抗吡虫啉单克隆抗体的制备及鉴定

Production and identification of monoclonal antibodies against pesticide imidacloprid

为制备灵敏度高,特异性强的抗吡虫啉单克隆抗体,建立经济、快速、准确的吡虫啉残留免疫学分析方法,采用分子模拟技术分析吡虫啉及其类似农药的电荷分布后,选择1-[6-(2-羧乙硫基-3-吡啶)甲基]-N-硝基-2-咪唑啉亚胺(H1)作为免疫半抗原,1-(6-氯-3-吡啶甲基)-3-羧丙基-N-硝基-2-咪唑啉亚胺(H2)作为包被半抗原,利用NHS酯法将H1和H2分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原与包被原。免疫BALB/c小鼠后,采用常规杂交瘤技术共获得2株稳定分泌抗吡虫啉单克隆抗体的细胞株2F11/A9和2G6/G12,其分泌的抗体类型分别为IgG3和IgG1,两细胞株的腹水抗体效价均达到了1∶128 000,IC50分别为5.3 ng/mL和28.3 ng/mL,检测限分别为1.1 ng/mL和7.7 ng/mL,两种抗体与6种吡虫啉的结构类似物均无明显的交叉反应。抗吡虫啉单抗的制备为吡虫啉残留免疫学检测方法的建立及免疫检测产品的研发奠定了基础。

To produce high-affinity monoclonal antibodies against pesticide imidacloprid,we synthesized the haptens 1-[（6-Carboxylethylthio-3-pyridinyl） methyl]-N-nitro-imidazolidinimine（named as H1） and 1-[（6-Chloro-3-pyridinyl） methyl]-3-carboxylpropyl-N-nitro-2-imidazolidinimine（termed as H2）.And then the haptens were coupled to bovine serum albumin（BSA） and ovalbumin（OVA） for immunogen（H1-BSA） and coating antigen（H2-OVA） respectively by NHS ester method.BALB/c mice were immunized with H1-BSA conjugate.We obtained two hybridoma cell lines 2F11/A9 and 2G6/G12 secreting antibody specific for imidacloprid from the conventional hybridoma technology.The result showed that the subtypes of obtained monoclonal antibodies were IgG3 and IgG1,respectively,and the titers of ascites were up to 1：128 000.The indirect competitive ELISA indicated the IC50 values of 5.3 and 28.3 ng/mL with detection limits of 1.1 ng/mL and 7.7 ng/mL,respectively.Two monoclonal antibodies had no apparent cross reactivity with six analogous compounds.Thus,two prepared monoclonal antibodies had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of imidacloprid residue and laid a solid foundation for research and development of products for immunoassay.