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目的制备人脱嘌呤脱嘧啶核酸内切酶(hAPE1)单克隆抗体(mAb)并对其生物学特性进行鉴定。方法以hAPE1融合蛋白为抗原免疫Balb/c小鼠,运用细胞融合、克隆化技术和间接ELISA法获得并筛选针对hAPE1的杂交瘤细胞,以体内诱生法产生腹水,并用Protein G柱亲和纯化得到hAPE1 mAb。采用秋水仙素阻断法、Western blot和间接ELISA法鉴定mAb的染色体核型、特异性、种属交叉反应、亚类、效价和亲和力,并应用于免疫荧光、免疫细胞化学和免疫组化实验中评价mAb性能。结果获得2株分泌稳定、高特异性的hAPE1 mAb的杂交瘤细胞株2-G1和4-F6,其效价均超过1:107,Ig亚类分别是IgG2b和IgG3,亲和常数分别为1.8×10-7和3.4×10-5,与兔、小鼠、大鼠无种属交叉性。2-G1mAb可应用于免疫细胞化学、免疫细胞荧光和免疫组织化学等实验技术中。结论成功获得2株稳定分泌抗hAPE1mAb的杂交瘤细胞株,产生的mAb具有高纯度、高效价和高特异性的特性,能特异性识别hAPE1天然蛋白和融合蛋白,为开展多种免疫学实验及研究提供必要的物质基础。
OBJECTIVE: To prepare and characterize human monoclonal apatase (hAPE1) monoclonal antibody (mAb). Methods Balb / c mice were immunized with hAPE1 fusion protein as antigen. The hybridoma cells targeting hAPE1 were obtained by cell fusion, cloning and indirect ELISA. Ascites was induced by in vivo method and affinity purified by Protein G column HAPE1 mAb was obtained. Cytokines, specificities, cross-reactions, subclasses, titers and affinity of mAbs were identified by colchicine blocking assay, Western blot and indirect ELISA, and applied to immunofluorescence, immunocytochemistry and immunohistochemistry The mAb performance was evaluated experimentally. Results Two hybridoma cell lines, 2-G1 and 4-F6, secreting stable and highly specific hAPE1 mAb were obtained. The titer of the two hybridomas was over 1: 107. The Ig subclasses were IgG2b and IgG3, respectively. The affinity constants were 1.8 × 10-7 and 3.4 × 10-5, no cross-species with rabbits, mice and rats. 2-G1 mAb can be used in experimental techniques such as immunocytochemistry, immunofluorescence and immunohistochemistry. Conclusion Two hybridoma cell lines stably secreting anti-hAPE1 mAb were successfully obtained. The mAbs produced showed high purity, high titer and high specificity. They could specifically recognize hAPE1 natural protein and fusion protein. To carry out a variety of immunological experiments and Research provides the necessary material basis.