摘要
目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。
Objective：Preparation and characterization of monoclonal antibodies against NSE protein,and establishment of a double-antibody sandwich ELISA assay.Methods：BALB/c mice were immunized by using purified recombinant NSE,and monoclonal antibodies were generated by hydridoma technique.These antibodies were characterized with ELISA,Western blot,Immunofluorescent and Immunohistochemical staining.The isotypes of these antibodies were determined with an antibody isotyping kit.With Horseradish Peroxidase labelled NSE monoclonal antibody,we were able to establish a double-antibody sandwich ELISA to detect NSE protein.Results：Two positive hybridoma cell lines were selected for test,the titers of these two monoclonal antibodies could reach 4.2×10^7-6.5×10^7,and their isotypes were IgG2b.Our NSE antibodies could detect not only endogernous NSE protein from cells,but also secreted NSE protein from cells in culture medium by Western blot,in addition,they could be used for immunofluorescent and immunohistochemical staining.The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8.85 ng/ml.Conclusion：Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers,and we established a double-antibody sandwich ELISA assay which could be used for clinical test in future.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第10期922-925,935,共5页
Chinese Journal of Immunology
基金
福建省科技重点项目(编号项目No.2011Y0050)
厦门市科技计划项目(No.3502Z20123009)
