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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。
In order to prepare an anti-imidacloprid monoclonal antibody with high sensitivity and specificity, an analytical method of imidacloprid residue immunoassay was established. After analyzing the charge distribution of imidacloprid and its similar pesticides by molecular simulation, 1- [6- (2-carboxyethylthio-3-pyridyl) methyl] -N-nitro-2-imidazolidinimine (H1) - carboxypropyl-N-nitro-2-imidazolidinimine (H2) as a coated hapten, H1 and H2 were coupled with bovine serum albumin (BSA) and ovalbumin (OVA) Into the original immunogen and coated. After immunization of BALB / c mice, 2 strains of stable secreting anti-imidacloprid monoclonal antibody-producing cell lines, 2F11 / A9 and 2G6 / G12, were obtained by conventional hybridoma technique. The secreted antibodies were IgG3 and IgG1. The antibody titer of ascites reached 1: 128 000 with IC50 of 5.3 ng / mL and 28.3 ng / mL, with the detection limits of 1.1 ng / mL and 7.7 ng / mL, respectively. Structural similarities of the two antibodies to 6 imidacloprid No significant cross-reaction. Preparation of anti-imidacloprid monoclonal antibody laid the foundation for the establishment of imidacloprid residue immunological detection method and the development of immunoassay products.