Zhang Xuexin, Li Na, Li Qi, Cheng Kun, Ma Jingchang, Wang Yuling, Zhang Yuan, Zhuang Ran
Department of Immunology, School of Basic Medicine, Air Force Medical University, Xi'an 710032, China.
Department of Immunology, School of Basic Medicine, Air Force Medical University, Xi'an 710032, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Oct;36(10):934-940.
Objective To prepare and preliminarily identify anti-mouse CD226 monoclonal antibodies (mAbs) using CD226 knockout (CD226 KO) mice as immunized animals. Methods Animals were immunized by recombinant mouse CD226 protein expressed by eukaryotic cells. Anti-mouse CD226 mAbs were prepared by conventional B-cell hybridoma technology. The application of the generated mAbs for flow cytometry and Western blotting was tested. A sandwich ELISA system was established for the detection of soluble CD226 in mice. And the concentrations of plasma soluble CD226 was determined by this sandwich ELISA system in an LPS-induced sepsis mouse model. Results Two hybridoma cell lines secreting mouse anti-mouse CD226 mAbs were successfully prepared. The clones were named mA1.1 and mA1.3, respectively. The antibody subclasses were both IgG1, and the light chains were κ. The obtained mAbs could be applied for flow cytometry to detect exogenous transfected CD226 on the cell surface and natural CD226 on the mouse platelet membrane. In Western blot assay, the mAb could bind to lymphocyte membrane proteins with a relative molecular mass (M) of 67 000 that was consistent with the M of CD226. In the sandwich ELISA system, the purified mAbs of mA1.3 were coated on ELISA plates as capture antibody, and the mAbs of mA1.1 were labeled with horseradish peroxidase or biotin as detection antibody. The detection sensitivities were 3.0 and 0.25 ng/mL, respectively. The concentration of plasma soluble CD226 of the septic mice was lower than that of the normal mice in the control group. Conclusion The mouse mAbs for identifying mouse CD226 have been prepared successfully and can be applied in various detection techniques.
目的 以CD226基因敲除（CD226 KO）小鼠为免疫动物，制备并初步鉴定抗小鼠CD226单克隆抗体（mAbs）。方法 用真核细胞表达的重组小鼠CD226蛋白免疫动物。采用常规B细胞杂交瘤技术制备抗小鼠CD226 mAbs。检测所产生的mAbs在流式细胞术和蛋白质印迹法中的应用。建立夹心ELISA系统检测小鼠可溶性CD226。并在脂多糖诱导的脓毒症小鼠模型中用该夹心ELISA系统测定血浆可溶性CD226的浓度。结果 成功制备了两株分泌小鼠抗小鼠CD226 mAbs的杂交瘤细胞系。克隆分别命名为mA1.1和mA1.3。抗体亚类均为IgG1，轻链均为κ链。所获得的mAbs可用于流式细胞术检测细胞表面外源性转染的CD226和小鼠血小板膜上的天然CD226。在蛋白质印迹分析中，该mAb可与相对分子质量（M）为67 000的淋巴细胞膜蛋白结合，与CD226的M值一致。在夹心ELISA系统中，将纯化的mA1.3 mAbs包被在ELISA板上作为捕获抗体，将mA1.1 mAbs用辣根过氧化物酶或生物素标记作为检测抗体。检测灵敏度分别为3.0和0.25 ng/mL。脓毒症小鼠血浆可溶性CD226的浓度低于对照组正常小鼠。结论 已成功制备出用于鉴定小鼠CD226的小鼠mAbs，可应用于多种检测技术。
