小鼠腹水JWA单克隆抗体的制备鉴定

Generation and Identification of Monoclonal Antibody of JWA from Mouse Ascites

建立小鼠腹水中JWA单克隆抗体(Monoclonalantibody,mAb)的制备方法及鉴定,获得高纯度、高效价的JWA单克隆抗体。对预先用弗氏不完全佐剂处理的小鼠腹腔接种JWA单克隆抗体杂交瘤细胞,收集小鼠腹水、离心,采用Protien G亲和柱层析法进行亲和层析纯化JWA单克隆抗体,并采用SDS-PAGE检测方法分析纯化抗体的纯度。分别运用细胞免疫荧光、免疫印迹技术鉴定纯化的抗体特异性。BCA法测定纯化后JWA单抗的浓度为6.79 mg/mL;SDS-PAGE电泳显示纯化的抗体只有IgG的重链和轻链,而没有其它杂蛋白带;纯化后的JWA单抗在免疫荧光实验可以检测到细胞中JWA蛋白在胞浆中呈丝状均匀分布,与前期实验结果一致。免疫印迹实验中1∶800、1∶400均可以得到清晰的目的条带。用Protein G亲和层析法获得了高纯度和较高效价的JWA单克隆抗体,这将为JWA蛋白的基础和应用研究提供技术支持。

To develop an affinity chromatography purification of anti JWA monoclonal antibody （mAb） from mouse ascites, the JWA specific hybridoma cells were developed by contract service. After filtrated by centrifugation the ascites sample was loaded on a Protein G affinity chromatography column and the purified JWA monoclonal antibody was collected. The purity of JWA mAb was identified by isolation of SDS PAGE. The intracellular distribution of JWA protein was determined by immunolluorescence staining on cultured HeLa cells, and the specificity of JWA mAb was determined by Western blot. The results showed that the average concentration of the obtained JWA mAbs in mouse ascites was 6.79 mg/mL after protein G affinity chromatography purification. The purity of JWA mAbs were generated in SDS-PAGE. The immunofluorescence staining showed clear intracellular linar distribution of JWA protein. The Western blot indicated a single specific band with correct molecular weight. Using the affinity chromatography purification method,high specific and purifed JWA mAb from mouse ascites ,was successfully obtained which could be effectively used for immunofluorescent and Western blot assays.