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制备Annexin A2特异性单克隆抗体(McAb),为Annexin A2的体内外功能的研究提供有力的抗体工具。以原核表达的全长p36蛋白(Annexin A2单体)免疫BALB/c小鼠,通过杂交瘤技术制备针对Annexin A2的McAb。以间接ELISA法筛选出目的杂交瘤细胞株并进行染色体计数;分析McAb的亚型,检测其效价、浓度及亲和常数;以细胞荧光染色,Western blot和RT-PCR鉴定McAb特异性。结果得到1株能稳定分泌特异性针对p36的McAb的细胞株F8,其抗体亚类重链为IgG2a,轻链为κ型;腹水纯化后效价为1∶4 000;亲和常数为3.4×109L/mol。成功获得了能稳定分泌AnnexinA2特异性McAb的细胞株。
Preparation of Annexin A2-specific monoclonal antibody (McAb), Annexin A2 in vitro and in vivo function provides a powerful antibody tools. BALB / c mice were immunized with the full-length p36 protein (Annexin A2 monomer) and the McAbs against Annexin A2 were prepared by hybridoma technology. The hybridoma cell lines were screened by indirect ELISA and the chromosome counts were counted. The subtypes of McAbs were analyzed for their potency, concentration and affinity constants. The McAb specificity was identified by fluorescence staining, Western blot and RT-PCR. Results A cell strain F8 was obtained that could stably secrete McAb against p36. The antibody subtype heavy chain was IgG2a, the light chain was κ type. The ascites titer was 1: 4000 and the affinity constant was 3.4 × 109L / mol. Successfully obtained a cell line that can stably secrete AnnexinA2-specific McAb.