基于亚结构半抗原的氰戊菊酯抗体制备及免疫分析方法研究

Preparation and Immunoassay of Fenvalerate Antibody Based on Substructure Hapten

目的:制备氰戊菊酯特异性抗体并建立免疫分析方法。方法:以氰戊菊酯的亚结构单元——氰戊菊酸为半抗原,通过活泼酯法偶联载体蛋白作为人工抗原并进行动物免疫,采用辛酸-硫酸铵沉淀法纯化处理抗血清得到多克隆抗体,最后,采用棋盘法和逐步优化方法确立实验参数并通过对数拟合法建立间接竞争酶联免疫分析方法标准曲线。结果:最佳反应条件为,包被抗原6000倍稀释(蛋白浓度为0.167μg/mL),抗体稀释倍数14000,最佳药物、抗体和二抗稀释液均为PBS,竞争反应时间30 min,二抗稀释倍数8000,二抗反应时间30 min。在该条件下,建立了氰戊菊酸的icELISA检测方法,其IC_(50)为67.08 ng/mL,线性检测范围为10.30~436.64 ng/mL,检测限(LOD)达3.44 ng/mL,且与甲氰菊酯等结构及功能类似物均无明显交叉反应,特异性良好。结论:该方法为氰戊菊酯免疫检测产品开发奠定了基础,对保障食品安全具有重要意义。

Objective:To prepare the needle of monoclonal antibody against fenvalerate and establish the immunoassay method.Methods:Fenvaleric acid,a substructural unit of fenvalerate,was used as the hapten,which was conjugated to the carrier protein by active ester method as the artificial antigen.And then the female mice were immunized for producing the antiserum from which the polyclonal antibody against fenvalerate was purified by octanoic acid-ammonium sulfate precipitation.Finally,the experimental parameters were optimized by chessboard method and stepwise optimization method,and the standard curve of ELISA method was established by logarithmic fitting method.Results:The optimal reaction conditions were determined as follows:6000-fold dilution of coated antigen(protein concentration was 0.167μg/mL),antibody dilution of 14000,optimal dilution solution of drug,antibody and secondary antibody of PBS,competitive reaction time of 30 min,dilution ratio of secondary antibody of 8000,reaction time of secondary antibody of 30 min.In this condition,the icELISA method for detecting florfenicol was established with the IC_(50) of 67.08 ng/mL,linear detection range of 10.30~436.64 ng/mL,the detection limit(LOD)of 3.44 ng/mL.There was no obvious cross reaction with fenvalerate and other structural and functional analogs,and the specificity was good.Conclusion:This method laid a foundation for the preparation of fenvalerate monoclonal antibody and the development of detection products.