Preparation of antibodies and development of enzyme-linked immunosorbent assay for nonylphenol

Polyclonal rabbit antibodies against nonylphenol (NP), the main product of nonionic surfactants destruction, were obtained and applied for immunoenzyme assay (ELISA). Two approaches to immunogen synthesis were compared. The first was direct coupling of a mixture of NP isomers to the carrier protein by Mannich reaction. The second was formation of amide bonds between 7-(p-hydroxyphenyl)heptanoic acid (HHA), a linear carboxylated analog of NP, and the carrier protein. Anti-HHA antibodies showed a low affinity to technical NP, whereas with Mannich synthesis it was possible to generate antibodies specific to branched NP molecules. An indirect competitive ELISA was developed based on the anti-NP antibodies. The detection limit of the analysis is 10 ng/mL, with a total duration of around 3 h. The developed ELISA can be applied for group-specific determination of nonionic surfactants and their toxic metabolites. The possibility of NP detection in environmental water matrices using the proposed ELISA without loss of sensitivity is explored.     

Hapten Design and Development of Polarization Fluoroimmunoassay for Nonylphenol

Polyclonal antibodies to nonylphenol (NP), raised against two different haptenic derivatives were characterized by a rapid, homogeneous polarization fluoroimmunoassay (PFIA). The first hapten, ω-(4-hydroxyphenyl)nonanoic acid (NP9), was designed to mimic the linear NP isomer and contains hydroxyphenyl and linear alkyl chain moieties of the NP molecule. The second hapten, 4-aminophenol (4AP), contains the hydroxyphenyl moiety of NP molecule alone and thus potentially mimics various phenolic compounds with different side chain structures. A number of fluorescent labeled antigens (tracers) with various structures of the spacer arm between the antigen and the fluorescent dye was synthesized and used to optimize the competitor structure for NP-specific PFIA. The most sensitive assay with limit of detection (LOD) and IC₅₀ values of 8 and 53 mg L^–1, respectively, was obtained when anti-NP9 antibody and NP9-labeled antigen were used. Anti-NP9 resulted in more specific assay, where the cross-reactivity toward the relative phenolic compounds did not exceed 5%. Anti-4AP displayed substantial recognition of several bis-substituted phenols, including 2-amino-4-chlorophenol and 2,4-dinitrophenol.     

Antibody-based methods for surfactant screening

This brief overview summarises the immunoassay-based results obtained in the course of two years of the European INCO-Copernicus project BIOTOOLS. The project is aimed at simplifying the procedures for detection of surface active compounds (SAC) using, among others, antibody-based methods, i.e., microtiter plate-based enzyme-linked immunosorbent assays (ELISA), polarisation fluoro immunoassays (PFIA), and enzyme flow injection immunoassays (FIIA). Thirty-three rabbits were immunised with five different sulphophenyl moieties and three p-hydroxyphenyl moieties conjugated to protein immunogens to produce analytical antibodies against linear alkylbenzene sulphonates (LAS) and nonylphenol (NP). Although most of the antibodies exhibited binding reaction in indirect ELISA, only a few showed the required assay sensitivity. The best antibodies for LAS exhibited a 50% binding inhibition at IC50 19.8 microg L(-1) in indirect ELISA. Similar inhibition was observed for direct ELISA using peroxidase tracers. Antibodies against NP allowed the establishment of an indirect assay operating in the mg L(-1) range. A rapid and simple protocol for the screening of NP and LAS using homogeneous PFIA is described. The assay time for 10 samples was 7 minutes, thus allowing fast detection of the selected SAC at the mg L(-1) level. A generic competitive FIIA system, using a protein G column for separation of free and antibody-bound beta-galactosidase (beta-Gal) tracer, was developed for the screening of LAS, NP, and nonylphenol decaethoxylate (NPEO10). The FIIA had a sample throughput (STP) of 5-10 samples per hour, with limits of detection (LOD) for LAS, NP, and NPEO10 of 19.5, 52, and 2.4 microg L(-1), respectively. The developed FIIAs were applied to spiked rain and surface water.     

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10 ng ml⁻¹ and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.     

Determination of acetone, 2-butanone, diethyl ketone and BTX using HSCC-UV-IMS

A combination of a custom-designed ion mobility spectrometer (IMS) with a UV ionization source and a high speed capillary column (HSCC) has been developed as an analytical device for the sensitive detection of volatile organic compounds (VOCs), e.g. 2-propanone (acetone), 2-butanone and 3-pentanone (diethyl ketone) in the gas phase. A fast separation of the three selected substances and benzene, toluene and m-xylene (BTX) - all of which occur in human breath - has been achieved within less than four minutes at a carrier gas flow rate of 4.5 mL x min(-1). Multi-dimensional correlations presented support the interpretation of the acquired spectra of mixtures. Method detection limits were 2.7 microg x L(-1) for acetone and 2-butanone and 3.0 microg x L(-1) for diethyl ketone in nitrogen, respectively. The assay linear dynamic range is 4-320 microg x L(-1).     

Colorimetric assay of propranolol tablets by derivatization: novel application of diazotized 4-amino-3,5-dinitrobenzoic acid (ADBA)

A novel colorimetric assay of propranolol tablets has been developed. The assay is based on chemical derivatization (aromatic ring derivatization technique) using diazotized 4-amino-3,5-dinitrobenzoic acid (ADBA) as the chromogenic derivatizing reagent and resultant formation of azo dyes. Optimization studies established an optimal reaction time of 5 min at 30 degrees C after mixing on a Vortex mixer the drug/reagent mixture for 10 s. A new absorption maximum (lambda(max)) was found at 470 nm, which was selected as the analytical wavelength. The assays were linear over 1-8 microg/mL propranolol, and the reaction occurred by a 1:1 reagent/drug stoichiometric ratio. The developed method has a low limit of detection of 0.76 microg/mL and is reproducible. It has been applied successfully to the assay of propranolol tablets and is of equivalent accuracy (p > 0.05) with the official (British Pharmacopoeia) ultraviolet spectrophotometric method. The new method has the main advantage of using more affordable instrumentation and could be applied to the in-process quality control of propranolol tablets.     

A simple and rapid determination of valproic acid in human plasma using a non-porous silica column and liquid chromatography with tandem mass spectrometric detection

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay was developed and validated to determine valproic acid in human plasma. The method involved a solid-phase extraction of valproic acid and betamethasone valerate, an internal standard, from plasma and detection using an LC-MS/MS system with electrospray ionization source in negative ion mode. Separation was achieved within 3 min on a non-porous silica column with mobile phase containing ammonium acetate and methanol. Multiple reaction monitoring was utilized for detection monitoring at 142.89-142.89 for valproic acid and 457.21-457.21 for the internal standard. The calibration curve for valproic acid was linear over the range of 0.5-150 microg/mL. The limit of detection was 0.17 microg/mL and the lower limit of quantification was 0.5 microg/mL, when 0.2 mL plasma was used for extraction. The percentage coefficient of validation for accuracy and precision (inter- and intra-day) for this method was less than 9.5% with recovery ranging from 82.3 to 86.9% for valproic acid.     

Stereoselective determination of propafenone enantiomers in transgenic Chinese hamster CHL cells expressing human cytochrome P450

An enantioselective assay for S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450 was developed. The method involved extraction of propafenone from the S9s incubates, and formation of propafenone diastereomeric derivatives with the chiral reagent 2,3,4, 6-tetra-O-beta-D-glucopranosyl isothiocyanate. Separation and quantitation of diastereomeric propafenone derivatives were carried out in a reverse-phase-HPLC system with UV detection. The assay was linear from 2 to 200 microg/mL for each enantiomer. The analytical method gave average recoveries of 97.5% and 97.0% for S(+)- and R(-)-propafenone, respectively. The limits of detection and quantitation for the method are 0.1 and 2.0 microg/mL for both S(+)- and R(-)-propafenone, respectively. The reproducibility of the assay was good (RSD <10%). The method allowed study of the depletion of S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450. The stereoselectivity of propafenone phase I metabolism via cDNA-expressed CYP3A4 was observed.     

Biological activity of two related triterpenes isolated from Combretum nelsonii (Combretaceae) leaves

Antifungal bioassay-guided fractionation of Combretum nelsonii leaf extracts afforded two closely related triterpenes, asiatic acid and arjunolic acid. Antifungal activities of the mixture of asiatic acid and arjunolic acid were determined against five fungal animal pathogens. The minimum inhibitory concentrations of the mixture to the different pathogens varied from 0.2 to 1.6 microg mL(-1); Candida albicans (0.9 microg mL(-1)), Cryptococcus neoformans (0.4 microg mL(-1)), Aspergillus fumigatus (1.6 microg mL(-1)), Microsporum canis (0.2 microg mL(-1)) and Sporothrix schenckii (0.2 microg mL(-1)). Microsporum canis and S. schenckii were the most susceptible followed by C. neoformans. Aspergillus fumigatus was the most resistant. The R(f) value of the mixture of asiatic acid and arjunolic acid was 0.27 in CEF (chloroform : ethylacetate : formic acid), 0.09 (BEA; benzene : ethanol : ammonium hydroxide) and 0.55 (EMW; ethylacetate : methanol : water) which was active against all pathogens. In vitro cytotoxicity of mixture gave an LC(50) of 10.58 microg mL(-1) towards Vero monkey kidney cells.     

高效液相色谱法同时测定尿中新喋呤和生物喋呤

建立了同时分离检测尿中新喋呤（neopterin,NP）和生物喋呤(biopterin,BP)的高效液相色谱（HPLC）-荧光检测方法。采用Hypersil BDS C18柱、甲醇-水(体积比为10∶90)流动相(流速0.5 mL/min)、激发波长360 nm、发射波长 440 nm、柱温20 ℃的色谱条件,同时分离测定了尿中的NP和BP。尿标本经三氯乙酸处理,在4 ℃下,以12000 r/min的速率离心15 min,上清液用碱中和后,取30　μL直接进样。研究结果表明,NP的线性范围为0.12～10     

Determination of natural olive fruit fly pheromone in insect samples by enzyme linked immunoassays

The olive fruit fly pheromone avidin-biotin ELISA immunoassay, based on the use of polyclonal G antibodies derived from rabbits (reported previously) and a newer assay, based on the use of polyclonal Y antibodies isolated from the eggs of laying hens (reported in this paper), were applied successfully for the analysis of natural pheromone in virgin adult female olive fruit flies. According to the results obtained, the pheromone content in the glands of adult female olive fruit flies increases from the third to the ninth day of their age. During the calling period, the female olive fruit flies seem to emit approximately 1.1microg pheromone/insect/day at least. The immunoassay, based on the Y antibodies, is slightly more sensitive (detection limit 40ng/mL) than the assay based on polyclonal anti-pheromone rabbit antiserum (detection limit 80ng/mL). As revealed by thorough cross-reactivity studies, including 14 structurally similar to the olive fruit fly pheromone molecules, the newer immunoassay is less selective than the previous one and seems to cross react with few molecules bearing the spiroketal moiety.     

Antimicrobial activity of some new thioureides derived from 2-(4-chlorophenoxymethyl)benzoic acid

We report here the characterisation of eight newly synthesized thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid and the evaluation of the in vitro antimicrobial activity of the new compounds against Gram-positive [Listeria monocytogenes,Staphylococcus aureus, Bacillus subtilis], Gram-negative [Psedomonas aeruginosa,Escherichia coli, Salmonella enteritidis], as well as Candida spp., using both reference and clinical multidrug resistant strains to establish the minimal inhibitory concentration (MIC)values. Our results showed that the tested compounds exhibited specific antimicrobial activities, both concerning the spectrum of antimicrobial activity and the corresponding MIC values, which ranged widely between 1024 and 32 mug/mL, depending on the nature and position of the substituents on the benzene ring. The most active compounds were N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(2,6-dichlorophenyl)-thiourea (5 g) and N-[2-(4-chlorophenoxymethyl)-benzoyl]-N'-(4-bromophenyl)-thiourea (5h), which showed a broad spectrum of antimicrobial activity against enterobacterial strains (E. coli and S. enteritidis),P. aeruginosa, S. aureus and Candida spp. All the tested compounds except 5f were highly active against S. aureus (MIC=32 mug/mL), suggesting their possible use in the treatment of MRSA infections. Four of compounds also exhibited antifungal activity (MIC =256-32 microg/mL) against C. albicans, but L. monocytogenes as well as B. subtilis were resistant to all tested compounds. Our studies thus demonstrated that among other biological activities,the thioureides of 2-(4-chlorophenoxymethyl)-benzoic acid also exhibit selective and effective antimicrobial properties that could lead to the selection and use of these compounds as efficient antimicrobial agents, especially for the treatment of multidrug resistant infections.     

纳米金标记抗体增强SPR检测大肠杆菌O157∶H7

将金纳米粒子(AuNPs)标记的大肠杆菌O157∶H7(E.coli O157∶H7)的多克隆抗体(PAb)作为二抗,采用氨基偶联法将PAb固定在传感器表面作为一抗,通过三明治方法用双通道表面等离子体子共振(SPR)传感器对E.coli O157∶H7进行检测,并与SPR直接法检测进行了比较.结果表明,直接法的检出限为103cfu/mL,线性范围为103～109cfu/mL;AuNPs增强三明治法的检出限为10 cfu/mL,线性范围为10～1010cfu/mL,灵敏度比直接法提高了100倍,且具有更宽的检测范围.本方法不仅检测时间短,而且具有良好的选择性和重现性.     

Development of a novel method based on liquid chromatography-evaporative light scattering detection for the direct determination of streptomycin and dihydrostreptomycin in raw materials, pharmaceutical formulations, culture media and plasma

A novel method for the non-derivatization liquid chromatographic determination of streptomycin (STR) and dihydrostreptomycin (DHSTR) was developed and validated based on evaporative light scattering detection (ELSD). Utilizing a ThermoHypersil BetaBasic C18 analytical column, evaporation temperature of 50 degrees C and pressure of nebulizing gas (nitrogen) of 3.5 bar, the optimized mobile phase was 1.25 mL L(-1) TFA aqueous solution, in an isocratic mode at a rate of 1.0 mL min(-1). STR was eluted at 5.6 min and DHSTR at 7.8 min with a resolution of 4.4. Linear calibration curves were obtained from 2 to 120 microg mL(-1) (r > 0.9990) for STR and 2-75 microg mL(-1) (r > 0.9994) for DHSTR, with a LOD equal to 0.7 and 0.5 microg mL(-1), respectively. The developed method was applied for the assay of STR and DHSTR (sulfate) in pharmaceutical raw materials and formulations, while the simultaneous direct determination of sulfate was feasible (tR = 2.5 min, LOD = 1.4 microg mL(-1), double logarithmic calibration curve in the range of 4-50 microg mL(-1), r > 0.9998). Modified isocratic mobile phase (H2O-ACN, 90:10, v/v, containing 1.25 mL L(-1) TFA), was used for the determination of streptomycin B impurity in STR sulfate raw material and a gradient mobile phase (H2O-ACN containing TFA) was used for the determination of DHSTR in the presence of penicillinG procaine. The developed method was also applied for the assay of commercial formulations (STR powder and DHSTR injection solution and suspension) (%recovery 98-102, %RSD < 1.3, n = 3 x 3), for the determination of STR in bacteria culture medium (%recovery 99.6, %RSD = 0.8, n = 3 x 3), and for the determination of DHSTR in human plasma (2.0-23.0 microg mL(-1)) after solid phase extraction using carboxylate cartridges (%recovery 98.4-101.8, %RSD = 3.2, n = 3 x 3).     

Detection of hazelnut in foods using ELISA: challenges related to the detectability in processed foodstuffs

Hazelnuts are widely used nowadays, and can pose a serious threat to allergic consumers due to cross-contamination that may occur during processing. This might lead to the presence of hidden hazelnut in foods. Therefore, reliable tests are needed to detect hazelnut, especially in processed foods. A hazelnut-specific indirect competitive ELISA based on polyclonal chicken antibodies was developed. The polyclonal antibodies were raised against modified hazelnut proteins in order to improve the detectability of hazelnut proteins in processed foods. The assay showed a detection limit of 1.36 microg hazelnut protein/mL of 5 mM urea in phosphate-buffered saline buffer (pH 7.4). Limited cross-reactivity with walnut and pecan nut was observed; no cross-reactivity was observed with other food ingredients. Blank cookies spiked before analysis showed recoveries of 73-107%. However, cookies spiked before baking showed that the detectability was severely decreased. Addition of lactose to the cookies, which led to more severe modification through the Maillard reaction, led to an increase in the detectability. These results indicate that using antibodies developed toward allergens modified through food processing-simulating reactions is a better approach for detection.     

Fluorescence‐based lateral flow assays for rapid oral fluid roadside detection of cannabis use

With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral‐fluid testing for Δ⁹‐tetrahydrocannabinol (THC). This proof‐of‐concept assay uses a fluorescent‐based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling—thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL.     

Design and synthesis of all diastereomers of cyclic pseudo-dipeptides as mimics of cyclic CXCR4 pentapeptide antagonists

The four diastereomers of 2,5-bis[(3-guanidino)propyl]-1-[3-(4-hydroxyphenyl)propionyl]-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (-) and of 2,5-bis[(3-guanidino)propyl]-1-(4-hydroxyphenylacetyl)-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (-) were synthesized by a divergent methodology from l- and D-glutamic acids. The 11-membered ring core was made by ring closing metathesis of linear bis(allylamines), and the guanidyl functions were introduced by a simultaneous double Mitsunobu reaction using bis(Boc)guanidine. These compounds were designed to mimic cyclic pentapeptide FC131 (c[Gly-D-Tyr-Arg-Arg-Nal]).     

Sulfonation of Novel Fluorine-containing Poly(arylene ether nitrile)s for Polymer Electrolyte Membranes

Sulfonation reaction of fluorine-containing poly(arylene ether nitrile) derived from 4-phenoxy-2,3,5,6-tetrafluorobenzonitrile (PTFB) and 2,2-bis(4-hydroxyphenyl)-1,1,1,3,3,3-hexafluoropropane (6FBA) by concentrated sulfuric acid at 30°C yielded quantitative introduction of one sulfonic group into phenoxy ring in the polymer at para-position with 68 mol% and at meta-position with 32 mol% due to the influence of fluorines at the ortho-positions of adjacent benzene ring. Membrane properties of the resulting polymer, thermal stability, proton conductivity and water uptake, were investigated for the application in fuel cells. While the membrane derived from the polymer showed similar level of proton conductivity at humidified condition to NAFION®112, its dimensional stability based on water uptake was lower than that of NAFION®112.     

Determination of organic compounds in water using dispersive liquid-liquid microextraction

A new microextraction technique termed dispersive liquid-liquid microextraction (DLLME) was developed. DLLME is a very simple and rapid method for extraction and preconcentration of organic compounds from water samples. In this method, the appropriate mixture of extraction solvent (8.0 microL C2Cl4) and disperser solvent (1.00 mL acetone) are injected into the aqueous sample (5.00 mL) by syringe, rapidly. Therefore, cloudy solution is formed. In fact, it is consisted of fine particles of extraction solvent which is dispersed entirely into aqueous phase. After centrifuging, the fine particles of extraction solvent are sedimented in the bottom of the conical test tube (5.0 +/- 0.2 microL). The performance of DLLME is illustrated with the determination of polycyclic aromatic hydrocarbons (PAHs) in water samples by using gas chromatography-flame ionization detection (GC-FID). Some important parameters, such as kind of extraction and disperser solvent and volume of them, and extraction time were investigated. Under the optimum conditions the enrichment factor ranged from 603 to 1113 and the recovery ranged from 60.3 to 111.3%. The linear range was 0.02-200 microg/L (four orders of magnitude) and limit of detection was 0.007-0.030 microg/L for most of analytes. The relative standard deviations (RSDs) for 2 microg/L of PAHs in water by using internal standard were in the range 1.4-10.2% (n = 5). The recoveries of PAHs from surface water at spiking level of 5.0 microg/L were 82.0-111.0%. The ability of DLLME technique in the extraction of other organic compounds such as organochlorine pesticides, organophosphorus pesticides and substituted benzene compounds (benzene, toluene, ethyl benzene, and xylenes) from water samples were studied. The advantages of DLLME method are simplicity of operation, rapidity, low cost, high recovery, and enrichment factor.     

Cytotoxic lignans from the stems of Styrax camporum (Styracaceae)

An ethanolic extract from the stems of Styrax camporum Pohl (Styracaceae), a plant traditionally used for gastrointestinal diseases, was fractionated and subjected to flash chromatography and afforded two benzofuran lignans, egonol and homoegonol, and one furofuran lignan, (+/-)syringaresinol, which were identified by spectral data interpretation. Their cytotoxic activities against Hep-2 (larynx epidermoid carcinoma), HeLa (human cervix carcinoma) and C6 (rat glioma) cell lines were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay at several concentrations for 24h. Activities could be observed for egonol against C6 (IC50 = 3.2 microg/mL) and Hep-2 (IC50 = 3.6 microg/mL) cell lines, and for homoegonol against C6 (IC50 = 4.9 microg/mL) and HeLa (IC50 = 5.3 microg/mL) cells.