Yang Yong-chang, Lai Chun-ning, Wang Yan, Shen Bei-fen, Li Yan
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Sep;20(5):563-7.
To develop a Fab antibody against platelet GPIIb/IIIa.
A mouse anti-human GPIIb/IIIa monoclonal antibody (mAb) P140 was selected by the indirect immunofluorescence assay (IFA) and platelet aggregation inhibition test. The Fd and light chain genes were cloned from the hybridoma cells secreting the mAb P140. The genes of P140 Fab were inserted into plasmid p3MH to construct recombinant expression plasmid p3MH/P140kappa-Fd. After digestion with the restriction enzyme, the recombinant plasmid was transformed into E.coli XLI-Blue. The expressed product was purified by TALON metal affinity resin. Purified P140 Fab was characterized by SDS-PAGE, ELISA, Western blot and platelet aggregation inhibition test.
SDS-PAGE analysis showed that relative molecular mass (M(r)) of P140 Fab was 47x10(3). The results of ELISA, Western blot and platelet aggragation inhibition test indicated that P140 Fab could specifically bind to platelet and inhibit platelet aggragation in dose-dependent manner. The mean value of IC(50) was 16.85 mg/L.
A soluble anti-platelet GPIIb/IIIa antibody P140 Fab was prepared successfully, which lays the foundation for further clinical application.
制备抗血小板糖蛋白IIb/IIIa的Fab抗体。
通过间接免疫荧光法（IFA）和血小板聚集抑制试验筛选出小鼠抗人GPIIb/IIIa单克隆抗体（mAb）P140。从分泌mAb P140的杂交瘤细胞中克隆Fd和轻链基因。将P140 Fab的基因插入质粒p3MH中构建重组表达质粒p3MH/P140kappa-Fd。用限制性内切酶消化后，将重组质粒转化到大肠杆菌XL1-Blue中。表达产物用TALON金属亲和树脂纯化。通过SDS-PAGE、ELISA、Western印迹和血小板聚集抑制试验对纯化的P140 Fab进行鉴定。
SDS-PAGE分析显示P140 Fab的相对分子质量（M(r)）为47×10(3)。ELISA、Western印迹和血小板聚集抑制试验结果表明，P140 Fab能特异性结合血小板并以剂量依赖方式抑制血小板聚集。IC(50)的平均值为16.85 mg/L。
成功制备了可溶性抗血小板GPIIb/IIIa抗体P140 Fab，为进一步临床应用奠定了基础。
