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目的制备鼠抗早孕因子(EPF)单克隆抗体,纯化并进行鉴定。方法用重组EPF免疫BALB/c小鼠,采用杂交瘤技术获得抗EPF杂交瘤细胞株,注入BALB/c小鼠腹腔制备腹水型单抗,Protein-G亲和层析纯化后,SDS-PAGE电泳鉴定其纯度,用间接ELISA法测抗体效价,ELISA相加试验测定抗原表位,用Western blotting鉴定其特异性,并进行类及亚类、细胞染色体核型分析。结果筛选出2株具有较高活性和良好稳定性的分泌抗EPF单克隆抗体的杂交瘤细胞株,其腹水的抗体效价均高于2×10-5,2株单抗均为IgG类IgG1亚类,轻链类型均为κ,细胞核型分析符合杂交瘤细胞特征,特异性良好,2株单抗有不同的抗原表位。SDS-PAGE电泳显示纯化后抗体有较高的纯度,Western blotting表明其与EPF抗原有特异结合性。结论成功制备2株具有较高活性的鼠抗EPF单克隆抗体,为建立检测EPF方法奠定了良好的基础。
Objective To prepare murine anti-pregnancy factor (EPF) monoclonal antibodies, purify and identify them. Methods BALB / c mice were immunized with recombinant EPF. Anti-EPF hybridoma cell lines were obtained by hybridoma technique. BALB / c mice were injected intraperitoneally to prepare ascites monoclonal antibody. Protein-G affinity chromatography and SDS-PAGE electrophoresis The purity of the antibody was identified. The antibody titer was measured by indirect ELISA. The epitopes were determined by ELISA. The specificity of the epitope was identified by Western blotting. The karyotypes were also analyzed. Results Two hybridoma cell lines secreting anti-EPF monoclonal antibody with high activity and good stability were screened. The antibody titer of ascites was higher than 2 × 10-5. Subtypes and light chain types were both kappa. The karyotype analysis accorded with the characteristics of hybridoma cells with good specificity. The two monoclonal antibodies had different antigenic epitopes. SDS-PAGE electrophoresis showed that the purified antibody has higher purity, Western blotting showed that it has specific binding with EPF antigen. Conclusion Two monoclonal anti-EPF monoclonal antibodies with high activity were successfully prepared, which laid a good foundation for the establishment of the method of detecting EPF.