【摘要】 目的：拟研制能特异性识别血浆中人血管性血友病因子前导肽（VWF propeptide,VWFpp）的单克隆抗体，并且通过获得的抗VWFpp的单克隆抗体利用双抗体夹心法建立快速而可靠的检测血浆中VWFpp抗原的方法。方法：使用真核表达的重组人VWFpp(D1、D2区)蛋白作为免疫原，以常规方法免疫BALB/c小鼠，获取融合细胞的生长克隆，经过筛选和鉴定后，挑选能特异性分泌VWFpp单克隆抗体的杂交瘤细胞株，然后应用ELISA双抗体夹心法构建VWFpp抗原检测试剂盒，用于人血浆VWFpp的测定，对收集的12例行骨髓移植的白血病患者血浆进行VWFpp抗原水平动态检测。结果：最终获得两株可持续传代并分泌VWFpp单克隆抗体的杂交瘤细胞株，并分别命名为SZ175和SZ176。经ELISA和Western blot鉴定，抗体能特异性识别VWFpp而不能识别成熟的VWF（不含前导肽）。利用双抗体夹心ELISA的原理，将单克隆抗体SZ175和SZ176成功制备成检测VWFpp抗原的试剂盒，动态检测白血病患者骨髓移植前后血浆中的VWFpp水平，结果显示移植后较移植前血浆中VWFpp水平有明显上升。结论：本研究成功制备两株抗VWFpp单克隆抗体，并且建立了VWFpp抗原双抗体夹心检测试剂盒，为进一步研究VWFpp的生物学功能以及血管性血友病临床诊断分型和内皮损伤相关疾病的预后监测提供了有力的工具。更多还原

【Abstract】 Objective: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor(VWF) propeptide(VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. Methods: The recombinant human VWFpp(D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. Results: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn’t recognize mature VWF(without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. Conclusion: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease(VWD), and the prognostic monitoring of endothelial injury-related diseases.更多还原