川楝素半抗原设计及其抗体制备.docVIP

川楝素半抗原设计及其抗体制备 罗 利1，张 静1，刘 媛3，余向阳3，马志卿1，2，冯俊涛1，2，刘贤进3，张 兴1，2 （1西北农林科技大学无公害农药研究服务中心，陕西杨凌 712100；2陕西省生物农药工程技术研究中心，陕西杨凌 712100；3 江苏省农业科学院食品质量安全与检测研究中心，南京 210014） 摘要：【目的】获得抗川楝素的高灵敏度，高特异性多克隆抗体，建立川楝素的间接竞争酶免疫测定（IC-ELISA）方法。【方法】采用酯化反应合成川楝素的半抗原并经HRMS，NMR及IR鉴定；用混合酸酐法和活化酯法分别合成人工免疫抗原与包被抗原；通过免疫动物获得多克隆抗体。【结果】合成了琥珀酸单酰川楝素（TS）和戊二酸单酰川楝素（TG）2种半抗原，与载体蛋白BSA和OVA分别偶联制备了2种免疫原TS-BSA和TG-BSA，及2种包被原TS-OVA和TG-OVA。免疫后获得的2种抗体效价达6.4×104和3.2×105。经4种不同包被原－抗体组合优化，以（TS-Ab，TG-OVA）组合建立的川楝素IC-ELISA检测方法，线性检测范围为0.01～100 μg·ml-1，检测限达0.03 μg·ml-1，抑制中浓度为1.52 μg·ml-1；以（TG-Ab，TG-OVA）组合建立的方法，线性检测范围为0.1～100 μg·ml-1，检测限达0.11 μg·ml-1，抑制中浓度为3.49 μg·ml-1。水样中基于（TS-Ab，TG-OVA）组合的川楝素平均回收率为82.4%～106.0%，精密度为1.1%～14.3%。【结论】通过川楝素半抗原的合理设计，获得了高效价的抗川楝素多克隆抗体，建立了快速有效的川楝素IC-ELISA检测方法。 关键词：川楝素；半抗原；多克隆抗体；ELISA Haptens Design and Production of Antibodies Against Toosendanin LUO Li1, ZHANG Jing1, LIU Yuan3, YU Xiang-yang3, MA Zhi-qing1,2, FENG Jun-tao1,2, LIU Xian-jin3, ZHANG Xing1,2 (1Research and Development Center of Biorational Pesticides, Northwest Agricultural and Forestry University, Yangling 712100, Shaanxi; 2Shaanxi Engineering and Technology Center of Biopesticides, Yangling 712100, Shaanxi; 3Food Safety Research and Inspection Center, Jiangsu Academy of Agricultural Sciences, Nanjing 210014) Abstract:【Objective】The aims were to produce the polyclonal antibodies against toosendanin, and establish an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). 【Method】The haptens were synthesized by the reaction of toosendanin with succinate and glutarate, respectively, and characterized by HRMS, NMR and IR. Artificial immunogens and coating antigens were prepared separately with the mixed-anhydride method and the active ester method. Polyclonal antibodies were produced through immunizing rabbits. 【Result】Two haptens of toosendanin, toosendanin-succinil (TS) and toosendanin- glutaryl (TG), were synthesized successfully, and conjugated with BSA and OVA to form immunogens (TS-BSA and TG-BSA) and coat