摘要
建立了定量测定溴氰菊酯间接竞争酶联免疫吸附分析方法(ic-ELISA)。合成了半抗原1-羧基-(3′-苯氧基苯基)甲基-3-(2′,2′-二溴乙烯基)-2,2-二甲基环丙基羧酸酯(Med)和N-2-(羧基丙基)氨基甲酰基-(3′-苯氧基苯基)甲基-3-(2′,2′-二溴乙烯基)-2,2-二甲基环丙基羧酸酯(Di)。分别采用碳二亚胺法和混合酸酐法将半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联制备了免疫原Di-BSA和包被原Di-OVA、Med-OVA。将制得的溴氰菊酯免疫原免疫动物获得多克隆抗体,经间接非竞争ELISA法测得其效价为2.5×105。通过对甲醇含量、离子强度、pH值等影响因素进行异源分析条件的优化,确立了溴氰菊酯间接竞争酶联免疫分析方法的最佳检测条件(30%甲醇、氯化钠0.4mol/L、pH7.5),并建立了标准竞争曲线。该方法的IC50值为0.55±0.05mg/L,检测限(IC10)为3.76±0.35μg/L,对大多数拟除虫菊酯无交叉反应。分别在自来水、河水和土壤样品中添加0.05～5.0mg/L(或mg/kg)的溴氰菊酯,回收率分别为89.7%～106.8%、82.4%～101.7%、75.6%～97.8%。
An indirect competitive enzyme-linked immunosorbent assay （ic-ELISA） was developed for the quantitative detection of deltamethrin. The haptens of deltamethrin 1-carboxy-（3＇-phenoxyphenyl） methyl-3- （ 2＇, 2＇-dibromoethenyl ） -2, 2-dimethylcyclopropanecarboxylate （ Med ） and N-2- （ carboxy- propyl ） carbamoyl-（ 3＇-phenoxyphenyl ） methyl-3-（ 2＇, 2＇-dibromoethenyl ）-2, 2-dimethylcyclopropane- carboxylate （Di） were synthesized. Di was conjugated with bovine serum albumin （BSA） as immunogen （Di-BSA） by carbodiimide method and coating antigens （Di-OVA, Med-OVA） of deltamethrin were prepared by conjugating these two haptens with ovalbumin （OVA） by mixed anhydride method. Polyclonal antibodies were obtained from rabbits immunized with immunogen and the titer of the freeze- dried powder （2.5 x 105） was determined by indirect non-competitive ELISA procedure. Standard competitive curve of the optimized ic-ELISA for deltamethrin was developed. Thus, the optimized assay conditions such as concentration of methanol （30%） , inonic strength （ NaCl 0.4 mol/L ） , pH value （7.5） were investigated and obtained by analysis of heterologous. The value of 50% inhibition of antibody binding （IC50） for deltamethrin was 0.55 ±0.05 mg/L,and the low detection limit （IC10） was 3.76 ± 0.35μg/L. This ELISA assay had no cross-reactions significantly with other major pyrethroids. Tap water, river water and soil samples were spiked with deltamethrin at 0.05 ～ 5.0 mg/L （ or mg/kg）, the average recoveries were in the range of 89.7% - 106. 8%, 82.4% ～ 101. 7%, 75. 6% - 97. 8% respectively.
出处
《农药学学报》
CAS
CSCD
北大核心
2009年第2期261-268,共8页
Chinese Journal of Pesticide Science
基金
国家高技术研究发展规划(“863”计划)项目(2006AA108447)
关键词
溴氰菊酯
酶联免疫吸附分析
多克隆抗体
deltamethrin
enzyme-linked immunosorbent assay
polyclonal antibody
作者简介
张帮红（1977-），男．安徽怀远人，硕士研究生，E-mail：2006102115@njau．edu．cn；
通讯作者（Author for correspondence）：王鸣华（1961-），男，山西垣曲人，教授，博士生导师，主要从事农药残留与环境毒理研究工作．联系电话：025-4395479；E-mail：wangmha@njau．edu．cn
