重组载体免疫小鼠制备抗人CD33单克隆抗体

Preparation of monoclonal antibody against human CD33 by immunizing mice with recombinant vector

目的利用基因免疫方法制备抗人CD33单克隆抗体(mAb),并对抗体的特性及临床应用进行初步研究。方法真核载体pcDNA3.1(+)/CD33免疫小鼠,通过杂交瘤技术制备抗人CD33 mAb,并进行性能评价和临床验证。结果成功获得1株分泌小鼠抗人CD33单克隆抗体的杂交瘤细胞株,克隆号命名为HI33a,抗体亚类为IgG2a,κ。流式细胞术检测其可与髓系细胞系反应,不与淋巴系细胞系反应;可完全抑制同类进口抗体与HL-60细胞的结合。Western blot证实可识别相对分子量(Mr)为67000的HL-60细胞膜蛋白(符合CD33蛋白分子的特征)。标记藻红蛋白(PE)后,作为抗体试剂与进口试剂对比,准确度、线性、精密度等评估均符合行业标准。558例临床骨髓样本检测结果与对比试剂高度一致。结论制备了可持续分泌抗人CD33 mAb的杂交瘤细胞株。

Objective To prepare a monoclonal antibody(mAb)against human CD33 by immunizing mice with recombinant vector and analyze its characteristics and clinical application.Methods The eukaryotic expression vector pcDNA3.1(+)/CD33 was constructed and used to immunize mice.The mouse monoclonal antibody against human CD33 was then harvested using the hybridoma technique.Its properties were evaluated and the clinical performance was validated.Results One hybridoma cell line capable of secreting mouse anti-human CD33 monoclonal antibody was successfully obtained,which was named HI33a for clone identification with a subclass of IgG2a,κ.Flow Cytometry analysis revealed that the antibody could stain myeloid cell lines but not lymphoid cell lines,and it could inhibit the binding of similar imported antibodies with HL-60 cells competitively.Western blotting verified that it could bind a Mr 67000 membrane protein extracted from HL-60 cells,which was a strong indication of the characteristics of CD33 protein molecule.Labeled with PE fluorescein,CD33-PE was tested as an antibody reagent in comparison with other similar imported products.Its overall performance including the accuracy,linearity,and precision all met the industrial standard.Further clinical evaluation of 558 bone marrow samples showed that the results were highly consistent with those by the imported reagents used as controls.Conclusion A hybridoma cell line stably secreting anti-human CD33 mAb was prepared.