抗人肺鳞癌单克隆抗体的制备和纯化

摘要

目的 制备并纯化前期工作建立的杂交瘤细胞株C5所分泌的抗人肺鳞癌单克隆抗体,为研究该单抗对应肺癌相关抗原的生物学功能奠定基础.方法 常规培养前期工作建立的C5杂交瘤细胞,有限稀释法进行克隆化培养,ELISA法检测培养上清,挑选检测阳性细胞继续克隆化培养,直至3次检测亚克隆均呈阳性.收集阳性培养上清,利用Protein A-sepharoseCL-4B亲和层析法纯化抗体,Western-Blot法对纯化后的抗体进行鉴定.结果 得到了来源相同的亚克隆杂交瘤细胞株,克隆阳性率达100％.成功纯化出了目的单克隆抗体,并证实纯化产物系目的单抗,纯化后的C5杂交瘤细胞单克隆抗体重链分子量约为75 kDa.结论 成功纯化出抗人肺鳞癌单克隆抗体,该单抗可能在肺癌的早期诊断、靶向治疗和肿瘤疫苗的研制等方面具有重要意义和潜在应用价值.%Objective To subclone C5 hybridoma cells and purify the monoclonal antibody (McAb) by the immunoaffinity chromatography so as to provide with purified McAb the future research,the early diagnosis of lung cancers and targeted therapy and to study the lung cancer associated with antigen and the antigen associated with vaccine.Method C5 hybridoma cells were thawed from liquid nitrogen.The culture of hybridoma cells was subcloned for 3 times,and the culture's supernatant from each clone was tested by ELISA assay.Positive clones underwent culture expansion and the McAb was purified from its supernatant using ProteinaA-SepharoseCL-4B column affinity chromatography procedure.The specificity of the McAb protein expression was measured using SDS-PAGE and Western-Blot assay.Results the McAb was purified from C5 lung cancer hybridoma cell line using affinity chromatography technique.SDS-PAGE and Western Blot assay of the McAb showed one single band was specifically against the second antibody and the molecular weight of which was about 75KDa.Conclusion The McAb against lung cancer cells can be successfully purified from C5 hybridoma cells by limited dilution and affinity chromatography using ProteinaA-SepharoseCL-4B column.This McAb may play an important role in the early diagnosis and targeted therapy of human lung cancer and may contribute to the development of tumor vaccine.