有机磷农药三唑磷单克隆抗体制备及鉴定

Production and Identification of Monoclonal Antibodies against Organophosphate Insceticide Triazophos

采用活泼酯法和混合酸酐法将三唑磷半抗原(TZPM-Hap)与牛血清蛋白(BSA)和卵血清蛋白(OVA)偶联,分别制备出一种免疫抗原(TZPM-A-BSA)和两种包被抗原(TZPM-A-OVA和TZPM-M-OVA)。通过免疫小鼠及细胞融合,并对杂交瘤细胞进行经多次筛选和亚克隆得到1株产三唑磷单克隆抗体阳性细胞株(FC3),其抗体亚类为IgM。辛酸-硫酸铵沉淀法纯化后的抗体经间接ELISA检测,该方法IC50=9.7μg/L,最低检测限(LOD)为1.0μg/L,定量检测线性范围(IC20～IC80)为2.5～100.0μg/L,与其他有机磷农药结构类似物无明显交叉反应,显示出高度的特异性。该抗体可进一步用于免疫分析试剂盒开发。

In this study,artificial antigens for organophosphate insecticide triazophos including one immunogen and two coating antigens were prepared.The hapten（TZPM-Hap） of organophosphate insecticide triazophos was coupled to bovine serum albumin（BSA） by the active ester method to prepare the artificial immunogen TZPM-A-BSA.Coupling of TZPM-Hap to ovalbumin（OVA） by the active ester method or the mixed anhydride method produced the two coating antigens TZPM-A-OVA and TZPM-M-OVA,respectively.Balb/c mice were immunized with the immunogen,and the splenocytes of the immunized mice were fused with Sp2/0 cells to generate monoclonal antibody（McAbs）.A hybridoma cell line（FC3） secreting anti-triazophos McAbs was obtained,whose subtype was IgM.Compared with TZPM-M-OVA,better sensitivity in the icELISA format using TZPM-A-OVA was found,which demonstrated 9.7μg/L of IC50,1.0μg/L of LOD,and 2.5-100.0μg/L of work range（IC20-IC80）.The McAbs showed no obvious cross-reactivity with other structural-related organophosphate pesticides.The result indicated that one hybridoma cell line with high specificity and sensitivity was obtained,which will be of importance to develop an ELISA kit for triazophos in the future.