人红细胞生成素单克隆抗体的制备、鉴定及应用研究

Preparation, Characterization and Application of Monoclonal Antibodies Against Human Erythropoietin

用rhEPO作为抗原,免疫BALB/c小鼠,取其脾细胞与X63Ag8.653小鼠骨髓瘤细胞融合,用碱性PAGE方法进一步分离并纯化的rhEPO,包被PVC板,对杂交瘤用ELISA方法进行筛选,获得两株稳定分泌抗hEPO单抗的杂交瘤细胞株。经鉴定分别属于IgG_1、IgG_(2b),轻链均为k链,K_d分别为5.53×10^(-10)mol/L和1.34×10^(-10)mol/L。用Western blot方法证明两者对hEPO具有高度的专一性,能特异地识别rhEPO和尿源hEPO。所制备单抗可作为亲和层析的配体,用于再生障碍性贫血病人尿中EPO及哺乳类工程细胞所表达的hEPO的分离、纯化,并可用于hEPO的定量检测。

Two monoclonal antibodies (Mabs) against hEPO are developed. We used rhEPO as antigen for immunization. The spleen cells of the immunized mice were isolated and fused with myeloma cells X63Ag8. 653. Hybridoma cell supernatant were screened for the production of anti-hEPO Mabs with ELISA, using purified rhEPO absorbed to 96-well PVC plates. One Mab is IgGl with Kd of 5. 53x10-10mol/L, while the other is IgG2b with Kd of 1. 34 x 10-10mol/L. Both Mabs can recognize rhEPO and human urine EPO of patients with aplastic anemia. They are proved to be specific to rhEPO by ELISA and Western- blot analysis. They can be used as ligands of immunoaffinity chromatography for the purification of hEPO from urine of aplastic anemia patients or the supernatant media of the transfected mammalian cells.