【摘要】 目的制备和鉴定抗胎儿血红蛋白(fetal hemoglobin,HbF)单克隆抗体(mAb),为相关疾病的研究提供支持与工具。方法用脐带血红细胞裂解物免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞(NS-1)融合制备杂交瘤,经筛选和三次克隆化,从制备的腹水中纯化单克隆抗体。用ELISA以及western blot等方法鉴定单克隆抗体的特性(效价、Ig亚类、特异性以及亲和力)。结果筛选到3株可稳定分泌抗HbF单克隆抗体的杂交瘤细胞6A5、5C8、2C8,腹水效价分别为1×10-6、2×10-6和4×10-6。单抗亚类均为IgG1,抗体亲和力分别为7.8×109、2.2×108和8.6×109。ELISA和western blot结果显示,6A5、5C8、2C8 mAb只与HbF的二聚体特异性结合,与其他类型血红蛋白不发生交叉反应。结论获得3株能特异性识别HbF的高亲和力mAb,可结合二聚体与单体HbF,为临床检测HbF水平及相关研究提供了有效的工具。更多还原

【Abstract】 Objective Preparation and characterization of monoclonal antibodies (mAbs) against human fetal hemoglobin(HbF) which can be used as useful tool for detection of HbF.Methods Balb/c mice were immunized with HbF from hemolysate of cord blood,and the hybridoma cell were generated by fusion with murine spleen cells and myeloma cells NS-1,followed by screening and cloning for three times.The mAbs were purified from ascites and characterized by indirect ELISA and western blot.Results Three of hybridoma cell lines secreting anti-HbF mAbs were obtained and named as 6A5,5C5 and 2C8 which all belonged to isotype IgG 1.The titer of the three mAbs in ascites were 1×10-6,2×10-6 and 4×10-6 and Kaffs were 7.8×109,2.2×108 and 8.6×109,repectively.All of mAbs could bind with HbF in diamer and monomer specifically,and without any cross-reaction with HbA and HbA2.Conclusion Three mAbs against HbF with high titer,specificity and affinity were prepared,which would provide a useful tool for detection of HbF leverl in clinic and research.更多还原