抗血管性血友病因子单克隆抗体的制备及其初步鉴定  

Preparation and Characterization of Monoclonal AntibodyAgainst Human Von Willebrand Factor

作　　者：邵晓雁[1] 沈飞[1] 邵波静[1] 李佩霞[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所血栓与止血研究室,苏州215006

出　　处：《苏州大学学报（医学版）》2003年第2期137-141,共5页Suzhou University Journal of Medical Science

摘　　要：目的　研制抗人血管性血友病因子 (vWF)单克隆抗体 (McAb) ,鉴定其各种生物学特性。方法　Ⅷ因子浓缩物经Sepharose 4B凝胶柱提纯出vWF蛋白 ,免疫Balb/c小鼠 ,按常规方法融合并经间接ELISA方法筛选 ,制备稳定分泌抗vWFMcAb的细胞株 ,并对McAb的亚型、免疫活性及组织特异性等进行鉴定 ,运用放射免疫方法等与单抗SZ - 2 9,SZ - 34进行比较。结果　分离纯化出vWF ,vWF抗原含量为 12 0IU/mg ,并成功制备了一株抗人血管性血友病因子McAb ,暂命名为 4E10。琼脂糖双扩散鉴定为IgG1类 ,ELISA法测定其腹水效价为 5× 10 -5。放射免疫分析证实McAb4E10能特异的与血浆中vWF蛋白结合 ,应用间接免疫荧光法显示出与人脐静脉内皮细胞内vWF所特有的颗粒型荧光反应。流式细胞仪及免疫组化法测定表明McAb4E10与人脐静脉内皮细胞反应呈阳性 ,与外周血红细胞、白细胞、未活化的血小板不反应 ,而与其他组织细胞没有反应。放射免疫测定证实McAb4E10与SZ - 2 9,SZ - 34识别vWF蛋白上不同的结构。还原条件下Westernblot法证实McAb4E10和SZ -2 9都能识别相对分子量为 2 2 5kD和 180kD左右的单链蛋白多肽。结论　制备出了抗人血管性血友病因子单克隆抗体 4E10 (McAb4E10 ) ,它能特异识别血浆及内皮细胞的vWF 。Objective To prepare monoclonal antibody (McAb) against Human Von Willebrand Factor. Methods vWF was purified from Factor Ⅷ concentrates. Balb/c mice were immunized by intraperitoneal and subcutaneous injection of human vWF. After fusing and screening, the anti-vWF McAb's characterizations were determined and compared with monoclonal antibody SZ-29 and SZ-34 by radioimmunoassy. Results A marine McAb4E10 was produced, which raised aganist von Willebrand factor protein, belonged to IgG1 subclass and had no reaction with fibronectin and fibrinogen. It was confirmed by immunofluorence method that McAb4E10 stained fixed cultured Human Umbilical Vascular Endothelial Cells (HUVECS) with a 'dotted' staining pattern. McAb4E10 recognized all small and large blood vessels wall cells in all tissue sections detected by immunohistochemical staining, showing that antigen was expressed in endothelial cells lining both arteries and veins. And McAb4E10 also reacted selectively with HUVECS, while human leucocytes, red blood cells and inactive platelets remained negative. Western blotting demonstrated that McAb reacted with the antigens whose molecular weight were 225kD and 180kD under reducing condition respectively. And radioimmunoassy confirmed that McAb4E10?SZ-29 and SZ-34 recognized different domain of vWF protein. Conclusion McAb4E10 could provide us an effective and reliable analytic method in vascular related diseases during several pathologic conditions.

关 键 词：血管性血友病因子 单克隆抗体 制备 生物学特性 间接ELISA 方法筛选 放射免疫方法 

分 类 号：R554.1[医药卫生—血液循环系统疾病] R392－33[医药卫生—内科学]