淋巴细胞脉络丛脑膜炎病毒GP2单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibody against GP2 of lymphocytic choriomeningitis virus

目的制备淋巴细胞脉络丛脑膜炎病毒(LCMV)单克隆抗体(m Ab)。方法采用杂交技术获得并筛选出杂交瘤细胞,收集含单克隆抗体的培养上清行免疫荧光(IFA)、dot-blot、ELISA法鉴定抗体重链类型和测定单克隆抗体的免疫效价。用蛋白G Sepharose亲和层析填料纯化抗体,纯化后的抗体采用SDS-PAGE检测纯化效果,并进一步用Western blot检查抗体对LCMV的作用部位。结果成功获得能稳定分泌抗LCMV的特异性杂交瘤细胞株,顺利获得纯化的单克隆抗体,命名为anti-GP2。免疫荧光、ELISA结果均证实获得的单克隆抗体能识别LCMV抗原。用ELISA法鉴定出该单克隆抗体重链类型为Ig G2b,培养液免疫效价大约为1.35×10~3。蛋白G Sepharose亲和层析填料纯化抗体后经SDS-PAGE检测证实纯化抗体成功。Western blot提示该单克隆抗体对LCMV的作用部位为病毒颗粒表面糖蛋白抗原GP2。结论成功制备出了针对LCMV病毒颗粒表面糖蛋白抗原GP2的单克隆抗体(anti-GP2 m Ab)。

The purpose of this study is to prepare monoclonal antibody（m Ab） against lymphocyticchoriomeningitis virus（LCMV）. We utilized the technical of cell hybridization to obtain hybridoma, and the mediumof hybridoma was collected. Then the subclass of m Ab CHand the immune potency were identified viaimmunofluorescence（IFA）, dot-blot and ELISA. Subsequently, the m Ab was purified by protein G Sepharoseaffinity chromatography protocol, and the purity of the m Ab was checked by SDS-PAGE. At last, Western-blot wasperformed to detect the site where the m Ab interacted with the LCMV antigen. As a result, we obtained a hybridomastrain which could secreted specific anti-LCMV m Ab stably, and a kind of purified m Ab named Anti-GP2 wascollected favorably. IFA and ELISA demonstrated that the m Ab could recognize the antigen from LCMV. ELISAresult indicated that the subclass of the m Ab CHis Ig G2 b, and the immune potency of the medium is 1.35×10-3approximately; SDS-PAGE showed that the m Ab was purified by protein G Sepharose affinity chromatographyprotocol successfully; Western-blot implied that the site where the m Ab combined specifically was GP2 on surfaceof the LCMV particle. In conclusion, we has prepared a kind of m Ab successfully, which could recognize GP2 onsurface of the LCMV particle specifically.