Evaluation and comparison of high-sensitivity immunoradiometric assay kits for thyroid stimulating hormone

Fundamental and clinical characteristics of 3 kinds of high-sensitivity immunoradiometric assay (IRMA) kits for thyroid stimulating hormone (TSH). i.e., RIA BEADS II (kit A), TSH kit Daiichi II (kit B) and Ab tube TSH 'Eiken' (kit C) and one conventional radioimmunoassay (RIA) kit, i.e., TSH kit Daiichi (kit D), were studied. In the recovery test and the reproducibility test, there was no significant difference between the 4 kits. The sensitivities of kits A, B and C were much higher than that of kit D, and those IRMA kits were sensitive enough to distinguish hyperthyroidism from normal samples. For low concentrations of TSH (less than 5 microU/ml), the data from kits D, B, C and A tended to show higher values in that order. The correlation between the data measured by kits B and D, and the tendency of kit A toward lower values agreed well with other reports.     

Basic evaluation of highly sensitive thyroid stimulating hormone immunoradiometric assay kits

Usefulness of three kinds of TSH kits by immunoradiometric assay (IRMA) was evaluated. They were able to measure low levels (less than 0.1 microIU/ml) in serum thyroid stimulating hormone (TSH) with incubation of short time (4 hours). In particular, RIABEAD II kit had a highly specific affinity for TSH and the normal range (+/- 2 S.D.) using it showed from 0.20 to 3.50 microIU/ml in 150 normal subjects. In patients with hyperthyroidism and in patients with hypothyroidism, the values of TSH were lower and higher than those of normal subjects, respectively. Another kits showed similar results. These results indicate that these TSH-IRMA kits are useful to evaluate serum TSH levels exactly.     

Level of Human Chorionic Gonadotrop in-Like Substance in Serum of Normal Subjects

Abstract

The level of human chorionic gonadotropin (hCG)-like substance in normal serum was measured using 50 μl of serum specimen by a highly specific and sensitive sandwich enzyme immunoassay for hCG which did not significantly cross-react with hLH. The level was calculated using a standard hCG and expressed in mIU/ml as that of hCG. In normal male (20–59 years of age) and normal, non-pregnant female (20–42 years of age) adults, the level was below 0.6 mIU/ml with mean levels of 0.16 mIU/ml (n=209) and 0.14 mIU/ml (n=36), respectively. In older subjects, however, the level tended to rise. In normal male adults, aged 60–91 years, the level was mostly below 0.8 mIU/ml (mean=0.56 mIU/ml, n=99), but levels of 15 specimens out of 99 were 1.1–5.5 mIU/ml. In normal, non-pregnant female adults, aged 45–88 years, the level was widely distributed between 0.1 and 11.7 mIU/ml (mean=1.6 mIU/ml, n=141). In these older female adults, levels of hCG-like substance were not well correlated to either levels of hLH and hFSH, while levels of hLH and hFSH were well correlated to each other.     

增强化学发光体系酶联免疫分析法测定人绒毛膜促性腺激素

应用活化鲁米诺,用优化的增强化学发光酶联免疫分析体系测定人绒毛膜促性腺激素,检测限为0.2mIU/ml。线性范围0～200mIU/ml,与放射免分析测定结果比较,相关性良好。进而又发展了一种半定量的照相测定法,通过实际血清样品测定,效果良好。     

A comparison of the several methods for determination of specific IgE antibodies

The accuracy of measurement using kits in the clinical laboratories is important for the patient diagnosis and treatment. In the present paper, the AL-18, AlaSTAT, CAP, FAST and RAST methods were investigated and were compared among kits the results obtained with serum sample, for determination of specific IgE antibodies. Significant differences among kits were observed from the results of those methods. One of the reasons, why the data discrepancy exists, is that each kit uses a different reference and a different inclusion method of allergen. For the evaluation of data discrepancy among those kits, it might be important that the clinical history of symptoms and in vivo tests against the different allergens compared with results of in vivo tests.     

Solid phase inclusive immunoradiometric assay for human chorionic gonadotropin using monoclonal antibodies

Summary A simple, one step, inclusive immunoradiometric assay for human chorionic gonadotropin (hCG) employing monoclonal antibodies is described. Commercially available monoclonal antibodies from various commercial sources were screened. Identified “detection” antibody was radiolabeled with ¹²⁵I and the selected “capture” antibody was chemically coupled to magnetizable cellulose to form a solid phase. In the procedure developed, standard/sample, radiolabeled antibody and capture antibody were incubated together for 3 hours at room temperature with shaking. After incubation, the bound complex was quantitated for the associated radioactivity. The analytical sensitivity observed was 1.0 mIU/ml with a wide concentration range up to 1000 mIU/ml of hCG. “High dose hook” of the developed assay was observed beyond 2000 mIU/ml. Results showed that the developed assay had a good precision: intra-assay CV less than 8%, inter-assay CV less than 10% and good analytical recovery of 97-109%. The clinical samples analyzed by the developed procedure showed a good correlation with that of the commercial kit (r = 0.92; y = 0.99x+0.51).     

A comparison of INAA and ise for the determination of calcium in human hair

The calcium concentration was determined in human hair of several normal individuals and of patients suffering from various bone diseases. A comparison of results achieved with an ion-selective electrode with those obtained with the neutron activation analysis demonstrated a good accuracy of the two methods. The precision of the determination with the ionselective electrode may be characterized by a relative standard error of 8.8%.     

Determination of active ingredients in cough-cold preparations by micellar liquid chromatography

The chromatographic behaviour of some active ingredients in cough-cold pharmaceutical preparations, the antihistamine chlorpheniramine (or the dextro enantiomer dexchlorpheniramine), and the phenethylamines phenylephrine, phenylpropanolamine and pseudoephedrine, has been studied using a C(18) column, micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, and with UV detection. All possible combinations of chlorpheniramine/phenethylamine were resolved and determined using a mobile phase of 0.15 M SDS-6% (v/v) pentanol at pH 7, with analysis time below 7 min. Repeatabilities and within laboratory precisions were evaluated at four different drug concentrations in the range 0.5-25 mug ml(-1) (n=5), resulting RSDs below 1.6%. The drug amounts found in the analysis of 14 commercialised preparations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using an aqueous 60% (v/v) methanol reference mobile phase. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, caffeine, codeine phosphate, diphenhydramine, lactose, paracetamol, and prednisolone. The studied combinations required a rather high amount of methanol in conventional RPLC to be eluted from the column. In contrast, the proposed procedure used a much lower amount of organic solvent (pentanol), which is highly retained in the SDS solution, being also less toxic than methanol.     

Rapid and sensitive detection of citrinin production during fungal fermentation using high-performance liquid chromatography

A rapid and sensitive assay was developed for the detection of the mycotoxin citrinin by reversed-phase chromatography. Citrinin was eluted from a radical-compression C18 column with a retention time of 3.86 min (flow-rate of 2.5 ml/min) with acetonitrile-water-acetic acid (40:59:1) containing tetrabutylammonium phosphate (0.0025 M) [corrected]. Comparative analysis revealed fluorescence detection to be 100 times more sensitive than detection by conventional ultraviolet absorbance. The fluorescence excitation and emission maxima of citrinin were 330 and 500 nm, respectively. The assay was linear over the concentration range between 0.01-100 micrograms/ml. Recovery experiments conducted by addition of citrinin to fermentation samples, revealed the assay quantitation efficiency to be 91-102%. Assay utility was demonstrated by using an Aspergillus niveus culture, propagated in complex liquid medium. Citrinin production was detected as early as 20 h following inoculation and increased dramatically when the culture entered the stationary phase of growth, analogous to other secondary metabolites. Unlike previously reported methods, this procedure has the advantage of enabling the direct quantitative analysis of citrinin in crude microbial fermentations without sample extraction.     

Derivatization of primary amines by 2-naphthalenesulfonyl chloride for high-performance liquid chromatographic assay of neomycin sulfate

A normal phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of neomycin sulfate. The method involves pre-column derivatization with 2-naphthalenesulfonyl chloride (NSCl) followed by extraction in chloroform and chromatography using a normal phase silica column with detection at 254 nm. The standard curve for the HPLC assay of neomycin sulfate is linear with a correlation coefficient of 0.9996 over the range of 0.02 to 0.4 mg/ml. Neomycins B, and C, and neamine can be separated and quantified isocratically with relative standard deviations of 0.92% and 1.4% for neomycin (B + 1/2C) and neamine, respectively. Prednisolone is used as an internal standard to aid in quantification. Mass spectrometric data confirms that neomycin is derivatized at all the six primary amines on the neomycin molecule. Eight lots of neomycin sulfate were used to compare the HPLC [NSCl and 1-fluoro-2,4-dinitrobenzene (DNFB)], gas-liquid chromatographic and microbiological assay methods. The average results of the NSCl-HPLC method fell between those of the microbiological and DNFB-HPLC methods. Also, good correlation of the neomycin C contents in neomycin were obtained between the NSCl-HPLC and DNFB-HPLC methods.     

Immunoreactivity and detection of wheat proteins by commercial ELISA kits

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.     

Determination of catechin and epicatechin in the peel of apple varieties resistant and non-resistant to apple scab

Catechin and epicatechin were analysed in the peel of six apple cultivars (three resistant and three non-resistant to apple scab). Two methods of analytical sample preparation following extraction were tested: solid phase extraction and column separation with Sephadex LH-20 coupled to a differential refractometric detector. Prior to GC and GC-MS analyses, these compounds were silylized. This permitted co-injection with standards and the comparison of retention values and mass spectra with those recorded for standards. The content of catechin and epicatechin in apple peel is discussed in relation to the resistance of apple trees to scab.     

非水滴定测定烟叶中烟碱含量实验的微型化

研制了一种微型滴定装置,利用该装置(WD-COⅡ型微型滴定装置)对烟叶中烟碱含量进行非水滴定,通过数理统计方法将微型滴定管与常规滴定管的平行测定结果进行了比较,得到相同样品的一对非常接近的测定结果。证明新型微量滴定管具有较好的操作性能和精密度。     

The comparison of parametric and nonparametric bootstrap methods for reference interval computation in small sample size groups

According to the IFCC, to determine the population-based reference interval (RI) of a test, 120 reference individuals are required. However, for some age groups such as newborns and preterm babies, it is difficult to obtain enough reference individuals. In this study, we consider both parametric and nonparametric bootstrap methods for estimating RIs and the associated confidence intervals (CIs) in small sample size groups. We used data from four different tests [glucose, creatinine, blood urea nitrogen (BUN), and triglycerides], each in 120 individuals, to calculate the RIs and the associated CIs using nonparametric and parametric approaches. Also for each test, we selected small groups (m = 20, 30,…, 120) from among the 120 individuals and applied parametric and nonparametric bootstrap methods. The glucose and creatinine data were normally distributed, and the parametric bootstrap method provided more precise RIs (i.e., the associated CIs were narrower). In contrast, the BUN and triglyceride data were not normally distributed, and the nonparametric bootstrap method provided better results. With the bootstrap methods, the RIs and CIs of small groups were similar to those of the 120 subjects required for the nonparametric method, with a slight loss of precision. For original data with normal or close to normal distribution, the parametric bootstrap approach should be used, instead of nonparametric methods. For original data that deviate significantly from a normal distribution, the nonparametric bootstrap should be applied. Using the bootstrap methods, fewer samples are required for computing RIs, with only a slightly increased uncertainty around the end points.     

The environmental stress cracking of linear ethylene copolymers

A new method of measuring the environmental stress cracking (ESC) of polymers at constant strain is proposed in which an occurrence of fracture is detected automatically. The new method showed a very good reproducibility within 10% compared with a few hundred percent by conventional methods. The ESC values obtained by the new method was found to be proportional to those by the conventional Bell Telephone Laboratory method in which the ESC was determined by the occurrence of small cracks, with a proportionality contant of 2.8. From the fact that the difference of both ESCs were also proportional to the ESC determined by the BTL method, it was concluded that the value of the ESC was proportional to the velocity of crack propagation. These conclusions were supported by the observation of the test pieces by a scanning electron microscope. The study of the blended polymers revealed that the additivity of logarithmical ESC with weight fraction holds for a wide range of polymes, which enables estimation of ESCs up to millions of hours. Using this technique, the ESCs of a wide range of molecular weights and a number of short chain branches were studied. It was found that the branches in the high molecular weight polymer were much more effective on the ESC than those in the low molecular weight polymer. This makes it possible to design a good resin with a good ESC.     

Characterisation of poly(N-isopropylacrylamide) by asymmetrical flow field-flow fractionation, dynamic light scattering, and size exclusion chromatography

Asymmetrical flow field-flow fractionation (AsFIFFF) was used to determine the hydrodynamic particle sizes, molar masses, and phase transition behaviour of various poly(N-isopropylacrylamide) (PNIPAM) samples synthesised by reversible addition--fragmentation chain transfer (RAFT) and conventional free radical polymerisation processes. The results were compared with corresponding data obtained by dynamic light scattering (DLS) and size exclusion chromatography (SEC). Agreement between the three methods was good except at higher molar masses, where the molar mass averages obtained by SEC were much lower than those obtained by AsFIFFF and light scattering. The aggregation of the polymers, which are thermally sensitive, was studied by DLS and AsFIFFF at various temperatures. In deionised water there was an abrupt change in the particle size due to phase separation at approximately equal to 32-35 degrees C. The critical temperatures determined by AsFIFFF were 3-5 degrees C higher than those obtained by DLS.     

The study of a chemiluminescence immunoassay using the peroxyoxalate chemiluminescent reaction and its application

The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H₂O₂ catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H₂O₂ chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0-625.0 pg/ml, and β-HCG in the range of 12.5-400.0 mIU/ml. The detection limits were 0.5 pg/ml for rHu IL-6 and 3 mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0 pg/ml rHu IL-6, and 3.9 for 50.0 mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results.     

反复呼吸道感染与血清中微量元素关系的研究

反复呼吸道感染是儿科的常见病、多发病 ,研究表明 ,RRTI患儿急性期血清铁、锌明显降低 ,且感染发作频率与铁、锌下降程度有关[1] 。作者旨在通过对 68例 RRTI患儿 ,67例营养不良患儿及 5 6例健康儿童血清硒、铁、铜、锌、铅、钙、镁 7种元素含量的测定分析及比较 ,为临床诊断及治疗提供参考。1　材料与方法1 .1　主要仪器与试剂WYX— 90 0 3型原子吸收分光光度计 (沈阳分析仪器厂 ) ,WHG— 1 0 2 A2型流动注射氢化物发生器 (北京瀚时制作所 ) ,管式自控电热消化器(北京化工研究院 ) ,硒、铅、铁、锌、铜、钙、镁 7种元素分析物质均属…     

Selective determination of urinary free cortisol by liquid chromatography after solid-state extraction

We have developed a selective and precise high-performance liquid chromatographic method for urinary free cortisol with an improved and efficient sample clean-up using C18 Sep-Pak cartridges. The urine sample (2 ml), with 11-deoxycortisol as internal standard, is applied to the Sep-Pak, which is then sequentially washed with acetone-water (1:4, v/v), water and hexane. Cortisol is eluted with diethyl ether, evaporated to dryness and redissolved in 2 ml of water. The wash cycle is repeated once using the same Sep-Pak cartridge. This double extraction greatly improves sample clean-up and allows modification of the mobile phase (tetrahydrofuran-methanol-water) so that cortisol is rapidly eluted as a single well resolved peak at 13 min. Chromatography is performed isocratically on a reversed-phase column with detection at 254 nm. Detection limits for urinary free cortisol by this procedure were two or three times lower than those obtained with two commercial radioimmunoassay kits. The chromatographic method was used successfully in the diagnosis of patients with hypercortisolism and Cushing's syndrome.     

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.