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目的:制备抗-lamp2单克隆抗体,并对其生物学特性进行初步鉴定。方法:根据已发表的文献通过人工合成多肽,并将此多肽分别连接KLH和BSA,以peptide-KLH作为抗原免疫BALB/c小鼠,取血清效价大于1:10000的小鼠的脾细胞与骨髓瘤细胞SP2/0进行融合,以peptide-BSA包被,通过酶联免疫吸附实验(ELISA)检测杂交瘤细胞的上清进行筛选;得到能稳定分泌单克隆抗体的杂交瘤细胞株;通过鼠单克隆抗体亚类分型试剂盒鉴定单克隆抗体的亚类,采用免疫组化方法证明新制备的单克隆抗体能识别目的蛋白。结果:得到一株能稳定分泌抗-LAMP2单克隆抗体的杂交瘤细胞株,压用免疫组化方法证明新制备的抗-LAMP2单克隆抗体能识别天然的LAMP2;此单克隆抗体的亚类为。结论:杂交瘤细胞株能稳定分泌目的单克隆抗体;免疫组化方法证明新制备的单克隆抗体能识别自己天然的目的蛋白,为更好地研究LAMP2的生物学功能奠定了基础。
Objective: To prepare anti-lamp2 monoclonal antibody and to identify its biological characteristics. METHODS: According to the published literature, BALB / c mice were immunized with peptide-KLH as an antigen by artificial synthesis of the polypeptide and then linked to KLH and BSA respectively. The spleen cells of mice with serum titer greater than 1: 10000 were incubated with The myeloma cells SP2 / 0 were fused and coated with peptide-BSA. The supernatants of hybridoma cells were screened by enzyme-linked immunosorbent assay (ELISA). The hybridoma cell lines that can stably secrete monoclonal antibodies were obtained. The monoclonal antibody subclassing kit was used to identify the subclass of monoclonal antibodies. Immunohistochemistry was used to demonstrate that the newly prepared monoclonal antibodies recognize the target protein. Results: A hybridoma cell line secreting anti-LAMP2 monoclonal antibody was obtained. Immunohistochemistry was used to confirm that the newly prepared anti-LAMP2 monoclonal antibody recognized the native LAMP2. The subclass of this monoclonal antibody was . Conclusion: The hybridoma cell line can stably secrete monoclonal antibody of interest. Immunohistochemistry proved that the newly prepared monoclonal antibody can recognize its natural target protein, which lays the foundation for better study of the biological function of LAMP2.