单 位：
南京工业大学理学院应用化学系;江苏省农业科学院食品质量安全与检测研究中心
关键词：
异丙隆;半抗原;人工抗原;多克隆抗体;酶联免疫吸附测定(ELISA)
摘 要：
【目的】为了建立测定脲类除草剂异丙隆残留的免疫分析方法(ELISA),对制备高效价抗异丙隆多克隆抗体的方法进行了研究。【方法】以对异丙基苯基异氰酸酯、N-甲基吡咯环酮和N-甲基己内酰胺为反应原料,经两步化学反应合成了两种异丙隆半抗原:1-(3-丙基羧基)-3-(4-异丙基苯基)-1-甲基脲(HAPTEN 4C)和1-(5-戊基羧基)-3-(4-异丙基苯基)-1-甲基脲(HAPTEN 6C)。通过活性酯法将半抗原与载体蛋白(BSA、OVA)偶联制备了两种异丙隆的人工抗原HAPTEN 6C-BSA和HAPTEN 4C-OVA。利用免疫抗原HAPTEN 6C-BSA免疫新西兰大白兔获得了高效价的异丙隆的多克隆抗体。建立了以HAPTEN 4C-OVA为包被原的间接竞争ELISA方法。【结果】合成的半抗原经薄层色谱、IR、1HNMR确证;人工抗原HAPTEN 6C-BSA和HAPTEN 4C-OVA的结合比分别为46﹕1和36﹕1。抗血清的最高效价为1.6×105。经优化确定的间接竞争ELISA分析方法中包被抗原的浓度为0.1μg.ml-1,抗血清的工作稀释倍数为1.0×105。根据异丙隆的标准抑制曲线,异丙隆对免疫反应的的IC50值为0.07μg.ml-1。抗体对氯溴隆、绿谷隆、特丁塞隆等6种取代脲类除草剂的交叉反应率都小于0.1%。【结论】异丙隆是小分子,本身没有免疫活性,也没有与载体蛋白偶联的活性基团。高效价、高特异性抗体的制备是利用免疫学方法进行异丙隆残留分析的最关键因素。本研究合成的两种异丙隆的相似物具有不同长度碳链的羧基,并且使异丙基和苯基充分暴露,具备了异丙隆半抗原的特点。将半抗原偶联到载体蛋白上获得的人工抗原经免疫兔子后获得了高效价、高特异性的抗异丙隆抗体。
译 名：
Development of Anti-Isoproturon Polyclonal Antibody
作 者：
LI Fang-shi,1 SUN Feng,1 LIU Xian-jin,2 CUI Heng-hua2(1Department of Applied Chemistry,College of Science,Nanjing University of Chemical Technology,Nanjing 210009;2Food Safety Research and Inspection Center,Jiangsu Academy of Agricultural Sciences,Nanjing 210014)
关键词：
Isoproturon;Hapten;Artificial antigen;Polyclonal antibody;Enzyme-linked immunosorbent assay(ELISA)
摘 要：
【Objective】 An competitive enzyme-linked immunosorbent assay(ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea,in food and environmental samples was developed.【Method】 Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea(HAPTEN 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea(HAPTEN 6C) were synthesized.The haptens were attached to bovine serum albumin(BSA) and ovalbumin(OVA) using the N-hydroxysuccinimide reaction.The conjugate HAPTEN 6C-BSA was used as immunogen,with which a high-titer anti-isoproturon polyclonal antibody(pAb) was successfully obtained by immunization of New Zealand white rabbits.The conjugate HAPTEN 4C-OVA was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established.【Result】The haptens were confirmed with TLC,IR,and 1H NMR.The conjugation molar ratio of HAPTEN 4C to OVA and HAPTEN 6C to BSA were 36:1 and 46:1,respectively,calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105.The optimal concentrations of the coating antigen and the dilution of the pAb used in the ELISA were 0.1 μg.ml-1 and 1.0×105,respectively.The concentration of isoproturon that inhibits 50% of antibody-antigen binding(IC50) was 0.07μg.ml-1.The cross-reactivities of six urea herbicides including chlorbromuron,fluometuron,monolinuron were lower than 0.1%.【Conclusion】 Isoproturon is a small molecule without immune activity and active functional group for attaching to a protein carrier.To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples.The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms and the phenyl and isopropyl groups are fully exposure.An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
