing correlated well (r = 0.63, n = 48), irrespective of clinical diagnosis. We conclude that the present assay readily discriminates between euthyroid and hyperthyroid patients and should replace conventional TSH RIAs in diagnostic laboratories.     

Evaluation of a new chemiluminescence technique for human thyrotropin (BeriLux hTSH): diagnostic value of five immunometric assay methods.

A new commercially available human thyrotropin immunochemiluminometric assay (ICMA) kit was evaluated. The BeriLux assay (Hoechst Co., Germany) was compared with two other non-radioisotopic methods (AIA-1200 and IMx) and two other immunoradiometric assays (RIA-gnost TSH IRMA and EIKEN IRMA kits) in 32 normal subjects and 104 patients with Graves' disease, divided into seven groups: 1) untreated hyperthyroidism; 2) hyperthyroidism during treatment; 3) euthyroid with negative thyroliberin test (subclinical hyperthyroidism); 4) euthyroid with low thyroliberin test; 5) euthyroid with normal thyroliberin test; 6) euthyroid with high thyrotropin level (subclinical hypothyroidism); and 7) primary hypothyroidism. Patients in groups 2-6 were undergoing treatment with mercazole and propylthiouracil. The new immunoluminometric assay (ILMA) BeriLux kit was shown to have a remarkably improved analytical and clinical sensitivity. The minimal detectable level of thyrotropin in the assay was 0.006 mU/l. The precision was 2.8% and 6.1% at 0.093 +/- 0.003 mU/l and 0.028 +/- 0.002 mU/l, respectively, whereas the precision of the other methods was above 17.2% and 59.4% respectively. Seven patients from the untreated hyperthyroid group were given 500 micrograms thyroliberin i.v. (the thyroliberin test). The thyrotropin pattern before and after thyroliberin administration was always less than 0.006 mU/l with the BeriLux kit, whereas the other methods showed random fluctuations indicating their low accuracy at this concentration. Using the BeriLux kit, 7 of the 16 overt hyperthyroid patients undergoing treatment showed a measurable thyrotropin level below 0.01 mU/l but a negative thyroliberin test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-linked immunosorbent assay (ELISA) for direct quantification of surface-bound platelet immunoglobulins

Surface-bound platelet IgG and IgM were measured by an enzyme-linked immunosorbent assay (ELISA) using washed platelets and commercially available alkaline phosphatase anti-human immunoglobulins (Fc-specific). With this technique platelets from normal donors had small amounts of platelet-bound IgG ranging from 0.00 to 0.16 A405 (absorbance at 405 nm wavelength) (10(7) platelets)-1 (0 to 124 ng) and of platelet-bound IgM ranging from 0.00 to 0.05 A405 (10(7) platelets)-1. Eight out of 10 (80%) thrombocytopenic patients with idiopathic autoimmune thrombocytopenic purpura (IATP) had values of both IgG and IgM exceeding the normal range. In addition, one patient (8%) had platelet-bound IgM only. An inverse relationship was demonstrated in patients with IATP between the blood platelet count and the amount of both IgG and IgM. Increased values were also demonstrated in patients with SLE and patients with monoclonal hypergammaglobulinaemia. The direct ELISA is a useful and reproducible technique for platelet-bound IgG and IgM, which requires standard laboratory equipment only.     

Detection and quantification of erythrocyte-associated immunoglobulins using immunoenzyme technic--enzyme immunometric assay (EIMA)

A new technique is described for the quantification of erythrocyte-associated immunoglobulin G (EAIgG). Enzyme immunometric assay (EIMA) measures the consumption of enzyme-labelled anti-human globulin. Thus, the release of cellular enzymes is avoided and, thereby also falsification of the results owing to the antigen-antibody reaction taking place in media of differing molarity and ionic strength (as is inevitable in red cell lysis assays). This consumption technique is more sensitive than simple "sandwich" procedures. The measurement of minimal amounts of physiologically bound EAIgG is, moreover, possible.     

D-二聚体检测在进展性脑梗死中的临床价值

目的探讨血浆D-二聚体在进展性脑梗死中的变化及其临床应用价值。方法检测并比较47例进展性脑梗死和162例非进展性脑梗死患者血浆中D-二聚体浓度,用ROC曲线评价D-二聚体的诊断性能。结果在脑梗死患者的众多监测指标中,进展性脑梗死患者的血浆D-二聚体浓度明显高于非进展性脑梗死患者(P0.01);在不同时间段(0、24、48、72h),两组间D-二聚体浓度的差异有统计学意义(P0.01);D-二聚体诊断进展性脑梗死的ROC曲线下面积为0.859(95%CI:0.745~0.973),当cut off值为1 510μg/L时,血浆D-二聚体诊断进展性脑梗死的敏感性为95.2%,1-特异性为65.7%。结论血浆D-二聚体检测在辅助诊断进展性脑梗死中具有重要的临床价值。     

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.     

Properties of thyrotropin (TSH) binding to plasma membranes prepared from rat fat tissues

Rat fat membranes were prepared from male Sprague-Dawley rats. At 37 degrees C, TSH binding to rat fat membranes was rapid and unstable, although the binding reached a steady state after 2 hr and unchanged up to 24 hr at 4 degrees C. The binding to rat fat membranes was significantly inhibited by 50 mM NaCl and almost completely inhibited by 150 mM NaCl. TSH binding to rat fat membranes was not affected by 10 mM dithiothreitol (DTT) or 1 mM diamide although the binding to human thyroid membranes was inhibited by 10 mM DTT significantly. Conventional Scatchard analysis revealed a single class of binding site which had lower Ka value (2.6 X 10(8) M-1) than that of high affinity binding site of human thyroid membranes (9.3 X 10(8) M-1). Immunoglobulin G (IgG) from patients with Graves' disease inhibited the binding of TSH to rat fat membranes. A significant correlation was observed between the inhibiting activity of Graves' IgG measured with rat fat and human thyroid membranes (r = 0.82, p less than 0.01). In conclusion, TSH receptors on rat fat membranes were not identical to those on human thyroid membranes, but TSH receptor antibodies crossreacted with TSH receptors in rat fat tissue.     

A sensitive and practical immunoradiometric assay of thyrotropin

We describe a two-site immunoradiometric assay for thyrotropin (TSH) in serum, based on use of two monoclonal antibodies directed against two separate antigenic determinants on the TSH molecule. One antibody is immobilized on polystyrene beads; the other is radioiodinated by a modified Chloramine T method. The detection limit of the assay is 0.02 milli-int. unit/L. The working range (CV less than 10%) is from 0.1 to greater than 50 milli-int. units/L. The log mean concentration of TSH in sera collected from 100 euthyroid subjects between 08:00 and 11:00 hours was 1.9 milli-int. units/L, the range 0.4-5.4 milli-int. units/L. Values for hyperthyroid patients and thyroid-cancer patients being treated with thyroxin were much lower than those for euthyroid persons. Results by this new assay correlated excellently with those by our conventional radioimmunoassay (r = 0.99) and also with a sensitive immunofluorometric TSH method (Delfia TSH) (r = 0.99).     

Innovance D-dimer 试剂在血栓形成早期的筛查价值

目的：评价 Innovance D-dimer 试剂性能及其在血栓形成早期的临床筛查价值。方法采用 Innovance D-dimer 检测试剂,分别对86例疑似血栓患者（血栓组）和40例门诊健康体检者（对照组）进行血浆 D-二聚体水平测定,并进行统计学 t 检验,受试者工作特征曲线（ROC）分析和筛检评价。结果血栓组 D-二聚体水平[（4．14±7．29）mg/L]显著高于对照组[（0．1±0．12）mg/L],差异有统计学意义（P ＝0．000）。ROC 曲线分析结果显示,D-二聚体水平用于血栓的临床筛查有显著统计学意义（AUC＝0．938,P ＝0．000）,D-二聚体水平越高,发生血栓的可能性越大;以 D-二聚体水平为0．50 mg/L 为截断值,其灵敏度、特异度和阴性预测值表现良好,分别为98．08％和100．00％和98．67％。结论Innovance D-dimer 试剂适用于排除血栓形成,但诊断价值有限。     

Use of a sensitive enzyme immunoassay for human thyrotropin in the diagnosis of hyperthyroidism

A sensitive, simultaneous sandwich enzyme immunoassay for TSH was evaluated especially for its ability to distinguish hyperthyroid patients from the euthyroid population. A total of 140 patient samples was analzyed by this assay as well as with a two-step sandwich radioimmunoassay. The diagnostic sensitivity of the thyrotropin assay was 92.5% and the specificity was 88%. False negatives by thyrotropin assay included two patients with Graves' disease who were being treated with propranolol at the time of testing and one patient who was considered hyperthyroid while receiving synthroid. Twelve patients with elevated free thyroxine index levels were considered euthyroid and 50% of these had thyrotropin values that were undetectable; most were elderly patients with nonthyroidal illnesses. Although the thyrotropin enzyme immunoassay had good sensitivity and precision for the detection of hyperthyroidism, our data suggest the limitation of a single thyrotropin determination in establishing the euthyroid state, especially in elderly patients with associated nonthyroidal illnesses and hyperthyroxinemia.     

Use of sensitive immunoradiometric assay for thyrotropin in clinical practice

Although measurement of thyrotropin (thyroid-stimulating hormone; TSH) by radioimmunoassay was a major advance in the laboratory diagnosis of thyroid failure--replacing the time-consuming TSH stimulation test--it was not sufficiently sensitive to discriminate reliably between euthyroid and hyperthyroid patients. Measurement of the TSH response to thyrotropin releasing hormone (TRH) served this purpose, however. The recent development of TSH assays that are severalfold more sensitive and more specific than conventional radioimmunoassays has allowed distinction of euthyroid from hyperthyroid patients and eliminated the need for the TRH test. Although undetectable levels of TSH, compatible with hyperthyroidism, are occasionally noted in euthyroid patients with severe nonthyroidal illness and during the first trimester of pregnancy, false-positive results are less often recorded for TSH than for free or total thyroid hormone measurements. Measurement of TSH by sensitive immunoradiometric assay is currently the most useful first-line test of thyroid function in patients with suspected thyroid disease and, in addition, has a valuable role in monitoring the dose of thyroxine replacement therapy.     

Rebamipide suppresses formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils

The purpose of the present work was to investigate the mechanism underlying the inhibitory action of rebamipide on superoxide anion (O2) production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a product of phosphoinositide 3-OH-kinase (PI 3-kinase) accumulated in response to fMLP and this accumulation was well correlated with O2 production in human neutrophils. Rebamipide inhibited PIP(3) production in parallel with the inhibition of fMLP-induced O2 production. PI 3-kinase activity in anti-PI 3-kinase p85 immunoprecipitates was not affected by the presence of rebamipide, therefore rebamipide did not have a direct inhibitory action on PI 3-kinase activity. Since rebamipide had no inhibitory effect on O2 production induced by NaF, a direct activator of G protein, the target of the inhibitory action of rebamipide appears to be a component of the signal transduction pathway upstream of G protein. Scatchard analysis of [3H]fMLP binding to human neutrophil membrane revealed that rebamipide increased the K(D) value of [3H]fMLP without altering the number of [3H]fMLP binding sites, suggesting that rebamipide has a competitive antagonistic action against the fMLP-receptor. The competitive antagonistic action was further confirmed by the finding that rebamipide caused a parallel shift to the right in the dose-response curve of O2 production induced by fMLP. These results provide evidence that the competitive inhibitory action of rebamipide on the fMLP-receptor plays a main role in its inhibitory action on fMLP-induced O2 production.     

Competitive inhibitor binding assay (CIBA) of captopril and other ACE inhibitors

We describe a new principle for measuring concentrations of pharmacologically active captopril or other angiotensin-converting enzyme (ACE) inhibitors in blood. Serum is incubated with 125I-labelled ACE inhibitor (351A, a p-hydroxy-benzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) in a nonequilibrated system, in which label and ACE inhibitor compete for binding to added serum ACE. Free label is separated by adsorption to coated charcoal. Sensitivity for captopril is 2 ng/ml, and for other tested ACE inhibitors 0.25-5 ng/ml. Results in healthy volunteers showed rapid absorption of captopril with maximal concentration of active drug within 1 h, and fast disappearance within 2.5 h. Stability of captopril was improved by immediate 1:100 dilution of blood samples with assay buffer. In spite of this precaution, analysis should be performed within two days to avoid loss of active drug due to polymerization and protein binding. Samples of other tested ACE inhibitors can be frozen and later analyzed at convenience. The new principle is simple, sensitive, and specific.