有机磷农药多残留免疫分析方法的研究
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摘要
本论文旨在研究有机磷农药的多残留酶联免疫(ELISA)分析方法。试验首先以对羟基苯丙酸和二甲氧基磷酰氯为原料,通过甲酯化、酰化、水解三个步骤合成了甲基对氧磷的半抗原O,O-二甲基-O-(4-丙酸基苯基)磷酸酯。将甲基对氧磷半抗原和对硫磷半抗原(氨基对硫磷)分别与不同的载体蛋白(BSA,OVA)偶联,制备了对应的免疫原和包被原。每种免疫原分别免疫两只新西兰大白兔获得多克隆抗体。通过方阵滴定法确定工作浓度及血清效价,并选择较优的血清供后面试验用。
以对硫磷为例建立了间接竞争性ELISA分析方法,并对ELISA分析的影响因素如有机溶剂的种类及含量、离子强度、pH进行了优化。在最优条件下,建立了对硫磷和甲基对氧磷的标准抑制曲线和回归方程。其中对硫磷的IC50为0.343μg/mL,LOD (IC10)为0.005μg/mL,线性范围(IC20～IC80)为0.014～8.24μg/mL;甲基对氧磷的IC50为0.368μg/mL,LOD (IC10)为0.004μg/mL,线性范围(IC20～IC80)为0.015～12.02μg/mL。以包菜为样本,同时添加了对硫磷和甲基对氧磷,在不同的孔中同时测得两种农药的回收率分别为79.00%～88.40%和74.00%～81.40%,变异系数分别为3.48%～8.53%和3.53%～7.89%。该分析方法重复性良好,相对标准偏差低于10%,可以满足对硫磷和甲基对氧磷同时检测的要求。
另外,还针对常用的二乙氧基有机磷农药进行了多残留酶联免疫分析方法的研究。首先对几种常用的二乙氧基磷酸酯农药毒死蜱、二嗪农、对硫磷、对氧磷、辛硫磷等的结构进行分析,发现它们的共性结构为(C2H5O)2P(O)或(C2H5O)2P(S),于是选择乙氧基膦酸乙酸((C2H5O)2P(O)CH2COOH)作为通用半抗原(DPA)。通过活性酯法将半抗原(DPA)与不同的载体蛋白(BSA、OVA)偶联,制备了人工抗原。以其BSA偶联物作为免疫原免疫新西兰大白兔,获得两种多克隆抗体。以半抗原(DPA)和对氧磷作为小分子竞争物,对抗体及其免疫分析条件进行了筛选和试验,在优化条件下建立了针对对氧磷的标准抑制曲线和回归方程。在此基础上进行了其它结构类似化合物的抑制试验,测定了它们的交叉反应。结果表明,由DPA抗原产生的抗体对对氧磷和嘧啶磷表现一定的识别,两者的IC50分别为1.69μg/mL和2.33μg/mL,交叉反应率分别为13.3%和9.6%;对其他有机磷药物(对硫磷、毒死蜱、二嗪农、马拉硫磷)等没有识别作用。以包菜为样本,分别添加了对氧磷和嘧啶磷,二者的添加回收率分别为75.02%～78.37%和75.02%～78.37%;变异系数分别为3.04%～3.79%和2.82%～3.36%。
The aim of the study was to develope a multi-residue immuoassay for the determination of organophosphorus pesticides. First of all, the hapten (O, O-dimethyl-O - (4 - propionyloxy) phosphate) of methyl-paraoxon was synthesized through three steps using 3-(4-Hydroxyphenyl) propionic acid and dimethyl chlorophosphate as the raw materials. NH2-Parathion was choosen for the hapten of Parathion. Then, the two haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen and coating antigen respectively. According to the immune protocol, four New Zealand white rabbits were immunized for six times by two immunogens during three months, and the antiserum with high titres were choosen for the experiment.
Using the parathion as the inhibitor, an indirect competitive ELISA was developed. For application to actual samples, the in?uence of several factors such as organic solvent, ionic strength and pH were studied. Under optimized conditions, for parathion, the quantitative working range was 0.014～8.24μg/mL with a limit of detection (LOD) of 0.005μg/mL and the IC50 was 0.343μg/mL; for methyl-paraoxon, the quantitative working range was 0.015～12.02μg/mL with a limit of detection (LOD) of 0.004μg/mL and the IC50 was 0.368μg/mL. There was negligible cross reactivity (CR) with other OP pesticides. The fortified recovery test of parathion and methyl-paraoxon was performed with the ic-ELISA method by spiking three level concentrations (0.1, 0.5, 1.0μg/mL) in cabbage. The recoveries were 79.00%～88.40% and 74.00%～81.40%; the C.V% were 3.48%～8.53% and 3.53%～7.89%, respectively.
Furthermore, the research of multi-residue immuoassy for diethoxy organophosphorus pesticides was also performed. First of all, the structures of some diethoxy organophosphorus pesticides such as chlorpyrifos, diazinon, parathion, paraoxon and phoxim were analyzed, and it was found that the common structure was (C2H5O)2P(O) or (C2H5O)2P(S), then diethylphosphonoacetic acid(DPA) was selected as the general-hapten.Then the hapten was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) using active ester method. New Zealand white rabbits were immunized with the hapten-BSA conjugates, and the polyclonal antibodies against the DPA-BSA were raised. Using the hapten (DPA) and paraoxon as the inhibitors, the indirect competitive EL1SA was developed and optimized, respectively. Under the optimal conditions, other similar organophosphorus pesticides were used as inhibitors to test for the cross-reactivity. The results of these assays showed that the pesticides which cause relative high inhibition to the antiserum obtained from DPA-BSA were paraoxon and pirimiphos.The IC50 were 1.69 and 2.33μg/mL; the cross-reaction rate were 13.3% and 9.6%. All the other Ops (parathion, chlorpyrifos, diazinon and malathion) tested showed no cross reactivity. The fortified recovery test of paraoxon and pirimiphos was performed with the ic-ELISA method by spiking three level concentrations (0.5, 1.0, 5.0μg/mL) in cabbage. The recoveries were 75.02%～78.37% and 75.02%～78.37%; the C.V% were 3.04%～3.79% and 2.82%～3.36%, respectively.
以对硫磷为例建立了间接竞争性ELISA分析方法,并对ELISA分析的影响因素如有机溶剂的种类及含量、离子强度、pH进行了优化。在最优条件下,建立了对硫磷和甲基对氧磷的标准抑制曲线和回归方程。其中对硫磷的IC50为0.343μg/mL,LOD (IC10)为0.005μg/mL,线性范围(IC20～IC80)为0.014～8.24μg/mL;甲基对氧磷的IC50为0.368μg/mL,LOD (IC10)为0.004μg/mL,线性范围(IC20～IC80)为0.015～12.02μg/mL。以包菜为样本,同时添加了对硫磷和甲基对氧磷,在不同的孔中同时测得两种农药的回收率分别为79.00%～88.40%和74.00%～81.40%,变异系数分别为3.48%～8.53%和3.53%～7.89%。该分析方法重复性良好,相对标准偏差低于10%,可以满足对硫磷和甲基对氧磷同时检测的要求。
另外,还针对常用的二乙氧基有机磷农药进行了多残留酶联免疫分析方法的研究。首先对几种常用的二乙氧基磷酸酯农药毒死蜱、二嗪农、对硫磷、对氧磷、辛硫磷等的结构进行分析,发现它们的共性结构为(C2H5O)2P(O)或(C2H5O)2P(S),于是选择乙氧基膦酸乙酸((C2H5O)2P(O)CH2COOH)作为通用半抗原(DPA)。通过活性酯法将半抗原(DPA)与不同的载体蛋白(BSA、OVA)偶联,制备了人工抗原。以其BSA偶联物作为免疫原免疫新西兰大白兔,获得两种多克隆抗体。以半抗原(DPA)和对氧磷作为小分子竞争物,对抗体及其免疫分析条件进行了筛选和试验,在优化条件下建立了针对对氧磷的标准抑制曲线和回归方程。在此基础上进行了其它结构类似化合物的抑制试验,测定了它们的交叉反应。结果表明,由DPA抗原产生的抗体对对氧磷和嘧啶磷表现一定的识别,两者的IC50分别为1.69μg/mL和2.33μg/mL,交叉反应率分别为13.3%和9.6%;对其他有机磷药物(对硫磷、毒死蜱、二嗪农、马拉硫磷)等没有识别作用。以包菜为样本,分别添加了对氧磷和嘧啶磷,二者的添加回收率分别为75.02%～78.37%和75.02%～78.37%;变异系数分别为3.04%～3.79%和2.82%～3.36%。
The aim of the study was to develope a multi-residue immuoassay for the determination of organophosphorus pesticides. First of all, the hapten (O, O-dimethyl-O - (4 - propionyloxy) phosphate) of methyl-paraoxon was synthesized through three steps using 3-(4-Hydroxyphenyl) propionic acid and dimethyl chlorophosphate as the raw materials. NH2-Parathion was choosen for the hapten of Parathion. Then, the two haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen and coating antigen respectively. According to the immune protocol, four New Zealand white rabbits were immunized for six times by two immunogens during three months, and the antiserum with high titres were choosen for the experiment.
Using the parathion as the inhibitor, an indirect competitive ELISA was developed. For application to actual samples, the in?uence of several factors such as organic solvent, ionic strength and pH were studied. Under optimized conditions, for parathion, the quantitative working range was 0.014～8.24μg/mL with a limit of detection (LOD) of 0.005μg/mL and the IC50 was 0.343μg/mL; for methyl-paraoxon, the quantitative working range was 0.015～12.02μg/mL with a limit of detection (LOD) of 0.004μg/mL and the IC50 was 0.368μg/mL. There was negligible cross reactivity (CR) with other OP pesticides. The fortified recovery test of parathion and methyl-paraoxon was performed with the ic-ELISA method by spiking three level concentrations (0.1, 0.5, 1.0μg/mL) in cabbage. The recoveries were 79.00%～88.40% and 74.00%～81.40%; the C.V% were 3.48%～8.53% and 3.53%～7.89%, respectively.
Furthermore, the research of multi-residue immuoassy for diethoxy organophosphorus pesticides was also performed. First of all, the structures of some diethoxy organophosphorus pesticides such as chlorpyrifos, diazinon, parathion, paraoxon and phoxim were analyzed, and it was found that the common structure was (C2H5O)2P(O) or (C2H5O)2P(S), then diethylphosphonoacetic acid(DPA) was selected as the general-hapten.Then the hapten was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) using active ester method. New Zealand white rabbits were immunized with the hapten-BSA conjugates, and the polyclonal antibodies against the DPA-BSA were raised. Using the hapten (DPA) and paraoxon as the inhibitors, the indirect competitive EL1SA was developed and optimized, respectively. Under the optimal conditions, other similar organophosphorus pesticides were used as inhibitors to test for the cross-reactivity. The results of these assays showed that the pesticides which cause relative high inhibition to the antiserum obtained from DPA-BSA were paraoxon and pirimiphos.The IC50 were 1.69 and 2.33μg/mL; the cross-reaction rate were 13.3% and 9.6%. All the other Ops (parathion, chlorpyrifos, diazinon and malathion) tested showed no cross reactivity. The fortified recovery test of paraoxon and pirimiphos was performed with the ic-ELISA method by spiking three level concentrations (0.5, 1.0, 5.0μg/mL) in cabbage. The recoveries were 75.02%～78.37% and 75.02%～78.37%; the C.V% were 3.04%～3.79% and 2.82%～3.36%, respectively.
引文
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2.高晓辉,朱光艳.蔬菜上农药残毒快速检测技术-酶抑制法检测有机磷和氨基甲酸酯类农药[J].农药科学与管理, 2000, 21(4):29-31.
3.纪淑娟,赵丽丽,冯辉.一种快速检测农产品有机磷农药残留的方法[J].农药, 2000, 39(10):17-18.
4.庄无忌,周显,刘胜利.毛细管色谱法测定果蔬中22种有机磷农药残留量[J].分析测试学报, 1995, 14 (3):68-72.
5. Oliva J, Barba A, Vela N, et al. J .Chromatography A, 2000, 882:213-220.
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