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目的 :研制抗人促红细胞生成素的特异性单克隆抗体 (mAb) ,对其生物学特性进行初步鉴定 ,并用于转基因羊乳中重组人促红细胞生成素 (rhEPO)的提纯。方法 :以自制的rhEPO粗品免疫BALB/c小鼠 ,制备抗rhEPO的mAb。以Westernblot和间接ELISA法 ,对所获mAb进行初步鉴定。以纯化的mAb同预活化的Sepharose 4B偶联 ,制得mAb亲和层析柱 ,并用于转基因羊乳中rhEPO的提纯。结果 :获得两株可分泌特异性抗rhEPO的mAb的杂交瘤细胞株 (1E7和 2E6 )。Ig亚类 (型 )的鉴定分别为IgG1和IgG2b ,轻链均为κ型。用Westernblot证实 ,获得的mAb可特异性地识别rhEPO。用自制的免疫亲和层析柱用于转基因羊乳中rhEPO的提纯 ,具有很好的吸附作用 ,回收率达 70 %。结论 :成功地获得两株可分泌特异性抗rhEPO的mAb的杂交瘤细胞。用自制的免疫亲和层析柱提纯转基因羊奶中的rhEPO具有较高的吸附效率
Objective: To develop a specific monoclonal antibody (mAb) against human erythropoietin and preliminary identification of its biological characteristics, and to purify recombinant human erythropoietin (rhEPO) in transgenic goat milk. Methods: BALB / c mice were immunized with homemade crude rhEPO to prepare anti-rhEPO mAb. The obtained mAbs were identified preliminarily by Western blot and indirect ELISA. A mAb affinity column was prepared by coupling purified mAb with pre-activated Sepharose 4B and used for the purification of rhEPO in transgenic goat’s milk. Results: Two hybridoma cell lines secreting mAbs specific for rhEPO (1E7 and 2E6) were obtained. Ig subclasses (type) were identified as IgG1 and IgG2b, respectively, and the light chains were all κ. Western blot confirmed that the mAb obtained specifically recognizes rhEPO. The self-made immunoaffinity column was used for the purification of rhEPO in transgenic goat’s milk with good adsorption and the recovery rate reached 70%. Conclusion: Two hybridoma cells secreting mAbs specific for rhEPO were successfully obtained. Purification of rhEPO in transgenic goat milk with self-made immunoaffinity chromatography column has higher adsorption efficiency