两株鼠抗人GL50单克隆抗体的制备及其生物学特性的初步研究

Anti-GL50 McAbs and their biological functions

目的 研制鼠抗人GL5 0分子单克隆抗体并对其生物学特性进行初步研究。方法 以天然高表达人GL5 0分子的Daudi细胞为免疫原 ,人GL5 0 L92 9转基因细胞为目的单克隆抗体(McAb)的筛选细胞 ,采用B淋巴细胞杂交瘤技术 ,获得分泌特异性鼠抗人GL5 0分子McAb的杂交瘤细胞株 ;免疫荧光标记和流式细胞术分析McAb识别GL5 0的表达 ;Westernblot检测抗体对特异性细胞膜蛋白的识别 ;台盼蓝 (Trypanblue)染色细胞计数法和MTT法检测McAb对Daudi细胞体外生长的抑制作用 ;3 H TdR法检测抗体对GL5 0 L转基因细胞介导的活化T细胞体外增殖的效应。结果 成功制备 2株分泌特异性抗人GL5 0抗体的杂交瘤细胞株 12B11和 11C4 ;两者皆为IgG2a亚型 ;腹水效价皆在 1∶10 0 0以上 ;12B11、11C4特异性地识别GL5 0分子。 11C4McAb可以明显抑制Daudi细胞在体外的生长 ,并可抑制GL5 0 L转基因细胞介导的活化T淋巴细胞的体外增殖效应。结论 成功获得了 2株特异性鼠抗人GL5 0抗体 ,其中 11C4McAb具有诱导表达GL5 0分子的B淋巴瘤细胞Daudi的体外生长抑制作用 ;在T淋巴细胞体外增殖反应中发挥了重要的调节作用。

Objective To prepare monoclonal antibodies against human GL50 and to identify their biological functions. Methods Splenocytes were isolated from the mice immunized with Daudi on which GL50 is highly expressed, and then hybridized with murine plasmocytoma cells Sp2/0. The hybridoma specifically secreating antibody against human GL50 molecule were obtained by immunofluorescence staining and cytometry analysis through human GL50 transfectants. The ascetics induced were purified with protein G affinity chromatography. Isotope was verified by fast isotope strip. The specific recognition of McAb with the membrane protein GL50 was determined by indirect immunofluorescence staining and Western blot analysis. The expression pattern of GL50 molecule on different cells and cell lines was explored by flow cytometry. Cell counting, Trypan blue staining and MTT assay were employed to study the biological effects of McAbs on the proliferation of Daudi in vitro. Results Two hybridoma cell lines (12B11, 11C4) specifically secreted IgG2a against GL50. GL50 was predominately expressed on B lymphocyte cell lines. McAb 11C4 inhibited the proliferation of Daudi cells and reversed the stimulation effect of GL50-L cells on the proliferation of preactivated T cells. Conclusion McAbs 11C4 and 12B11 were specific anti-GL50 McAbs with high affinity to its receptor. McAb 11C4 manifested certain role in inducing the proliferation inhibitory effect of Daudi cells and preactivated T cells in vitro. They would be of significant value in basic research and clinical application.