Zhou Chun-gang, Tian Xiao-jing, Ju Song-guang, Xu Ying, Yu Ge-hua, Zhang Xue-guang, Gu Zong-jiang
Department of Immunology, School of Medicine, Soochow University, Suzhou 215007, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Sep;23(9):850-2.
To Prepare three functional monoclonal antibodies(mAbs) against human FL molecule and analyze their bioactivity.
The cell line L929-FL transfected with human FL gene was used as immunogen. The hybridomas secreting the antibodies against human FL were obtained by fusing splenoecytes from the immunized mice with murine myeloma cells(Sp2/0). Their subclasses were analyzed using fast-strip method. The monoclonal antibodies were produced in mouse peritoneal cavity and purified by Protein G affinity chromatography. The inhibitory effect of mAbs against FL on leukemia cell lines U937 and HL-60 was detected by MTT. The apoptosis of U937 and HL-60 cells stained by annexin-V/PI was determined by FCM.
Three hybridomas named 3C2, 3C6 and 8D10 were successfully obtained, which secreted monoclonal antibodies against human FL molecule stably. Their subclasses were the mouse IgG2a with kappa light chains. The three monoclonal antibodies recognized the FL molecule on U937 and HL-60 cells that also coexpressed Flt3 molecule. When U937 and HL-60 cells were cultured in presence of 3C2, 3C6 and 8D10, their proliferation was reduced as compared to that in control in MTT assay(P < 0.05). The analysis of annexin-V/PI binding to U937 and HL-60 cells by FCM showed the mAbs had the apoptotogenic activity of the monoclonal antibodies against human FL molecule.
3C2, 3C6 and 8D10 are three funtional monoclonal antibodies against human FL molecule. They may be of some value in the study of the roles of FL/Flt3 interaction in leukemia pathogenesis.
制备三种抗人FL分子的功能性单克隆抗体（mAb）并分析其生物活性。
将转染人FL基因的细胞系L929-FL用作免疫原。通过将免疫小鼠的脾细胞与鼠骨髓瘤细胞（Sp2/0）融合，获得分泌抗人FL抗体的杂交瘤。使用快速检测法分析其亚类。单克隆抗体在小鼠腹腔中产生，并通过蛋白G亲和层析纯化。采用MTT法检测mAb对白血病细胞系U937和HL-60的抑制作用。通过流式细胞术（FCM）测定annexin-V/PI染色的U937和HL-60细胞的凋亡情况。
成功获得了三种名为3C2、3C6和8D10的杂交瘤，它们稳定分泌抗人FL分子的单克隆抗体。其亚类为带有κ轻链的小鼠IgG2a。这三种单克隆抗体识别U937和HL-60细胞上同时共表达Flt3分子的FL分子。在MTT试验中，当U937和HL-60细胞在3C2、3C6和8D10存在的情况下培养时，与对照组相比其增殖受到抑制（P<0.05）。通过FCM分析annexin-V/PI与U937和HL-60细胞的结合情况表明，这些单克隆抗体具有抗人FL分子单克隆抗体的促凋亡活性。
3C2、3C6和8D10是三种抗人FL分子的功能性单克隆抗体。它们在FL/Flt3相互作用在白血病发病机制中的作用研究中可能具有一定价值。
