抗ANXA2单克隆抗体的制备及初步鉴定

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∶8 000~1∶256 000,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。

Preparation and characterization of monoclonal antibody against ANXA2

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.