消减免疫法制备抗HL60、HL60/ADR表面抗原差异抗体

Production of discrepant monoclonal antibody against HL60 and HL60/ADR by SI technique

目的:建立可特异识别HL60和HL60/ADR细胞膜表面差异蛋白的抗体库,制备单克隆抗体并研究其生物学活性。方法:通过Cp诱导的消减免疫法免疫BALB/c小鼠,采用传统杂交瘤技术制备可特异识别两种细胞差异膜蛋白的单克隆抗体;采用FACS和激光共聚焦显微镜鉴定其和两种靶细胞结合的特异性和差异性。结果:获得51株候选的差异抗体,成功筛选并纯化其中一株单抗(5F6)。5F6与可特异稳定的识别HL60/ADR细胞,结合率扣除本底为68.2%,而该抗体与HL60细胞的结合率为17%。结论:SI结合差异筛选的方法是制备差异抗体便捷可行的方法,所获得的单克隆抗体与HL60和HL60/ADR细胞膜蛋白结合有特异性和差异性,实验中所获得的单抗是寻找肿瘤耐药的新靶点和发现肿瘤耐药新机制的工具,为研究这些问题开拓了新思路。

Objective： To prepare and characterize specific and discrepant mouse hyhfidoma antibodies on membrane of HL60 and HL60/ADR cell lines. Methods： BALB/c mice were immunized by subtractive immunization induced Cp（ Cyclophosphamide）. McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM. Results：51 candidates and discrepant antibodies were found, and one of them （5F6） was purified and identified. Conclusion： Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant MeAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR, which is a significative and potential method in the research and target therapy associated drug-resistance.