抗ANXA2单克隆抗体的制备及初步鉴定  

Preparation and characterization of monoclonal antibody against ANXA2

作　　者：尹江丽[1] 陈少琅[1] 万勇[1] 李明[1] 

机构地区：[1]南方医科大学生物技术学院,广东广州510515

出　　处：《热带医学杂志》2014年第5期578-581,618,共5页Journal of Tropical Medicine

基　　金：十二五国家高技术研究发展计划(863计划)(2012AA020205)

摘　　要：目的制备ANXA2特异性单克隆抗体（McAb），为研究ANXA2的生物学功能和检测该蛋白提供有力的抗体工具。方法用经IPTG诱导表达的基因重组PGEX-4T-1-ANXA2抗原免疫BALB／c小鼠．利用常规杂交瘤技术和间接ELISA法筛选抗ANXA2杂交瘤细胞，用ELISA法鉴定所得单抗腹水的效价及Western blot鉴定McAb特异性。单抗纯化和Eu3＋标记后，分别包被聚苯乙烯板及作为铕标单抗，利用时间分辨荧光免疫分析法（TRFIA）筛选出检测ANXA2蛋白的最佳配伍单抗。结果经细胞融合、筛选、克隆化，获得了16株可分泌高效价单抗的杂交瘤细胞，间接ELISA显示16株McAb与空载体PGEX．4T．1均未发生交叉反应．抗体特异性良好．16株抗体腹水效价为1：8000，1：256000，培养上清效价为1：256～1：512，纯化的腹水单抗经配对试验，2D6和9H9-Eu3＋配对最佳。结论获得16株能稳定分泌高特异性抗ANXA2的杂交瘤细胞．能用于后续研究。Objective To prepare monoclonal antibodies against ANXA2 for further study of biological function of ANXA2 and the detection of the protein. Methods BALB/c mice were immunized with recombinant human PGEX-4T-ANXA2 protein. Hybridoma cell lines secreting monoclonal antibodies against human PGEX-4T-1-ANXA2 protein were screened by regular cell fusion and subeloning approach. Positive hybridomas were selected by indirect enzyme-linked immunosorbent assay （ELISA）. The titer and specificity of the McAbs were determined by ELISA and Western blot respectively. An doubleantibody sandwich Time-resolved fluoroimmunoassay（TRFIA） method for insitu detection of ANXA2 was established by using the purified McAbs and the McAbs labelled with Eu3＋, and selected the optimum antibody pairs. Results After the fusion, screening and subeloning, sixteen McAbs against ANXA2 were obtained, none of which had cross reaction with PGEX-4T-1. The titer of these McAbs was 1 ： 8 000 to 1 ： 256 000 in the aseites and 1 ： 256 to 1 ： 512 in supernatant. By matching test, the 2D6 and 9H9-Eu3＋ was the optimum antibody pairs. Conclusion Sixteen hybridoma cell lines secreting the McAbs against ANXA2 with high specificity were successfully established, and can be used for follow-up study.

关 键 词：ANXA2 单克隆抗体 ELISA 

分 类 号：R735.7[医药卫生—肿瘤]