抗HoxA1单克隆抗体的制备、鉴定

发布时间：2018-04-27 11:34

本文选题：HoxA1 + 单克隆抗体　；参考：《第四军医大学》2011年硕士论文

【摘要】：[目的] 利用合成抗原GST-HoxA1免疫BALB/c小鼠,制备针对HoxA1蛋白的单克隆抗体,通过Westen bloting检测其特异性,FITC荧光素标记抗体,免疫荧光染色,研究其在HoxA1蛋白检测中的应用价值。 [方法与结果] GST-HoxA1合成抗原免疫BALB/c小鼠,采用传统单克隆抗体制备方法,通过细胞融合,HAT选择,有限稀释法克隆扩增,获得稳定分泌高纯度抗HoxA1单克隆抗体的杂交瘤细胞株。利用小鼠体内接种杂交瘤细胞制备含特异性抗体的腹水,通过辛酸—硫酸铵法纯化,得到优质单克隆抗体。通过间接ELISA法测定抗体效价,Westen bloting检测其的特异性。经测定抗HoxA1单克隆抗体4D5效价为1:102400,Westen bloting结果发现4D5可以特异性结合靶细胞Hela中HoxA1蛋白,与载体蛋白GST无交叉反应;利用FITC荧光素标记抗体,免疫荧光染色结果显示实验组肿瘤细胞明显荧光表达,对照组细胞仅见极微弱荧光表达。 [结论] 1.成功制备抗HoxA1单克隆抗体。 2.通过Westen bloting证实所制备单克隆抗体可与相应HoxA1蛋白特异性结合,用该抗体检测Hela细胞裂解液,结果在相对分子质量约36×10~3处有反应条带;FITC荧光素标记抗体,免疫荧光染色检测靶细胞Hela,证明该抗体可初步用于HoxA1蛋白检测。
[Abstract]:[purpose] The monoclonal antibody against HoxA1 protein was prepared by immunizing BALB/c mice with synthetic antigen GST-HoxA1. The specific FITC fluorescein labeled antibody was detected by Westen bloting and immunofluorescence staining was used to study its application value in the detection of HoxA1 protein. [methods and results] GST-HoxA1 synthetic antigen was used to immunize BALB/c mice. The hybridoma cell lines secreting high purity monoclonal antibody against HoxA1 were obtained by using traditional monoclonal antibody preparation method, by cell fusion with hat selection and cloning by limited dilution method. Ascites containing specific antibodies were prepared by inoculating hybridoma cells in vivo and purified by octanoic acid-ammonium sulfate method to obtain high quality monoclonal antibodies. The antibody titer was determined by indirect ELISA method and the specificity of the antibody was detected by Westen bloting. The titer of anti HoxA1 monoclonal antibody 4D5 was 1: 102400 HoxA1 bloting. It was found that 4D5 could specifically bind HoxA1 protein in target cell Hela and had no cross reaction with carrier protein GST, and the antibody was labeled with FITC fluorescein. The results of immunofluorescence staining showed that the tumor cells in the experimental group showed obvious fluorescence expression, while in the control group, only very weak fluorescence expression was observed. [conclusion] 1. Monoclonal antibody against HoxA1 was successfully prepared. 2. It was confirmed by Westen bloting that the monoclonal antibody could specifically bind to the corresponding HoxA1 protein. The Hela cell lysate was detected by the antibody. The results showed that there were FITC-labeled antibodies at the relative molecular weight of 36 脳 10 ~ (-3). The detection of Hela by immunofluorescence staining showed that the antibody could be used for the detection of HoxA1 protein.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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