阻断血管性血友病因子与胶原结合的抗人vWF—A3单抗的鉴定和功能研究  

Characterization and functional studies of vWF A3 domain monoclonai antibodies that inhibit binding of vWF to collagen

作　　者：赵益明[1,2] 董宁征[1,2] 沈飞[1,2] 谢丽倩[1,2] 阮长耿[1,2] 

机构地区：[1]苏州大学附属第一医院、江苏省血液研究所,215006 [2]卫生部血栓与止血重点实验室

出　　处：《中华血液学杂志》2008年第3期171-174,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金（30670904）,江苏省卫生厅“135”医学工程（SK200206）

摘　　要：目的制备阻断血管性血友病因子（von Willebrand因子）A3区（vWF-A3）与胶原结合的抗vWF-A3单抗，并对其进行生化鉴定和功能研究。方法用重组vWF-A3蛋白免疫BALB／c小鼠，经标准的方法制备单抗。用ELISA法鉴定单抗特异性，经vWF胶原结合抑制试验筛选获得抑制性单抗，用免疫印迹技术确定单抗与重组（r）vWF-A3和还原性人vWF识别的抗原分子。经胶原结合试验确定单抗对vWF与胶原结合的影响。结果获得一组共30株抗vWF-A3单抗，其中两株确定为抑制vWF与胶原结合的单抗，分别命名为SZ-123和SZ-125，两株单抗分别与rvWF-A3和vWF强反应，而不与rvWF-A1、A2反应。免疫印迹分析显示SZ-123和SZ-125分别识别相对分子质量为27×10^3的rvWF-A3、250×10^3还原性人vWF单体和170×10^3的vWF酶切片段。单抗SZ-123和SZ-125不仅剂量依赖性地抑制纯化的人血浆vWF与人胎盘和牛皮肤Ⅲ型胶原结合，而且分别抑制人、猕猴或Beagle犬血浆vWF与人胎盘和牛皮肤Ⅲ型胶原结合。结论抗vWF-A3单抗SZ-123和SZ-125是两株抑制性单抗，有望成为具有治疗潜力的抗血栓药物。Objective To prepare anti-yon Willebrand factor A3 （vWF-A3） domain monoclonal antibodies（mAbs） which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions. Methods BALB/c mice were immunized with purified recombinant vWF-A3 protein （rvWF- A3）. Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen Ⅲ was studied with collagen binding inhibition test. Results A group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ- 123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 × 10^3 band of rvWF-A3 and 2 reduced human vWF bands at 250 × 10^3 and 170 × 10^3. SZ-123 and SZ-125 not only inhi-bited the binding of purified human vWF （ 1.5 and 3.0 μg/ml） to human type Ⅲ collagen and to calf skin collagen Ⅲ in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens. Conclusion SZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.

关 键 词：von WILLEBRAND因子 胶原 单克隆抗体 

分 类 号：R686[医药卫生—骨科学]