噻虫嗪单克隆抗体制备及酶联免疫吸附分析方法的建立

通过化学修饰合成了噻虫嗪人工半抗原,采用碳二亚胺法将该半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,成功制备了分子结合比合理的免疫原和包被原。经过免疫原免疫6周龄Balb/c小鼠、PEG介导免疫鼠脾细胞和骨髓瘤细胞的融合和阳性杂交瘤细胞的筛选和克隆化,获得了效价高达1∶6.4×105的抗噻虫嗪单克隆抗体,抗体亚类为IgG1型。优化了ELISA实验条件,建立了基于单克隆抗体技术的噻虫嗪残留间接竞争ELISA方法。本方法的抑制中浓度(IC50)为0.0255mg/L,检测灵敏度(IC20)为0.0022mg/L,检出限(IC10)为0.001mg/L。除噻虫胺外,该抗体与其它噻虫嗪结构类似物无交叉反应。以自来水为基质的噻虫嗪添加回收实验显示,0.01,0.5和10.0mg/L添加水平的回收率均大于75%,且各添加水平重复测定8次的相对标准偏差均小于8%,说明所建立的间接竞争ELISA准确度高,重复性好,适合水中噻虫嗪残留的检测。

Preparation of Monoclonal Antibodies Against Thiamethoxam and Establishment of Enzyme Linked Immunosorbent Aaasy Detection Method

Chemical modification method was employed to synthesize the thiamethoxam hapten,which was then coupled with BSA and ovalbumin(OVA) to prepare immunogen and coating antigen through EDC method,respectively.And satisfied molecule coupling ratios between the thiamethoxam hapten and carrier proteins were eventually obtained.In order to obtain the monoclonal antibodies against thiamethoxam,the 6-week old Balb/c mice were immunized with the prepared immune antigen,and cells hybridization,positive screening and cloning.The related experimental results showed that the titer of the monoclonal antibodies was reached 1∶6.4×105 and the antibody subclass was all IgG1.After optimization of the ELISA conditions,an indirect competitive ELISA for quantitative analysis of thiamethoxam was established.The limit of detection(IC10),limit of quantification(IC20) and median inhibitory concentration(IC50) were 0.001 mg/L,0.0022 mg/L and 0.0255 mg/L,respectively.The produced monoclonal antibodies had no cross-reaction with some other analogues of thiamethoxam except for Lothianidin.The recovery rates were determined in Xinxiang city tap water spiked at 0.01 mg/L,0.5 mg/L and 10 mg/L.The overall recovery rates were over 75%,with a relative standard deviation(RSD) less than 8%.This developed method has the characteristics of high accuracy and precision,and can used to determine the thiamethoxam residue in water.