【作者】 王璇； 王亚； 刘冉； 尹立红；

【Author】 WANG Xuan, WANG Ya, LIU Ran, YIN Lihong (Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, China)

【机构】 东南大学公共卫生学院环境医学工程教育部重点实验室；

【摘要】 目的:建立毒死蜱间接ELISA检测方法。方法:以毒死蜱原药与3-巯基丙酸为起始原料,合成半抗原并对其进行结构鉴定。利用该半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联制备毒死蜱人工免疫抗原和包被抗原,再取已免疫的经过筛选的Balb/c小鼠脾细胞与小鼠骨髓瘤细胞SP2/0融合,用间接ELISA和间接竞争ELISA方法对2株细胞进行鉴定,并测定与其他农药的交叉反应率。结果:获得两株稳定分泌毒死蜱单克隆抗体的细胞株,初步建立了毒死蜱间接ELISA检测方法,其检测范围为0.04~0.42μg/mL,半数抑制浓度(IC50)为0.135μg/mL,最低检测限为0.03μg/mL,与杀螟硫磷、对硫磷和马拉硫磷几乎没有交叉反应。结论:成功制备两株分泌毒死蜱抗体的单克隆细胞株,初步建立了毒死蜱间接ELISA检测方法。更多还原

【Abstract】 Objective:To establish determination of chlorpyrifos by ELISA. Methods Hapten of chlorprifos was synthesized by using technical grade imidacloprid reacted with 3-Mercaptopropionic acid and the structure was identified. The hapten was covalently conjugated to BSA and OVA to prepare immunogen and coating antigen. Two hybridoma cell lines secreting monoclonal antibodies (McAb) against chlorpyrifos had been established by fusing mouse myeloma cells SP2/0 and splenocytes from the Balb/c mouse immunized with immunogen. Two cell lines were identified by indirect ELISA and indirect competitive ELISA; Cross-reactivity with other pesticides was also determined. Results Two monoclonal cell lines that secreted chlorprifos antibodies steadily were obtained. Based on the calibration curve, the linear detection was 0.04～0.42μg/mL and the IC50 was 0.135μg/mL. No cross-reactivity of the antibody with Sumithion parathion and malathion was observed, indicating that the antibody is highly specific for chlorprifos. Conclusions Two chlorpyrifos monoclonal antibody cell lines are prepared successfully. A ELISA is preliminarily developed using the monoclonal antibody to detect chlorprifos.更多还原