抗人微管不稳定蛋白单克隆抗体的制备及鉴定
Preparation and Identification of Monoclonal Antibodies Against Human Stathmin
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摘要: 目的 制备人微管不稳定蛋白（Stathmin）的单克隆抗体,检测Stathmin蛋白在真核细胞中的表达,为探讨Stathmin 生物学功能奠定基础。 方法 利用重组Stathmin蛋白为免疫原,免疫BALB/C小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过间接ELISA的筛选和有限稀释克隆化,获得稳定分泌抗Stathmin单克隆抗体的杂交瘤细胞株,通过ELISA,Western blot和免疫组织化学实验等方法分别对其效价和特异性进行鉴定。 结果 成功地建立了2株稳定分泌抗Stathmin的单克隆抗体杂交瘤细胞株,分别命名为F001和F002。两株单克隆抗体的免疫球蛋白亚类均为IgG1。两株单克隆抗体通过免疫组织化学实验和Western blot实验都能特异性地结合真核细胞内源性的Stathmin蛋白。 结论 成功建立了两株效价高、特异性好的抗Stathmin蛋白的单克隆抗体,为进一步研究Stathmin的生物学功能及其与肿瘤的关系创造了条件。Abstract: Objective To obtain monoclonal antibodies against stathmin for further study of the biological function of stathmin protein. Methods Balb/c mice were immunized with recomb inant stathmin,hybridoma cell lines secreting monoclonal antibodies against stathmin were screened by regular cell fusion and subcloning approach.The specificities of these monoclonal antibodies were determined by ELISA,Western blot,and immunohistochemistry. Results Two hybridoma cell lines( F001 and F002）stable insecreting specific monoclonal antibodies were successfully obtained.The subtype of the two monoclonal antibodies belong to the IgG1.Western blot analysis and immunohistochemistry showed that the two antibodies can specifically bind with stathmin antigen derived from human eucaryotic cells or tissue. Conclusion Monoclonal antibodies against stathmin with high titers and specificity have been successfully prepared,providing foundation for further study of stathmin protein and the relationship between stathmin and tumors.
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