环戊二烯类有机氯农药单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against chlorinated cyclodiene pesticide

中文关键词:  环戊二烯类农药  间接ELISA  单克隆抗体

英文关键词:chlorinated cyclodiene pesticides  ic ELISA  monoclonal antibody

中文摘要:

      目的 制备并鉴定环戊二烯类有机氯农药单克隆抗体。方法 以环戊二烯类农药的特征部分为基础设计合成半抗原4-(4,5,6,7,8,8-六氯-3a,4,7,7a-四氢-4,7-亚甲基-1H-聚-1-氧) 4-丁酮酸(CCD), 并通过活泼酯法将其与载体蛋白BSA、OVA分别偶联制备免疫抗原CCD-BSA和包被抗原CCD-OVA; 再经过动物免疫、细胞融合, 筛选、克隆, 得到1株能稳定分泌环戊二烯类农药抗体的单克隆细胞株。细胞株经扩大培养后, 注射小鼠体内产生腹水, 并将其用辛酸-硫酸铵和protein A柱子纯化出单克隆抗体。结果 用间接ELISA方法测定其效价为2.5×104, 亲和力Ka为6.7×1010 L/mol; 以β-硫丹为竞争物, 采用间接竞争ELISA法建立标准曲线, 测得其 IC50 为16.2 ng/mL, LOD为2.1 ng/mL, 定量检测线性范围为4.7~117.5 ng/mL, 与其他5种结构类似物的交叉反应率高于10%。结论 本研究成功获得了抗环戊二烯类农药单克隆抗体, 该抗体可实现果蔬中环戊二烯类农药的多残留快速检测。

英文摘要:

      Objective To prepare and identify a monoclonal antibody against chlorinated cyclodiene pesticide. Methods The hapten 4-(4,5,6,7,8,8-Hexachloro-3a,4,7,7a-tetrahydro-4,7-methano-1H-in-denyl-1- oxy)4-Oxobutanoic Acid (CCD) was designed and synthesized on the basis of characteristics part of chlorinated cyclodiene pesticide, and artificial antigens CCD-BSA (immmue antigen) and CCD-OVA (coating antigen) were prepared by coupling with the carrier proteins using active ester method. The monoclonal antibody (MAb) against chlorinated cyclodiene pesticide was produced by animal immunity, cell fusion, screening, and cloning. The MAb was prepared from ascetic fluids of Balb/c, which was purified with saturated ammonium sulfate followed by affinity chromatography on protein A. Results Indirect ELISA experiments showed that the titer of of ascitic fluids was up to 2.5×104 , the affinity Ka was 6.7×1010 L/mol. The standard curve of β-endosulfan was established, and its IC50 was 16.2 ng/mL, LOD was 2.1 ng/mL, and linearity range was from 4.7 to 117.5 ng/mL. The cross reactivities of 5 similar structure analogus were higher than 10%. Conclusion This research has successfully obtained the MAb against chlorinated cyclodiene pesticides, which could be used to detect multiple residues in fruits and vegetables.