环境中毒死蜱的酶联免疫分析方法研究

Study on Enzyme-linked Immunosorbent Assay for Chlorpyrifos in Environment

【目的】毒死蜱广泛使用及具有较好的稳定性,在很多地区的环境介质中均已检测到其残留。为实现土壤、水等环境基质中毒死蜱残留的现场监测,保障环境质量安全,研究采用酶联免疫法快速检测环境中的毒死蜱。【方法】以抗原抗体的特异性结合为原理,建立间接竞争ELISA酶联免疫检测环境中的毒死蜱。对试验条件进行优化,研究方法的灵敏度、交叉反应及有机溶剂对抗体的影响等。【结果】在最优条件下,毒死蜱质量浓度对数与吸光度值呈线性关系,回归方程为B/B_(0)=-24.87 logC+97.55,相关系数R^(2)>0.99,毒死蜱的IC_(50)=81.65μg/L,检出限IC_(20)为5.1μg/L。根据不同的基质选择合适的前处理方法并进行验证,毒死蜱回收率在70%~115%之间,相对偏差在11%以内;方法的精密度相对偏差均小于10%,与气相色谱法比对证明方法的准确性。在180 d内试剂盒储存于4℃或-20℃下均不影响检测效果。【结论】建立的间接竞争ELISA酶联免疫法具有一定的特异性、稳定性和精确性,可应用于检测不同类型的土壤及水样中的毒死蜱,能够实现快速、灵敏的毒死蜱农药残留检测。

【Objective】Chlorpyrifos is widely used and has good stability,and its residue has been detected in the environmental medium in many areas.In order to monitor the residues of chlorpyrifos in soil,water and other environmental substrates,and to ensure the quality and safety of environment,enzyme-linked immunosorbent assay (ELISA) was used to detect chlorpyrifos rapidly in the environment.【Method】Based on the principle of specific binding of antigen and antibody,an indirect competitive ELISA was established to detect chlorpyrifos in the environment.The test conditions were optimized,and the sensitivity,cross-reaction and effect of organic solvents on the antibody were studied.【Result】Under the optimal conditions,the logarithm of chlorpyrifos concentration was linearly related to the absorbance value,the regression equation was B/B_(0)=-24.87 logC+97.55 and the correlation coefficient (R^(2)) was over 0.99.The IC_(50)of chlorpyrifos was 81.65 ng/mL,and the detection limit IC_(20)was 5.1μg/L.The method was validated by selecting appropriate pretreatment methods according to different substrates.The recovery rates of chlorpyrifos ranged from 70%to 115%and the relative standard deviation was less than 11%.The relative standard deviation of precision was less than 10%.The accuracy of the method was verified by comparison with the gas chromatography.The prepared reagents shall not be stored for more than 6 months.The detection effects were not affected by reagents at 4℃and-20℃within 180 days.【Conclusion】With certain specificity,stability and precision,the established indirect enzyme-linked immunosorbent assay can be used for rapid and flexible detection of chlorpyrifos residues in different types of soil and water samples.