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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (5): 513-521    DOI: 10.3785/j.issn.1008-9209.2012.12.254
农业科学     
糙米、土壤和田水中三唑磷残留的化学发光酶免疫分析方法
田洁， 梁晓， 桂文君， 朱国念*
(浙江大学农药与环境毒理研究所，杭州310029)
Development of chemiluminescent enzyme immunoassay for the determination of triazophos residues in rice grain, soil and paddy water
TIAN  Jie, LIANG  Xiao, GUI Wenjun, ZHU Guonian*
(Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029,  China)
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摘要： 建立三唑磷间接竞争化学发光酶免疫法(indirect competitive chemiluminescent enzyme immunoassay， icCLEIA)。以鲁米诺和过氧化氢作为化学发光底物，通过对工作浓度、化学发光增敏液、反应缓冲液、有机助溶剂与浓度等条件的优化，确立方法的最佳工作条件。该方法对三唑磷检测线性范围为0.097~12.5 ng/mL，IC50为1.23 ng/mL，检出限可达0.14 ng/mL。将优化的方法应用到稻田水样、土样、糙米样品中三唑磷残留检测，样品的添加回收率为67%~113%，变异系数为0.08%~24.53%。研究表明该方法可用于上述样品中三唑磷残留的快速检测，并可为三唑磷化学发光试剂盒的开发应用提供依据。
Abstract: 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 H2O2. 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 H2O2. 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 (IC10) was 0.14 ng/mL, and the standard curve  midpoint (IC50) 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.
出版日期: 2013-09-20
CLC:  R 392.11  
基金资助:

公益性行业（农业）科研专项经费项目（2012030943）;教育部博士点新教师基金（20110101120085）;浙江省自然科学基金资助项目（Y5110109）。
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田洁
梁晓
桂文君
朱国念*

引用本文:

田洁， 梁晓， 桂文君， 朱国念*. 糙米、土壤和田水中三唑磷残留的化学发光酶免疫分析方法[J]. 浙江大学学报(农业与生命科学版), 2013, 39(5): 513-521.

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. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(5): 513-521.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.12.254    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I5/513

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