抗CP4-EPSPS单克隆抗体的制备和生物学特性的鉴定

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。

Preparation and characterization of monoclonal antibody against CP4-EPSPS

AIM: To prepare monoclonal antibody (mAb) against CP4-EPSPS, which could be applied to the development of gold colloidal rapid diagnostic kit for the specific detection of GMO. METHODS: BALB/c mice were immunized with CP4-EPSPS. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted CP4-EPSPS mAb antibodies were cloned with limited dilution method. The immunoglobulin (Ig) subtype, the ascites titers, the binding site, and the affinity of the obtained mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis. RESULTS: From over 80 positive hybridomas which secreted anti-cp4-EPSPS mAbs, a pair of hybridomas were screened out, designated III5A3 and III13A2. Chromosome analysis revealed that the obtained hybridomas were with the universal characteristics of the monoclonal hybridoma cells which secreted mAb, and the Ig subtype of III5A3 and III13A2 mAb was both IgG1. The ascites titers of III5A3 and III13A2 mAb were 1:10(6) and 1:10(8), respectively. Our study also demonstrated that these two mAbs, which recognized the same epitope, could both specifically bind to the CP4-EPSPS protein. The relative affinity constant of III5A3 and III13A2 mAb was determined as 10(5) and 10(6) respectively. CONCLUSION: A pair of high titer, specific mAbs against CP4-EPSPS have been successfully prepared and primarily identified, which may be useful in the development of a rapid and convenient diagnostic kit for detection of GMO.