毒死蜱单克隆抗体的制备及ELISA检测方法研究
Preparation of Monoclonal Antibody against Chlorpyrifos and Development of ELISA for Detection of Chlorpyrifos Residues
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摘要:
目的 制备毒死蜱单克隆抗体,为建立毒死蜱残留检测方法奠定基础。 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,其检测范围为0.04~0.42 mg/L,半数抑制浓度为0.135 mg/L,最低检测限为0.03 mg/L,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。 Abstract:Objective To produce monoclonal antibodies (McAb) against chlorpyrifos and to establish a rapid assay for detecting the residues of chlorpyrifos. Methods Hapten of chlorpyrifos was synthesized by technical grade imidacloprid reacted with 3-mercaptopropionic acid and the structure of synthesized product was identified.Then,the hapten was covalently conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare immunogen and coating antigen.Two hybridoma cell lines secreting McAb against chlorpyrifos were 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 enzyme-linked immunosorbent assay (ELISA) and indirect competitive ELISA; cross-reactivity with other organophosphate pesticides was also tested. Results Two monoclonal cell lines that secreted Chlorpyrifos antibodies steadily were obtained.Based on the calibration curve,the range of detection was 0.04-0.42 mg/L,the median inhibitory concentration was 0.135 mg/L,and the limit of detection was 0.03 mg/L.No cross-reactivity of the antibody with sumithion,parathion and malathion was observed. Conclusion Two chlorpyrifos monoclonal antibody cell lines are prepared successfully.A ELISA is preliminarily developed using the monoclonal antibody to detect chlorpyrifos.
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