农药残留免疫检测技术及其应用研究
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摘要
农药残留免疫检测技术是近年国内外小分子污染物快速检测的重要研究内容。本文针对我国目前重要农产品和环境污染药剂，开展了多克隆和单克隆抗体制备研究，并针对不同抗原-抗体反应特点建立了间接竞争ELISA方法，在此基础上，对建立的检测技术在农药的田间降解动态分析以及不同残留消解方法对农药的降解作用研究方面开展了实测应用工作，取得结果主要包括以下几方面。
1．以氟虫腈类似物FH作为半抗原，与蛋白偶联后免疫Balb／c小鼠。运用杂交瘤技术将激活脾细胞与瘤细胞系SP2／0融合，经多次筛选获得产生阳性抗体的杂交瘤细胞株3G_3，通过腹水制得高效价氟虫腈单克隆抗体。经检测抗体与定虫隆、三氟氯氰菊酯、氟乐灵、氟虫脲的交叉反应率均小于0.67％。在抗原稀释2000倍、抗体稀释4000倍时为最佳工作浓度，建立的间接竞争ELISA对氟虫腈最低检测浓度为0.074ng·mL~(-1)，抑制中浓度为5.99ng·mL~(-1)，最低检出量为0.0037ng。对两种不同基质中的氟虫腈进行添加试验，结果变异度为1.8-9.8％，回收率为94.7-108％。
2．利用单克隆抗体间接竞争ELISA监测氟虫腈在田间的残留降解动态。发现其在青菜中的降解动态符合指数方程C=0.1927e~(-0.0666t)，降解半衰期为10.4天，r=-0.9787；在土壤中的降解动态符合指数方程C=0.0990e~(-0.0459t)，半衰期为15.1天，r=-0.9826。在室内从紫外线、臭氧、PH值、原子辐照4个方面对氟虫腈降解因子进行了研究，结果发现紫外线照射25h时，氟虫腈降解率最高可到90％；通入臭氧时间60min时，氟虫腈降解率最高可达48.5％；溶液PH为12时，氟虫腈在48h时降解率高达88％。
3．选用二乙基膦酸乙酸作为半抗原，以NHS-DCC法合成的人工抗原所制得抗血清的最高滴度为25600，以EDC法合成的抗原制得的抗血清滴度最高为6400。以二乙基膦酸乙酸为对象建立了优化的间接竞争ELISA检测技术。对二乙基膦酸乙酸最低检测浓度为0.003536μg／mL，抑制中浓度为0.1820μg／mL，最低检出量为0.1782ng。对13种常见磷酸酯类有机磷农药进行交叉反应试验，结果发现：所得抗体对毒死蜱、氧乐果、二嗪农、乙基对硫磷、丙溴磷、辛硫磷等农药有特
农药残留免疫检测技术及其应用研究
异性反应，抑制中浓度 IC。③分另为 0．12，0．15，0．ZI，0．8 8，0．9 7和 2．5卜咖L。
上述结果表明该检测技术可用于毒死婢、氧乐果、二嘻农、乙基对硫磷、丙决磷
等药剂的快速定性或半定量检测。
Immunological analysis technique was developed and employed efficiently for the pesticide residue screening in different kinds of samples. In this work, polycolonal and monocolonal antibodies of some of the chemical pesticides widely used in our country were developed and enzyme-linked immunosorbent assay methods were standardized, which were approved in the residue detection of the pesticide in the fields and residue elimination determination of pesticides in the vegetables. The results were described as bellow.
1. One hybridoma-cell line 3C_3 secreting monoclonal antibodies against insecticide fipronil were produced by fusing mouse myeloma cells (sp2/0) with spleen cells from Balb/c mice immunized by the hapten fipronil analygue conjugated to bovine serum albumin. The production and characterization of hybridoma cell line secreting positive antibodies permanently are described. The effect of several physicochemical factors on the monoclonal antibodies for fipronil was studied and the results were used to optimize the assay system, when incorporated into a competitive indirect ELISA, the antibody provided a detection limit of 0.074ngmL-1 and I50 5.99ng
·mL-1 of fipronil. The specificity of the resultant fipronil-specific monoclonal antibodies was tested with several insecticides including Chlorfluazuron, Cyhalothin, Trifluralin and Flufenoxuron, the cross-reaction was less than 0.67%, indicating there no interfering comfounds was found.In different matrix, the fipronil recovery is in the range of 94.7-108% and the coefficient of variation 1.8-9.8%.
2. The indirect competitive ELISA based on monoclonal antibody introduced previously was applied to moitor the degradation kinetics of fipronil in vegetable and soil. The results showed that the semi-life of fipronil in vegetable is 10.4 d., and 15.1 d. in soil. The immunological assay method was applied to evaluate the effect of PH value, ozone in aqueous solution, Co-60 irradiation and UV-light irradiation on the
degration of fipronil in the lab. The reduction of the amount of the pesticide indicated that dissipation rate of fipronil was significantly accelerated by PH value, ozone into aqueous solution and UV-light irradiation.
3. Diethylphosphono acetic acid (DPA) was used as a generic hapten in order to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. In an indirect enzyme-linked immunosorbent assay (ELISA) format, the monoclonal-antibody has a high affinity to DPA , chlorpyrifos , diazinon , omethoate , parathion-ethyl and profenofos, showed preferential binding towards pesticides containing diethyl-phosphate and p=o functionalities rather than those containing dimethyl-structure and p=s group. The potential for using DPA was discussed.
1．以氟虫腈类似物FH作为半抗原，与蛋白偶联后免疫Balb／c小鼠。运用杂交瘤技术将激活脾细胞与瘤细胞系SP2／0融合，经多次筛选获得产生阳性抗体的杂交瘤细胞株3G_3，通过腹水制得高效价氟虫腈单克隆抗体。经检测抗体与定虫隆、三氟氯氰菊酯、氟乐灵、氟虫脲的交叉反应率均小于0.67％。在抗原稀释2000倍、抗体稀释4000倍时为最佳工作浓度，建立的间接竞争ELISA对氟虫腈最低检测浓度为0.074ng·mL~(-1)，抑制中浓度为5.99ng·mL~(-1)，最低检出量为0.0037ng。对两种不同基质中的氟虫腈进行添加试验，结果变异度为1.8-9.8％，回收率为94.7-108％。
2．利用单克隆抗体间接竞争ELISA监测氟虫腈在田间的残留降解动态。发现其在青菜中的降解动态符合指数方程C=0.1927e~(-0.0666t)，降解半衰期为10.4天，r=-0.9787；在土壤中的降解动态符合指数方程C=0.0990e~(-0.0459t)，半衰期为15.1天，r=-0.9826。在室内从紫外线、臭氧、PH值、原子辐照4个方面对氟虫腈降解因子进行了研究，结果发现紫外线照射25h时，氟虫腈降解率最高可到90％；通入臭氧时间60min时，氟虫腈降解率最高可达48.5％；溶液PH为12时，氟虫腈在48h时降解率高达88％。
3．选用二乙基膦酸乙酸作为半抗原，以NHS-DCC法合成的人工抗原所制得抗血清的最高滴度为25600，以EDC法合成的抗原制得的抗血清滴度最高为6400。以二乙基膦酸乙酸为对象建立了优化的间接竞争ELISA检测技术。对二乙基膦酸乙酸最低检测浓度为0.003536μg／mL，抑制中浓度为0.1820μg／mL，最低检出量为0.1782ng。对13种常见磷酸酯类有机磷农药进行交叉反应试验，结果发现：所得抗体对毒死蜱、氧乐果、二嗪农、乙基对硫磷、丙溴磷、辛硫磷等农药有特
农药残留免疫检测技术及其应用研究
异性反应，抑制中浓度 IC。③分另为 0．12，0．15，0．ZI，0．8 8，0．9 7和 2．5卜咖L。
上述结果表明该检测技术可用于毒死婢、氧乐果、二嘻农、乙基对硫磷、丙决磷
等药剂的快速定性或半定量检测。
Immunological analysis technique was developed and employed efficiently for the pesticide residue screening in different kinds of samples. In this work, polycolonal and monocolonal antibodies of some of the chemical pesticides widely used in our country were developed and enzyme-linked immunosorbent assay methods were standardized, which were approved in the residue detection of the pesticide in the fields and residue elimination determination of pesticides in the vegetables. The results were described as bellow.
1. One hybridoma-cell line 3C_3 secreting monoclonal antibodies against insecticide fipronil were produced by fusing mouse myeloma cells (sp2/0) with spleen cells from Balb/c mice immunized by the hapten fipronil analygue conjugated to bovine serum albumin. The production and characterization of hybridoma cell line secreting positive antibodies permanently are described. The effect of several physicochemical factors on the monoclonal antibodies for fipronil was studied and the results were used to optimize the assay system, when incorporated into a competitive indirect ELISA, the antibody provided a detection limit of 0.074ngmL-1 and I50 5.99ng
·mL-1 of fipronil. The specificity of the resultant fipronil-specific monoclonal antibodies was tested with several insecticides including Chlorfluazuron, Cyhalothin, Trifluralin and Flufenoxuron, the cross-reaction was less than 0.67%, indicating there no interfering comfounds was found.In different matrix, the fipronil recovery is in the range of 94.7-108% and the coefficient of variation 1.8-9.8%.
2. The indirect competitive ELISA based on monoclonal antibody introduced previously was applied to moitor the degradation kinetics of fipronil in vegetable and soil. The results showed that the semi-life of fipronil in vegetable is 10.4 d., and 15.1 d. in soil. The immunological assay method was applied to evaluate the effect of PH value, ozone in aqueous solution, Co-60 irradiation and UV-light irradiation on the
degration of fipronil in the lab. The reduction of the amount of the pesticide indicated that dissipation rate of fipronil was significantly accelerated by PH value, ozone into aqueous solution and UV-light irradiation.
3. Diethylphosphono acetic acid (DPA) was used as a generic hapten in order to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. In an indirect enzyme-linked immunosorbent assay (ELISA) format, the monoclonal-antibody has a high affinity to DPA , chlorpyrifos , diazinon , omethoate , parathion-ethyl and profenofos, showed preferential binding towards pesticides containing diethyl-phosphate and p=o functionalities rather than those containing dimethyl-structure and p=s group. The potential for using DPA was discussed.
引文
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