DPPs类有机磷农药宽谱酶联免疫吸附分析方法的建立  

Establishment of broad-spectrum enzyme-linked immunosorbent assay for organophosphorus pesticides in the DPPs group

作　　者：吕丽兰 张娅 陆覃昱 邹承武[2] 甘志勇 李冬桂 吴静娜 王艺霖 李鑫生 何丽珊[2] LYU Li-lan;ZHANG Ya;LU Qin-yu;ZOU Cheng-wu;GAN Zhi-yong;LI Dong-gui;WU Jing-na;WANG Yi-lin;LI Xin-sheng;HE Li-shan(Guangxi Subtropical Crops Research Institute/Laboratory of Quality Risk Assessment for Agro-products of Ministry of Agriculture and Rural Affairs(Nanning)/Quality Supervision and Testing Center of Subtropical Fruit and Vegetable of Ministry of Agriculture and Rural Affairs,Nanning 530001,China;College of Agriculture,Guangxi University/Guangxi Key Laboratory for Agro-Environment and Agro-Product Safety,Nanning 530004,China)

机构地区：[1]广西亚热带作物研究所/农业农村部农产品质量安全风险评估实验室(南宁)/农业农村部亚热带果品蔬菜质量监督检验测试中心,南宁530001 [2]广西大学农学院/广西农业环境与农产品安全重点实验室,南宁530004

出　　处：《西南农业学报》2022年第11期2659-2666,共8页Southwest China Journal of Agricultural Sciences

基　　金：广西重点研发计划项目(桂科AB17292070);广西农业科学院基本科研业务专项(桂农科2021YT148)。

摘　　要：【目的】制备识别二乙氧基硫代磷酸酯类有机磷农药(DPPs)的单克隆抗体,建立其间接竞争酶联免疫吸附分析法(ic-ELISA),为实现农产品有机磷农药残留快检提供技术支持。【方法】以化合物4-(二乙氧基硫代磷酰氧基)-肉桂酸为半抗原,制备完全抗原。筛选免疫小鼠,制备稳定分泌可识别DPPs抗体的单克隆细胞株,筛选出抗原和抗体最佳工作浓度组合,建立该单抗的ic-ELISA标准曲线,并分析该单抗与6种DPPs的交叉反应率,评估ic-ELISA的广谱性。利用所得抗体与喹硫磷交叉反应最强的特性对采购的柑橘和鲜橙样品进行喹硫磷添加回收试验。【结果】成功合成识别DPPs的人工抗原,包括包被抗原DPPs-OVA和免疫抗原DPPs-BSA。通过有限稀释法筛选获得灵敏度高、广谱性最强的单克隆细胞株5G_(8)。以0.5μg/mL包被抗原DPPs-OVA、1.0μg/mL抗体5G_(8)2D_(5)为最佳工作浓度组合,将细胞株筛选过程中抗体识别率较高的有机磷农药——对硫磷选定为抑制标准物,建立对硫磷ic-ELISA及其标准曲线,其半抑制浓度(IC_(50))为41.78 ng/mL,检测范围(IC_(20)~IC_(80))为3.21~239.59 ng/mL;所建立的ic-ELISA与5种DPPs(蝇毒磷、喹硫磷、甲拌磷、甲基对硫磷和倍硫磷)均存在不同程度的交叉反应,其中以喹硫磷的交叉反应率最高,达19.79%。比较ic-ELISA和气相色谱法(GC)对柑橘和鲜橙样品中喹硫磷的添加回收结果发现,ic-ELISA的添加回收率为75.80%~116.12%,GC的添加回收率介于92.5%~115.00%,2种方法的检测结果一致性较好。【结论】建立的ic-ELISA经GC交叉验证准确性较高,可用于快速检测农产品中的喹硫磷残留。【Objective】The present paper aimed to prepare a monoclonal antibody against O,O-diethyl organophosphorus pesticides(DPPs)and establish an indirect competitive enzyme-linked immunosorbent assay(ic-ELISA),so as to provide technological support for rapid detection of organophosphorus pesticide residues.【Method】Compound(E)-3-[4-(diethoxyphosphorothioyloxy)phenyl]acrylic acid was used as hapten to make antigen.Immunized mice were screened to prepare monoclonal cell lines that stably secreted antibodies that recognized DPPs.The positive rate and recognition rate of the fusion cell lines were evaluated and the monoclonal cell lines stably secreting antibodies against DPPs were screened.Then,the best concentration combination of antigen and antibody was selected,and the standard curve of ic-ELISA was established.Based on the cross-reactivity rate of the antibody with 6 DPPs,the broad-spectrum of ic-ELISA was evaluated.The cross reaction between the antibody and quinalphos was the strongest,thus the addition and recovery test of quinalphos was carried out on citrus and fresh orange samples.【Result】Artificial antigens were synthesized successfully,including coating antigen DPPs-OVA and immune antigen DPPs-BSA.Sensitive and broad-spectrum cell line 5G_(8)was obtained by limited dilution method.0.5μg/mL coating antigen DPPs-OVA and 1.0μg/mL antibody 5G_(8)2D_(5)made up the best working concentration combination.Parathion,a pesticide with high antibody recognition rate in this cell line screening,was selected as the inhibition standard,and its ic-ELISA and standard curve were established.The half inhibitory concentration(IC_(50))was 41.78 ng/mL,and the detection range(IC_(20)-IC_(80))was 3.21-239.59 ng/mL.The established ic-ELISA showed varying degrees of cross reactions with 5 DPPs(coumaphos,quinalphos,phorate,methyl parathion and fenthion).The cross-reactivity rate of quinalphos was the highest,which was 19.79%.The recoveries of quinalphos in citrus and orange samples by ic-ELISA and GC were compared.The recov

关 键 词：二乙氧基硫代磷酸酯类有机磷农药(DPPs) 单克隆抗体 间接竞争酶联免疫吸附分析法(ic-ELISA) 气相色谱法(GC) 农药残留检测 

分 类 号：S481.8[农业科学—农药学]