Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3',5,5'-tetramethylbenzidine as substrate

An immunosensing system for Schistosoma Japonicum antibody (SjAb) assay has been developed which is useful for the diagnosis of schistosomaisis. To circumvent the difficulty of regeneration of the immunosensing device, the sol-gel technique is used which results in a considerable retention of the activity of the encapsulated antigen (SjAb) and easily diffusing into the pores of the polymeric silica matrix. The surface of the immunosensing device prepared can easily be renewed by simply polishing. The regenerated surface serves as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg bound at the surface. By using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as the substrate, the amount of HRP-SjAb bound is quantitated fluorimetrically, which is in turn related with the SjAb content. An amplification effect is obtained by using the enzymatic reaction, and an improved detection limit of 4.5 ng ml(-1) is thus realized. The optimum analytical conditions such as pH, amount of the labeled antibody and flow rates of substrate carrier solution were established. The immunosensing procedure shows a pseudo linear response range from 4.5 to 55 ng ml(-1). The proposed procedure has been employed to determine SjAb in serum samples.     

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the thermostable glucose-6-phosphate dehydrogenase as a label enzyme in steroid enzyme immunoassays.

A thermostable glucose-6-phosphate dehydrogenase was assessed as a label enzyme in a testosterone enzyme immunoassay system. Enzyme labeling of a steroid with the enzyme was carried out by the N-succinimidyl ester method. A dose-response curve with a satisfactory sensitivity could be obtained by the use of the enzyme-labeled antigen. This enzyme should be useful in hapten enzyme immunoassays because of its relatively high stability.     

Colorimetric method for determination of chlorine with 3,3',5,5'-tetramethylbenzidine

A new method is developed for the calorimetric determination in water of chlorine (free and combined) with 3,3′,5,5′-tetramethylbenzidine. The procedure proposed achieves a detection limit of 2 ng/ml and its sensitivity is greater than that of the methods used at present.     

Determination of trace phosphate ion with 3,3',5,5'-tetramethylbenzidine using a flotation-extraction preconcentration method and spectrophotometric detection

A flotation-extraction method for sensitivity enhancement in spectrophotometry is proposed. The method is based on the reaction among molybdate, phosphate and 3,3',5,5'-tetramethylbenzidine (TMB) in acidic medium to form an color charge transfer complex ion. It forms an ion associate with positive ions and floated at the water/benzene interface simultaneously, and then extracted by the DMSO-formic acid directly and determined by spectrophotometry. The proposed method is simple and convenient. The apparent molar absorptivity is 4.03x10(5) L mol(-1) cm(-1) at 458 nm. The proposed method has been applied to the determination of phosphate in wastewater with satisfactory results.     

Enzyme labeling in steroid enzyme immunoassays. The p-nitrophenyl ester method for alkaline phosphatase and glucose oxidase labelings.

The p-nitrophenyl ester method was assessed as an enzyme labeling technique. The active ester of a carboxylated testosterone derivative was treated with alkaline phosphatase and glucose oxidase to give labeled antigens, using various molar ratios of steroid to enzyme. Satisfactory immunoreactivities with an anti-testosterone antibody in an enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30 and 10, respectively, in the alkaline phosphatase and glucose oxidase labelings.     

Flow injection determination of iron by its catalytic effect on the oxidation of 3,3',5,5'-tetramethylbenzidine by hydrogen peroxide

A flow injection-photometric method has been developed for the determination of iron(II+III). The method is based on the catalytic effect of iron(III) on the hydrogen peroxide oxidation of 3,3',5,5'-tetramethylbenzidine to form a blue compound (lambda(max)=650 nm). In this catalyzed reaction, 1,10-phenanthroline acted as an effective activator. Iron(II) is also determined, being oxidized by hydrogen peroxide. Calibration graphs for iron(II) and iron(III) obtained under the optimized conditions were identical with each other and linear in the range 0.2-200 ng ml(-1) with a detection limit of 0.05 ng ml(-1) iron. The reproducibility was satisfactory with a relative S.D. of 1.0% for ten determinations of 5 ng ml(-1) iron(III). The proposed method was successfully applied to the determination of iron in river and lake water samples and can be determined free iron species.     

Catalytic flow injection determination of vanadium by oxidation of N-(3-sulfopropyl)-3,3',5,5'-tetramethylbenzidine using bromate

A catalytic flow-injection (FI) method was developed for the determination of 10⁻⁹ mol l⁻¹ levels of vanadium(IV, V). The method is based on the catalytic effect of vanadium(V) on oxidation of N-(3-sulfopropyl)-3,3′,5,5′-tetramethylbenzidine (TMBZ·PS) using bromate as oxidant to form a yellow dye (λ_max=460 nm). The use of 5-sulfosalicylic acid (SSA) as an activator enhanced the sensitivity of the method. The calibration graphs with a working range 0.05–8.0 ng ml⁻¹ were obtained for vanadium(V). Vanadium(IV) was also determined, being oxidized by bromate. The detection limit (signal/noise, S/N=3) was 0.01 ng ml⁻¹ (ca. 2×10⁻¹⁰ mol l⁻¹) vanadium. The relative standard deviations (R.S.D.) for 15 determinations of 0.5 ng ml⁻¹ vanadium, and for ten determinations of 0.1 and 1.0 ng ml⁻¹ vanadium were 0.41, 2.6 and 0.25%, respectively, with a sampling rate of 15 samples h⁻¹. The proposed method was successfully applied to the determination of vanadium in natural waters.     

Solvent effect on the photochemical properties of symmetrically substituted trans-3,3',5,5'-tetramethoxystilbene

Photochemical properties of trans-3,3',5,5'-tetramethoxystilbene (TMST) have been studied in various polar solvents. The Stokes shift of trans-TMST was found to be increased with increasing solvent polarity. The fluorescence lifetime of trans-TMST experienced a large solvent effect changing from 2.3 ns in cyclohexane to 16.6 ns in acetonitrile. These results indicate that the excited singlet state of trans-TMST has a charge-transfer (CT) character. On the basis of the obtained results, the interior polar environment of a water-soluble TMST dendrimer is discussed.     

Enzyme labeling in steroid enzyme immunoassays. Comparison of the p-nitrophenyl ester and N-succinimidyl ester methods.

Enzyme labeling of steroids by the p-nitrophenyl ester method was investigated in comparison with the N-succinimidyl ester method. The active ester of a testosterone or 11-deoxycortisol derivative was treated with beta-galactosidase and horseradish peroxidase to give labeled antigens. Various molar ratios of steroid to enzyme and pH conditions were tested. Satisfactory immunoreactivities with an anti-steroid antibody in each enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30. The enzyme labeling method should be useful in the case of polar steroids or drugs, since the p-nitrophenyl ester is relatively stable when compared with the N-succinimidyl ester.     

基于1，1′-苯乙炔-3，3′，5，5′-四羧酸的配位聚合物：水热合成、结构和荧光性质

合成并表征了配合物1,[Zn2(EBTC)(BPDO)(DMSO)(H2O)2]n,其中EBTC为1,1′-苯乙炔-3,3′,5,5′-四羧酸根,BPDO为4,4′-二吡啶基1,1′-二氧化物,DMSO为二甲亚砜。1沿着b轴的方向具有类似于三腿梯子状的链状结构,链与链之间通过氢键作用和π-π堆积作用形成了三维超分子结构。在室温条件下1表现出了荧光性质。     

Investigation of the origin of the glucose response in a glucose oxidase|polyaniline system

The origin of the signal seen in response to glucose in a polyaniline|glucose oxidase system is explored by immittance spectroscopy, by comparing data from an equivalent circuit model and the parameters obtained from a solution of the faradaic branch of the frequency dispersion for a coupled chemical—electrochemical reaction mechanism. It was shown that an RC subcircuit in the equivalent circuit model was sensitive to peroxide concentration, and the interaction of peroxide with polyaniline at potentials where it either oxidised or reduced the polyaniline was discussed. This information was used to compare the data obtained in a bulk and entrapped glucose oxidaselglucose system, and it was seen that the origin of the response could not be fully attributed to peroxide interaction in the latter case. Under anaerobic conditions with entrapped enzyme, it was proposed that a complex between the gluconolactone product of the enzyme reaction and the polymer leads to a more conducting polymer, with inherent charge compensation, and this results in the observed enhanced current signal.     

Amperometric enzyme electrodes: Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase

Results are reported for the direct oxidation of the enzyme glucose oxidase on 7 different one-dimensional conducting donor acceptor salts. Experiments conducted with the enzyme in bulk solution are shown to be in good agreement with theory. Three salts, made of the cations tetrathiafulvalinium (TTF⁺), N-methylphenazinium or quinolinium with the anion tetracyanoquinodimethanide (TCNQ^?) had the lowest background currents and were used to make membrane sensors for glucose. Analysis of the variation of current with glucose concentration identified the rate limiting processes as transport of gluycose through the membrane and electrochemical kinetics under unsaturated and saturated conditions respectively. The electrochemical rate constants for these three materials were all greater than 10^?2 cm s^?1. TTF⁺TCNQ^? is the material of choice and linear calibration plots were obtained for glucose concentrations between 50 μmol dm^?3 and 10 mmol dm^?3.