110 research outputs found

    BPA Endocrine Disruptor Detection at the Cutting Edge: FPIA and ELISA Immunoassays

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    BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20–30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten–protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 ÎŒg/L, working range from 2 to 155 ÎŒg/L. The validation of the methods was conducted using actual samples compared to LC–MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.BAM’s MI-1 programPeer Reviewe

    Fluorescence polarization immunoassay for the determination of diclofenac in wastewater

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    Pharmacologically active compounds are often detected in wastewater and surface waters. The nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the European watch list of substances that requires its environmental monitoring in the member states. DCF may harmfully influence the ecosystem already at concentrations ≀ 1 Όg L−1. The fast and easy quantification of DCF is becoming a subject of global importance. Fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read method which does not require the immobilization of reagents. FPIA can be performed in one phase within 20–30 min, making it possible to analyse wastewater without any complicated pre-treatment. In this study, new tracer molecules with different structures, linking fluorophores to derivatives of the analyte, were synthesized, three homologous tracers based on DCF, two including a C6 spacer, and one heterologous tracer derived from 5-hydroxy-DCF. The tracer molecules were thoroughly assessed for performance. Regarding sensitivity of the FPIA, the lowest limit of detection reached was 2.0 Όg L−1 with a working range up to 870 Όg L−1. The method was validated for real wastewater samples against LC-MS/MS as reference method with good agreement of both methods. Graphical abstractBundesanstalt fĂŒr Materialforschung und -PrĂŒfung http://dx.doi.org/10.13039/100009553Peer Reviewe

    Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat

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    [EN] Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and tetraconazole were synthesized and the tracers' structures were confirmed by HPLC-MS/MS. The 4-aminomethylfluorescein-labeled tracers allowed achieving the best assay sensitivity and minimum reagent consumption in comparison with aminofluorescein-labeled and alkyldiaminefluoresceinthiocarbamyl-labeled tracers. Measurements of fluorescence polarization were performed using a portable device. The developed FPIA methods were applied for the analysis of wheat. Fast and simple sample preparation technique earlier developed by authors for pesticides was adapted for thiabendazole and tetraconazole. The limits of detection of thiabendazole and tetraconazole in wheat were 20 and 200g/kg, and the lower limits of quantification were 40 and 600g/kg, respectively. The recovery test was performed by two methodsFPIA and HPLC-MS/MS. The results obtained by FPIA correlated well with those obtained by HPLC-MS/MS (r(2)=0.9985 for thiabendazole, r(2)=0.9952 for tetraconazole). Average recoveries of thiabendazole and tetraconazole were 744% and 723% by FPIA, and average recoveries of thiabendazole and tetraconazole were 86 +/- 2% and 74 +/- 1% by HPLC-MS/MS (n=15).The work was financially supported by the Russian Science Foundation (project No. 14-16-00149).Boroduleva, AY.; Manclus Ciscar, JJ.; Montoya, Á.; Eremin, SA. (2018). Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat. Analytical and Bioanalytical Chemistry. 410(26):6923-6934. https://doi.org/10.1007/s00216-018-1296-zS6923693441026Robinson HJ, Stoerk HC, Graessle OE. Studies on the toxicologic and pharmacologic properties of thiabendazole. Toxicol Appl Pharmacol. 1965;7:53–63.Abbassy MA, Marzouk MA, Nasr HM, Mansy AS. Effect of imidacloprid and tetraconazole on various hematological and biochemical parameters in male albino rats (Rattus norvegious). J Pol Sci Pub Aff. 2014;2:7.European Commission, Regulation (EC) No 2017/1164 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for acrinathrin, metalaxyl and thiabendazole in or on certain products. Off J EU L170. 2017:3–30.Hygienic standard GN 1.2.3539-18. Hygienic standards for pesticide residues in environmental samples (list). 2018. In Russian. 〈 http://docs.cntd.ru/document/557532326 . Accessed 07.07.2018).European Commission, Regulation (EC) No 822/2009 amending Annexes II, III and IV to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for azoxystrobin, atrazine, chlormequat, cyprodinil, dithiocarbamates, fludioxonil, fluroxypyr, indoxacarb, mandipropamid, potassium tri-iodide, spirotetramat, tetraconazole, and thiram in or on certain products. Off J EU L239. 2009:5–45.GarcĂ­a-FernĂĄndez M, DĂ­az-Álvarez M, MartĂ­n-Esteban A. Molecularly imprinted magnetic nanoparticles for the micro solid-phase extraction of thiabendazole from citrus samples. J Sep Sci. 2017;40:2638–44.Yu QW, Sun H, Wang K, He HB, Feng YQ. Monitoring of carbendazim and thiabendazole in fruits and vegetables by SiO2@ NiO-based solid-phase extraction coupled to high-performance liquid chromatography-fluorescence detector. Food Anal Methods. 2017;10:2892–901.Alves AA, Rodrigues AS, Barros EBP, Uekane TM, Bizzo HR, Rezende CM. Determination of pesticides residues in Brazilian grape juices using GC-MS-SIM. Food Anal Methods. 2014;7:1834–9.Zhang H, Qian M, Wang X, Wang X, Xu H, Qi P, et al. Analysis of tebuconazole and tetraconazole enantiomers by chiral HPLC-MS/MS and application to measure enantioselective degradation in strawberries. Food Anal Methods. 2012;5:1342–8.Bordagaray A, GarcĂ­a-Arrona R, MillĂĄn E. Development and application of a screening method for triazole fungicide determination in liquid and fruit samples using solid-phase microextraction and HPLC-DAD. Anal Methods. 2013;5:2565–71.Aquino A, Navickiene S. MSPD procedure for determination of carbofuran, pyrimethanil and tetraconazole residues in banana by GC–MS. Chromatographia. 2009;70:1265–9.Dankwardt A, Pullen S, Hock B. Immunoassays: applications for the aquatic environment. In: Wells PG, Lee K, Blaise C, editors. Microscale testing in aquatic toxicology. Boca Raton: CRC Press; 2018. p. 13–29.Wells MJM, Bell KY, Traexler KA, Pellegrin M-L, Morse A. Emerging pollutants. Water Environ Res. 2011;82(10):2095–70.Abad A, ManclĂșs JJ, Moreno MJ, Montoya A. Determination of thiabendazole in fruit juices by a new monoclonal enzyme immunoassay. J AOAC Int. 2001;84:156–61.΀sialla Z, Ucles-Moreno A, Petrou P, Fernandez-Alba AR, Κakabakos SE. Development of an indirect enzyme immunoassay for the determination of thiabendazole in white and red wines. Int J Environ Anal Chem. 2015;95:1299–309.UclĂ©s A, GarcĂ­a AV, GarcĂ­a MDG, del Real AMA, FernĂĄndez-Alba AR. Benzimidazole and imidazole fungicide analysis in grape and wine samples using a competitive enzyme-linked immunosorbent assay. Anal Methods. 2015;7:9158–65.BlaĆŸkovĂĄ M, Rauch P, Fukal L. Strip-based immunoassay for rapid detection of thiabendazole. Biosens Bioelectron. 2010;25:2122–8.Estevez MC, Belenguer J, Gomez-Montes S, Miralles J, Escuela AM, Montoya A, et al. Indirect competitive immunoassay for the detection of fungicide thiabendazole in whole orange samples by surface plasmon resonance. Analyst. 2012;137:5659–65.Cairoli S, Arnoldi A, Pagani S. Enzyme-linked immunosorbent assay for the quantitation of the fungicide tetraconazole in fruits and fruit juices. J Agric Food Chem. 1996;44:3849–54.ManclĂșs JJ, Moreno MJ, Plana E, Montoya A. Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices. J Agric Food Chem. 2008;56:8793–800.Plana E, Moreno MJ, Montoya Á, ManclĂșs JJ. Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices. Food Chem. 2014;143:205–13.Feng J, Hu Y, Grant E, Lu X. Determination of thiabendazole in orange juice using an MISPE-SERS chemosensor. Food Chem. 2018;239:816–22.Smith DS, Eremin SA. Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules. 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    Development of Competitive ELISA and CLEIA for Quantitative Analysis of Polymyxin B

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    Polymyxin B (PMB), a member of polypeptide antibiotics, is widely used for the treatment of infection in animals such as cattle, sheep, pigs, and chickens. However, it is toxic on the kidneys and nervous system, and polymyxin resistance is increasingly reported, which leaves a serious threat to human health. Therefore, it is essential to establish rapid methods for detecting PMB with high sensitivity and specificity. In this study, an anti-PMB polyclonal antibody (pAb) was obtained by immunizing New Zealand white rabbits with PMB conjugated with glycosylated bovine serum albumin (GBSA). Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) were developed. Under the optimal conditions, inhibitory concentrations (IC50) of PMB were 257.1 ng/mL (ic-ELISA) and 250.8 ng/mL (ic-CLEIA); the limits of detection (LOD) were 17.4 ng/mL (ic-ELISA) and 14.5 ng/mL (ic-CLEIA), respectively. Cross-reactivity of the pAb toward polymyxin E (PME) was 257.1%, and no response was found with other antibiotics. The recovery rates in spiked meat samples were 77.4 similar to 106.1% (ic-ELISA) and 84.1 similar to 107.1% (ic-CLEIA), respectively.Polymyxin B (PMB), a member of polypeptide antibiotics, is widely used for the treatment of infection in animals such as cattle, sheep, pigs, and chickens. However, it is toxic on the kidneys and nervous system, and polymyxin resistance is increasingly reported, which leaves a serious threat to human health. Therefore, it is essential to establish rapid methods for detecting PMB with high sensitivity and specificity. In this study, an anti-PMB polyclonal antibody (pAb) was obtained by immunizing New Zealand white rabbits with PMB conjugated with glycosylated bovine serum albumin (GBSA). Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) were developed. Under the optimal conditions, inhibitory concentrations (IC50) of PMB were 257.1 ng/mL (ic-ELISA) and 250.8 ng/mL (ic-CLEIA); the limits of detection (LOD) were 17.4 ng/mL (ic-ELISA) and 14.5 ng/mL (ic-CLEIA), respectively. Cross-reactivity of the pAb toward polymyxin E (PME) was 257.1%, and no response was found with other antibiotics. The recovery rates in spiked meat samples were 77.4 similar to 106.1% (ic-ELISA) and 84.1 similar to 107.1% (ic-CLEIA), respectively

    Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

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    International audienceA Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia

    Exploring the anti‐α‐amylase activity of flavonoid aglycones in fabaceae plant extracts: a combined MALDI‐TOF‐MS and LC–MS/MS approach

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    A combination of TLC-bioautography, MALDI-TOF-MS and LC–MS/MS methods was used to identify flavonoids with anti-α-amylase activity in extracts of Lathyrus pratensis L. (herb), L. polyphillus L. (fruits), Thermopsis lanceolata R. Br. (herb) and S. japonica L. (buds). After the TLC-autobiography assay, substances with anti-amylase activity were identified by MALDI-TOF-MS followed by confirmation of the result by LC–MS/MS. Results of the study revealed that the flavonoids apigenin, luteolin, formononetin, genistein and kaempferol display marked anti-α-amylase activity. Formononetin showed the largest activity. Compared with LC–MS/MS, MALDI-TOF-MS is a quick and convenient method; results can be obtained within minutes; and only minor sample amounts are required which allows us to analyse mixtures of substances without preliminary separation. However, the inability to distinguish between isomers is the main limitation of the method.BAM/BMWi http://dx.doi.org/10.13039/501100002765German Research Foundation http://dx.doi.org/10.13039/501100001659Peer Reviewe

    Express Detection of Pentachlorophenol as Dioxins Precursor in Natural Water

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    A rapid detection method for the pesticide pentachlorophenol (PCP) — polarization fluoroimmunoassay (PFIA) — in the dynamic range of 10–9,000 ppb was developed. PCP may form polychlorinated dibenzo-p-dioxins, making environmental monitoring of this compound an issue of great importance. In order to optimize the PFIA procedure, a number of fluorescein-labeled PCP derivatives and similar compounds (tracers) were synthesized, and the influence of their structure on PFIA characteristics was studied. Also, two antisera were tested in developing PFIA for PCP. The developed method is highly specific for PCP and can be used for its determination in water samples at a level down to 10 ppb. Total time of the assay for 10 samples is about 7 min. The assay provides a useful and a highly practical screening tool for the processing of large numbers of samples and for the preliminary estimation of potential dioxins contamination in water resources

    Hematological parameters that determine the course of labor and the postpartum period in highly productive Holstein cows

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    The aim of the study is to determine the effect of hematological parameters of cows on the course of labor and the postpartum period in highly productive Holstein cows. For this purpose, one group of cows was formed on the principle of analogue pairs in the amount of 30 animals with a gestation period of 7.5–8.0 months, followed by blood sampling from them using the Monovet system in vacuum test tubescontainers to determine morphological and biochemical parameters. Blood was taken 25–30 days before delivery in the morning 2 hours before feeding. Blood values were studied according to generally accepted methods using certified equipment. Subsequently, depending on the nature of the course of the birth, the animals were divided into two groups. The first group included 24 cows without pathology of the course of labor and the postpartum period, the second group included 16 cows with pathologies of the course of labor and the postpartum period. In the process of research, it was found that in 60.0 % of the cows the birth and the postpartum period passed without pathologies, and in 40.0 % of the birth and postpartum complications were revealed (retention of the placenta, acute postpartum endometritis, uterine subinvolution). A comparative analysis of the blood values of cows 25–30 days before birth the course of the labor process and postpartum period showed that in animals without pathology — compared to the animals with pathologies — the hematological parameters are considerably higher (hemoglobin content by 16.42 g/l, total protein by 10.96 g/l, albumin by 7.7 %, calcium by 0.98 mmol/l, glucose by 0.91 mmol, immunoglobin A by 59.77 mg/M), while the content of beta-globulins is lower by 8.51 % and that of enzyme AcT is lower by 25.94 units/l. A decrease in blood biochemical parameters contributes to the manifestation of the pathology of labor and the postpartum period. The data obtained can be used to develop an algorithm for the prevention of postpartum complications in highly productive cows
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