酶联免疫法检测新型除草剂双甲胺草磷  

An Indirect Enzyme-Linked Immunosorbent Assay for Determination of New Herbicide H-9201 in Water

作　　者：张存政[1] 杨春龙[2] 刘贤进[1] 陈敏[2] 俞杰[1] 

机构地区：[1]农业部食品安全监控重点开放实验室江苏省食品质量安全重点实验室省部共建国家重点实验室培育基地,南京210014 [2]南京农业大学理学院,南京210095

出　　处：《分析化学》2011年第2期225-230,共6页Chinese Journal of Analytical Chemistry

基　　金：江苏省农业科技自主创新项目(No.CX(10)236);国家支撑计划子课题(No.2006BAD08A03)资助

摘　　要：以甲醇与三氯硫磷为原料,人工合成了半抗原结构类似物,与琥珀酸酣反应引入羧基,利用活性酯法使之与载体蛋白偶联,制得完全抗原,免疫动物获得多克隆抗体,建立了新型除草剂双甲胺草磷(H-9201)的间接竞争ELISA检测方法,并进行了水体中除草剂H-9201残留的实际样品检测。结果表明:所获得的多克隆抗体可以特异性的识别目标化合物H-9201,抗体的效价为1/102400,抗体的工作浓度为1/18000倍,抗原的工作浓度为1/512倍。在此工作浓度条件下建立的ELISA方法对H-9201的抑制中浓度(IC50)为1.331mg/L;检出限(IC10)为0.017 mg/L;线性范围为0.05～20.0 mg/L。交叉反应表明,该抗体对结构类似化合物2,4-二甲基-6-硝基苯酚、硝基苯酚、甲胺磷、甲拌磷、毒死蜱、对硫磷均无识别作用;微弱识别S-羧乙基-O,O-二甲基二硫代磷酸酯、乐果、马拉硫磷;对半抗原及其结构类似物具有较高的识别作用。对田间水样的添加样本进行检测,方法的回收率范围为(80.5±4.5)%～(95.2±3.2)%,可用于水体样本中H-9201残留的ELISA检测。A sensitive indirect enzyme-linked immunosorbent assay（ELISA）was developed for the detection of new herbicide H-9201,and their suitability for the determination of this analyte in environmental water was studied.Starting with phosphorus thiochloride and methanol,an analogs with similar structure of hapten was synthesized,and carboxyl group was conducted by succinicanhydride,then the derivative was conjugated with bovine serum albumin or ovalbumin by means of active ester as immunoagent and coating agent,respectively;IC-ELISA method was developed based on the polyclonal antibody generated in rabbits by immunizing with synthesized hapten.For standard,50% reduction of the maximum ELISA signal（IC50）was 1.331 mg/L,whereas the detection limit was 0.017 mg/L in the competitive standard curve ranged from 0.05 to 20.0 mg/L and the combination of the concentrations of coating solution and primary antibody was optimized to 1/512 and 1/18000,respectively.No significant cross-reactivity for related compounds was observed,such as 2,4-dimethyl-6-nitrophenol,p-nitrophenol,Methamidophos,phorate,chlorpyrifos,parathion,the antibody showed negligible cross-reactivity with S-carboxylethyl-O,O-Dimethyl-phosphorodithioate,dimethoate,malathion,but high cross-reactivity with analogs of hapten.Recovery of H-9201 in environmental water was（80.5±4.5）%-（95.2±3.2）% based on the developed IC-ELISA method.

关 键 词：除草剂 双甲胺草磷 酶联免疫 多克隆抗体 

分 类 号：X839.2[环境科学与工程—环境工程]