Wenbao Chang

A new competitive enzyme-linked immunosorbent assay (ELISA) for determination of estrogenic bisphenols

Bisphenol A and other hydroxylated diphenylalkanes (generally known as bisphenols) have been identified as potential estrogenic substances. In this paper, a specific polyclonal antibody was produced against these compounds by immunization of rabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serum albumin (BHPVA-BSA). The polyclonal antibody showed specific recognition of the bisphenol structure, while the cross reactions of other common phenolic compounds such as phenol, hydroquinol and p-hydroxybenzoic acid were all lower than 1%. The linear range of bisphenol A calibration curve was 1-10 000 ng ml(-1). Real water samples and mouse serum samples were spiked with known amount of bisphenol A and measured by the competitive ELISA. Recoveries were between 92 and 105%. The detection limits were found to be 0.1 ng ml(-1) for real water samples and 2 ng ml(-1) for serum samples. The method is very useful for monitoring bisphenol compounds in environmental and biological samples.

Micelle-mediated extraction of tanshinones from Salvia miltiorrhiza bunge with analysis by high-performance liquid chromatography

The feasibility of employing non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) as an alternative and effective solvent for the extraction of tanshinones from Salvia miltiorrhiza bunge was studied for the first time. Various experimental conditions were investigated to optimize the extraction. Under optimum conditions, i.e. 10% Genapol X-080 (w/v), liquid/solid ratio of 20:1 (mlg(-1)), ultrasonic-assisted extraction for 45min, the extraction recovery of the tanshinones reached the highest value. When compared with commonly used solvents, 10% Genapol X-080 yielded almost the same extraction efficiency as methanol and dichloromethane-methanol (1:4). For the pre-concentration of tanshinones by cloud-point extraction (CPE), sodium chloride was added to the solution to facilitate the phase separation and increase the pre-concentration factor by reducing the volume of the surfactant-rich phase.

Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples.

An immunoaffinity column (IAC) was developed by covalently coupling polyclonal antibodies against estrogenic bisphenols to CNBr-activated Sepharose 4B. The IAC showed high affinity for bisphenol A, while phenol was barely retained. Proteins in the sample matrix showed little nonspecific adsorption on the column. The best binding solvent for bisphenol A was found to be 0.01 mol l(-1) phosphate-buffered saline (PBS) and the optimal operating temperature was 4 degrees C. The bound bisphenol A could be quantitatively recovered by 1 ml of methanol-water (80:20) with an average recovery of 91.8% and a relative standard deviation of 7.1% (n=6). The immunoaffinity column has been successfully used for the isolation and purification of bisphenol A from serum samples.

Biotin-avidin amplified enzyme-linked immunosorbent assay for determination of isoflavone daidzein.

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed and optimized for the determination of a weakly estrogenic isoflavone daidzein in serum, urine and Puerariae radix. Specific polyclonal antibody was produced against daidzein by immunization of rabbits with a conjugate of 7-O-(carboxymethyl)-daidzein and bovine serum albumin (BSA). The polyclonal antibody showed specific recognition of daidzein, while cross-reactivities to coumarin, 4-hydroxycoumarin, phenol, and other isoflavones such as puerarin and rutin were all lower than 1%. The linear range of daidzein calibration curve was 0.1-1000ngmL(-1). The detection limit was found to be 0.04ngmL(-1), and the intra-assay and inter-assay coefficients of variation were 7 and 16%, respectively. Human serum and urine samples were spiked with known amounts of daidzein and measured by the established BA-ELISA. Recoveries were between 91 and 107%. Daidzein in P. radix was determined by the BA-ELISA method and HPLC method, and the content of daidzein was determined to be 0.0219 and 0.0194%, respectively. The results indicated that there was a good agreement between the two methods. The established method is very useful for monitoring daidzein in biological samples and traditional Chinese medicine.

Development of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K(aff)) of 7.3x10(7)L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2mug/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06ng/mL, respectively.

Direct immunoassay of estriol in pregnancy serum by capillary electrophoresis with laser-induced fluorescence detector

A simple and sensitive capillary electrophoretic immunoassay (CEIA) was described for the determination of estriol (E(3)) in pregnant womens serum. The method was based on the competitive reaction of fluorescein-labeled E(3) antigen and E(3) with limited amounts of monoclonal antibody. The addition of the thermally reversible hydrogel, poly-N-iso propylacrylamide (pNIPA) in the buffer serving as a replaceable packing material, improved the reproducibility of the method. With laser-induced fluorescence detector (LIF), this method can be applied to determine E(3) at concentrations lower to 31.6 pg ml(-1). Recoveries from human steroid-free serum matrix were greater than 94% with relative standard deviation (R.S.D.) values less than 3.5%. Serum E(3) levels of ten normal pregnant women were measured at the range of 10.2-15.6 ng ml(-1).

Voltammetric studies of the effect of Cisplatin-liposome on Hela cells

In this paper, the voltammetric method was used for the first time to study the effect of Cisplatin-liposome on Hela cells. The results showed the voltammetric behavior of Hela cells was irreversible and the peak current had linear relationship with the cell number. With both Cisplatin-liposome concentration and treating time increasing, the peak current decreased. The peak current decreasing was in accordance with the nuclear damage and loss of mitochondrial membrane potential revealed by two-photon laser scanning microscopy and confocal laser scanning microscopy. This voltammetric method may provide a simple way to study the electron-transfer mechanism in drug-treating cells.

Comparison of Microwave‐Assisted and Ultrasound‐Assisted Extraction for Determination of Main Water‐Soluble Bioactive Constituents in Traditional Chinese Medicinal Preparation Tongmaichongji by HPLC‐DAD

Abstract Microwave‐assisted extraction (MAE) and ultrasound‐assisted extraction (UAE) were compared as sample preparation methods for the determination of three main water‐soluble bioactive constituents (danshensu, puerarin, and ferulic acid) in traditional Chinese medicinal preparation (TCMP) Tongmaichongji by HPLC‐DAD. The optimal conditions of the two extraction methods were obtained. MAE could be completed in 2 min at 750 W of radiation power and 30% ethanol (pH 2.5) was used as extraction solvent. The time needed for UAE was 30 min, with 30% ethanol (pH 2.5) as extraction solvent. However, UAE allowed extraction at lower temperature. The present HPLC system used a Diamonsil C18 column (150 × 4.6 mm2, I.D. 5 µm) connected with Zorbax Ext‐C18 guard column (12.5 × 2.1 mm2, 4‐Pack) at 30°C, employing gradient elution with acetonitrile/water (pH 3) as mobile phase at a flow rate of 1 mL/min. The detection wavelength was set as: 0 min, 205 nm; 12 min, 254 nm; and 21 min, 320 nm. The linear calibration ranges were 6.8–81.9, 10.6–137.8, 4.9–77.8 µg/mL for danshensu, puerarin, and ferulic acid, respectively. The detection limits were 0.03, 0.10, 0.04 µg/mL for danshensu, puerarin, and ferulic acid, respectively. The contents of these three bioactive constituents in Tongmaichongji were successfully determined by the proposed method. The results indicated that there is a good agreement between the two extraction methods.

Rapid and sensitive determination of protein by light-scattering technique with Eriochrome Blue Black R

Based on the interaction between Eriochrome Blue Black R (EBBR) and proteins, which causes a strong light-scattering signal with the maximum scattering peak located at 398 nm, a simple, rapid, sensitive and selective method is developed for the determination of proteins by the light-scattering technique using a common spectrofluoremeter. Under proper experimental conditions, the protein determination can be performed in the range of 0.1-25, 0.1-20 and 0.25-25 mugml(-1) for bovine serum albumin (BSA), human serum albumin (HSA) and human immunoglobulin G (IgG), respectively. The detection limit, calculated as 3 times the S.D. of nine blank measurements, are 33 mugl(-1) for BSA, 25 mugl(-1) for HSA and 38 mugl(-1) for IgG. Moreover, there is no significant difference among the scattering signals yielded by HSA, IgG and BSA, and almost no interference of many amino acids and metal ions. The method has been satisfactorily applied to the direct determination of the total protein in human serum, saliva and urine samples. The results obtained from the studies on the binding characteristics of EBBR to BSA indicated that an electrostatic force existed in the binding system, and the binding constant (K) and the number of the binding sites (n) at 25 degrees C are 1.69x10(5) lmol(-1) and 0.946, respectively.

Simultaneous determination of iron(III) and vanadium(V) by use of a kinetic-spectrophotometric and rapid mixing flow system.

The quantitative spectrophotometric determination of V(V) by its catalytic effect on the oxidation of chromotropic acid [the disodium salt of 4,5-dihydroxynaphthalene-2,7-disulphonic acid (CS)] by potassium bromate has been adapted to a flow system employing a rapid sample and reagent introduction manifold, in which valves and carrier streams are omitted without decreasing the precision or increasing the sample consumption relative to flow-injection analysis (FIA). Measurements are made at 420 nm. Coexisting Fe(III) can be determined simultaneously at the same wavelength by its more rapid colour chelate formation with CS. An accurate determination of V(V) and Fe(III) in the mixture was developed with a sample throughput of about 60 hr.