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目的制备可用于胶体金快速检测试条的抗CP4-EPSPS(5-enolpyruvlshimimate-3-phosphate synthase)单克隆抗体(mAb),并鉴定其特性。方法以重组蛋白CP4-EPSPS免疫BALB/c小鼠,采用杂交瘤技术制备抗CP4-EPSPS的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类,检测mAb的效价及相对亲和力,并进行mAb结合表位分析。结果获得2株可分泌特异性mAb的杂交瘤细胞(Ⅲ5A3,Ⅲ13A2)。其抗体亚类均为IgG1;腹水效价分别为1∶106和1∶108;相对亲和力Ⅲ5A3在105以上,Ⅲ13A2在106以上。ELISA相加实验结果显示2株mAb识别相同或相近的抗原表位。结论成功地制备出抗CP4-EPSPS的2株mAb,为建立快速特异检测转基因植物(GMO)的实验方法提供了有力的工具。
Objective To prepare anti-CP4-EPSPS (mAb) monoclonal antibody (mAb) for rapid detection of colloidal gold and identify its characteristics. Methods BALB / c mice were immunized with the recombinant protein CP4-EPSPS. Anti-CP4-EPSPS mAbs were prepared by hybridoma technique. The mAb was identified by indirect ELISA and Western blot. Meanwhile, indirect ELISA was used to identify the Ig subclass of mAb , The potency and relative affinity of the mAb were tested and mAb binding epitope analysis was performed. Results Two hybridoma cells secreting specific mAb (Ⅲ 5A3, Ⅲ13A2) were obtained. The antibody subtypes were IgG1; ascites titers were 1:106 and 1:108 respectively; relative affinity III5A3 was above 105; ELISA results show that two mAbs recognize the same or similar antigenic epitopes. Conclusion Two mAbs against CP4-EPSPS were successfully prepared, which provided a powerful tool for the establishment of a rapid and specific method for the detection of transgenic plants (GMO).