Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water
Triazophos is now widely used in agriculture. Its residues may cause health implications for living organisms including humans. There has been a greater demand for developing analytical methods with very high sensitivity. Immunochemical techniques provide a simple and economical alternative to tradi...
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Zhejiang University Press
2013-09-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
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| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.12.254 |
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| author | TIAN Jie LIANG Xiao GUI Wenjun ZHU Guonian |
| author_facet | TIAN Jie LIANG Xiao GUI Wenjun ZHU Guonian |
| author_sort | TIAN Jie |
| collection | DOAJ |
| description | Triazophos is now widely used in agriculture. Its residues may cause health implications for living organisms including humans. There has been a greater demand for developing analytical methods with very high sensitivity. Immunochemical techniques provide a simple and economical alternative to traditional methods in order to meet such demands for determination of triazophos residues.The aim of this work was to develop an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) with an anti-mouse secondary antibody conjugated to peroxidase for the residual determination of triazophos. Luminol solution was used as the substrate of horseradish peroxidase. First, the optimal coating conjugate concentration and anti-triazophos monoclonal antibodies dilution were studied. Then the chemiluminescence substrate solution was obtained after a variety of conditions were optimized, including the selection of buffer and enhancer, concentrations of luminol, p-iodophenol and H<sub>2</sub>O<sub>2</sub>. On the basis of it, the effects of several physicochemical parameters on icCLEIA performance were studied and optimized, such as the effects of ionic strength and pH, and the tolerance to organic solvents (methanol, acetonitrile, acetone). After the condition optimization, the standard curve of CLEIA competitive reaction for triazophos was established. Afterwards, the optimized method was applied to the detection of triazophos residues in paddy water, soil, and rice grain samples, and the precision and accuracy were analyzed as well.As a result, the optimal concentration of conjugate concentration and monoclonal antibodies were 0.125 and 2 mg/L respectively. The optimum conditions were given as followed: 0.1 mol/L, pH 8.5 Tris-HCl buffer was chosen as the chemiluminescence substrate solution buffer, containing 1 mmol/L p-iodophenol, 0.5 mmol/L luminol and 1 mmol/L H<sub>2</sub>O<sub>2</sub>. Then we chose the 0.05 mol/L, pH 7.4 PBS buffer as the reaction buffer, which could improve the method's sensitivity and get the largest light-emitting count. Meanwhile, 5% methanol was proved to be suitable for the competitive reaction. Under the optimum conditions, the linear range of the icCLEIA was from 0.097 to 12.5 ng/mL. The limit of detection values (IC<sub>10</sub>) was 0.14 ng/mL, and the standard curve midpoint (IC<sub>50</sub>) was 1.23 ng/mL. The assay performance indicated that the CLEIA could offer the possibility of improving the sensitivity of immunoassays by 12 orders of magnitude compared with conventional ELISA which is widely used to detect triazophos. The matrix effects of the paddy water, soil, and rice grain samples were studied. According to the results, different dilution ratios were determined: the paddy water, soil, and rice grain samples were diluted with reaction buffer by 10, 100, 50-fold respectively. The assay was then used to detect triazophos in the three samples at above-mentioned dilution ratios. The results showed that when the spiked level was from 5800 ng/g, the recoveries of triazophos from spiked samples ranged from 67% to 113% with coefficients of variation of 0.08% 24.53%.In summary, the established icCLEIA is a simple, rapid and economical tool which is suitable for the rapid screening of triazophos residues in environmental samples such as paddy water, soil and rice grain samples with a high sensitivity. It can be used as the developing foundation of CLEIA test kits in the future. |
| format | Article |
| id | doaj-art-166fc1f8ada04cf7baf01030cb231f14 |
| institution | Kabale University |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2013-09-01 |
| publisher | Zhejiang University Press |
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| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-166fc1f8ada04cf7baf01030cb231f142025-08-20T03:58:18ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552013-09-013951352110.3785/j.issn.1008-9209.2012.12.25410089209Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy waterTIAN JieLIANG XiaoGUI WenjunZHU GuonianTriazophos is now widely used in agriculture. Its residues may cause health implications for living organisms including humans. There has been a greater demand for developing analytical methods with very high sensitivity. Immunochemical techniques provide a simple and economical alternative to traditional methods in order to meet such demands for determination of triazophos residues.The aim of this work was to develop an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) with an anti-mouse secondary antibody conjugated to peroxidase for the residual determination of triazophos. Luminol solution was used as the substrate of horseradish peroxidase. First, the optimal coating conjugate concentration and anti-triazophos monoclonal antibodies dilution were studied. Then the chemiluminescence substrate solution was obtained after a variety of conditions were optimized, including the selection of buffer and enhancer, concentrations of luminol, p-iodophenol and H<sub>2</sub>O<sub>2</sub>. On the basis of it, the effects of several physicochemical parameters on icCLEIA performance were studied and optimized, such as the effects of ionic strength and pH, and the tolerance to organic solvents (methanol, acetonitrile, acetone). After the condition optimization, the standard curve of CLEIA competitive reaction for triazophos was established. Afterwards, the optimized method was applied to the detection of triazophos residues in paddy water, soil, and rice grain samples, and the precision and accuracy were analyzed as well.As a result, the optimal concentration of conjugate concentration and monoclonal antibodies were 0.125 and 2 mg/L respectively. The optimum conditions were given as followed: 0.1 mol/L, pH 8.5 Tris-HCl buffer was chosen as the chemiluminescence substrate solution buffer, containing 1 mmol/L p-iodophenol, 0.5 mmol/L luminol and 1 mmol/L H<sub>2</sub>O<sub>2</sub>. Then we chose the 0.05 mol/L, pH 7.4 PBS buffer as the reaction buffer, which could improve the method's sensitivity and get the largest light-emitting count. Meanwhile, 5% methanol was proved to be suitable for the competitive reaction. Under the optimum conditions, the linear range of the icCLEIA was from 0.097 to 12.5 ng/mL. The limit of detection values (IC<sub>10</sub>) was 0.14 ng/mL, and the standard curve midpoint (IC<sub>50</sub>) was 1.23 ng/mL. The assay performance indicated that the CLEIA could offer the possibility of improving the sensitivity of immunoassays by 12 orders of magnitude compared with conventional ELISA which is widely used to detect triazophos. The matrix effects of the paddy water, soil, and rice grain samples were studied. According to the results, different dilution ratios were determined: the paddy water, soil, and rice grain samples were diluted with reaction buffer by 10, 100, 50-fold respectively. The assay was then used to detect triazophos in the three samples at above-mentioned dilution ratios. The results showed that when the spiked level was from 5800 ng/g, the recoveries of triazophos from spiked samples ranged from 67% to 113% with coefficients of variation of 0.08% 24.53%.In summary, the established icCLEIA is a simple, rapid and economical tool which is suitable for the rapid screening of triazophos residues in environmental samples such as paddy water, soil and rice grain samples with a high sensitivity. It can be used as the developing foundation of CLEIA test kits in the future.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.12.254triazophosresiduechemiluminescent enzyme immunoassay |
| spellingShingle | TIAN Jie LIANG Xiao GUI Wenjun ZHU Guonian Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water 浙江大学学报. 农业与生命科学版 triazophos residue chemiluminescent enzyme immunoassay |
| title | Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water |
| title_full | Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water |
| title_fullStr | Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water |
| title_full_unstemmed | Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water |
| title_short | Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water |
| title_sort | development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain soil and paddy water |
| topic | triazophos residue chemiluminescent enzyme immunoassay |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.12.254 |
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