抗凝血因子ⅧC2区单克隆抗体的制备及功能研究

Development of a monoclonal antibody to factor vm C2 domain and its functional study

目的 制备并鉴定抗凝血因子Ⅷ（FⅧ）C2区单克隆抗体（单抗），研究其对FⅧ活性的影响。方法体外表达rFⅧC2区蛋白，免疫BALB／c小鼠制备鼠源性单抗。不同剂量的单抗与正常人新鲜混合血浆孵育后测定FⅧ活性及活化部分凝血活酶时间（APTT）等；ELISA法测定该单抗对rhFⅧ与血管性血友病因子（vWF）、磷脂酰丝氨酸（PS）及血小板结合实验的影响。结果获得一株抗FⅧC2区单抗，命名为SZ-132。与血浆孵育后呈剂量依赖方式抑制FⅧ活性，APTT明显延长；抗体浓度超过25μg/ml时，FⅧ活性为0；该抗体能够抑制rhFⅧ-vWF、rhFⅧ-PW及rhFⅧ与血小板的结合。结论SZ-132是一株特异性针对FⅧC2区的抑制性单抗。

Objective To develop a monoclonal antibody （mAb） directed to Fvm C2 domain and investigate its effect on FⅧ activity. Methods FⅧ C2 protein was expressed in E. coli and purified. A murine antihuman FⅧ C2 domain mAb SZ-132 was developed by standard hybridoma technology and characterized. In coagulation assays, different concentrations of SZ-132 were incubated with freshly collected pooled human plasma and the residual activity of FⅧ and activated partial thromboplastin time （AFTT） were determined. The effects of SZ-132 on rhFⅧ binding to purified human vWF, phosphatidylserine （PS） and platelets were assessed by enzyme linked immunosorbent assays （ELISA）. Results SZ-132 could inhibit FⅧ procoagulant activity in a dose-dependent manner within the concentrations of 0 - 25μg/ml and the FⅧ activity was completely inhibited on above 25μg/ml. It could also prevent rhFVm from binding to vWF, PS and platelets. Conclusions SZ-132 is a neutralizing mAb against Fvm C2 domain and can inhibit FⅧ procoagulant activity by preventing FⅧ from binding to vWF and PS.