Li Gang, Ji Xianyong, Qian Guoliang, Hua Xiude, Qin Na, Wang Jie, Liu Fengquan
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Jun;27(6):943-51.
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.
为制备抗农药吡虫啉的高亲和力单克隆抗体，我们合成了半抗原1-[(6-羧基乙硫基-3-吡啶基)甲基]-N-硝基咪唑烷亚胺（命名为H1）和1-[(6-氯-3-吡啶基)甲基]-3-羧基丙基-N-硝基-2-咪唑烷亚胺（称为H2）。然后通过NHS酯法将半抗原分别与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联，制备免疫原（H1-BSA）和包被抗原（H2-OVA）。用H1-BSA偶联物免疫BALB/c小鼠。通过传统的杂交瘤技术，我们获得了两株分泌对吡虫啉具有特异性抗体的杂交瘤细胞系2F11/A9和2G6/G12。结果表明，获得的单克隆抗体亚型分别为IgG3和IgG1，腹水效价高达1:128 000。间接竞争ELISA表明，其IC50值分别为5.3和28.3 ng/mL，检测限分别为1.1 ng/mL和7.7 ng/mL。两种单克隆抗体与六种类似化合物均无明显交叉反应。因此，所制备的两种单克隆抗体具有很高的亲和力和特异性，可用于开发ELISA法快速测定吡虫啉残留，为免疫分析产品的研发奠定了坚实基础。
