抗重组人红细胞生成素单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody against Recombinant Human Erythropoietin

目的 :获得能特异性识别人红细胞生成素 (hEPO)的单克隆抗体 (McAb)。方法 :用重组人红细胞生成素 (rHuEPO)作抗原免疫Balb/c小鼠 ,以细胞融合、酶联免疫吸附实验 (ELISA)筛选和克隆化技术获得抗hEPO的杂交瘤细胞株。用生物学方法鉴定杂交瘤细胞 ,用免疫学方法鉴定McAb的特异性。结果 :获得 3株稳定分泌抗hEPO McAb的杂交瘤细胞株 (1H7、4G11、1B4 )。培养上清的ELISA效价分别为 :1∶10 2 4、1∶5 12、1∶5 12 ;腹水效价为 :1× 10 7、1× 10 6、2× 10 4。阻断法表明 1H7识别的hEPO上的抗原决定簇和 4G11、1B4识别的不同。结论 :成功建立了 3株稳定分泌抗hEPO McAb的杂交瘤细胞株 ,它们分别识别hEPO上 2个不同的抗原位点 ,有望作为EPO定量检测的特异性抗体。

AIM:To obtain monoclonal antibody (McAb) which can specificly identify human erythropoietin(hEPO).METHOD:Spleen cells from Balb/c mice immunized with recombinant human erythropoietin (rHuEPO) protien were fused with Sp2/0 myeloma cells.The specific McAb were screened by ELISA and identified by western blot.RESULT:Four hybridoma cell lines (3E_ 9,5A_ 6,4G_ 12and 2H_ 2) secreting specific McAb to human cytomegalo-virus (HCMV) were selected and determinded to recognize different epitops on gp52 protein.The titers of the four clones were as the follows:1∶2 048,1∶1 024,1∶1 024,1∶512(supernant);1×10 -7,1×10 -7,1×10 -6,2×10 -6 (AF).CONCLUSION:McAbs 2H_ 2 recognize one epitope on rhCMV gp52 protien,whereas 3E_ 9,5A_ 6and 4G_ 12 recognize another.