抗人尿激酶单克隆抗体制备和鉴定

Preparation and characterization of monoclonal antibodies to human urokinase

用人高分子量尿激酶(HMW-UK)为抗原,通过杂交瘤技术获得了两株阳性杂交瘤细胞(2B8、3A2)。采用葡萄球菌 A 蛋白(SPA)亲和层析或 Water's650快速蛋白分离系统纯化抗 UK 单克隆抗体(McAb).3A2McAb 为 IgG 1亚类,特异地识别 HMW-UK 和低分子量 LMW-UK,抑制 UK 活性,表明3A2McAb 所作用的位点为 UK 的 B 链,即活性中心。EIA 测定3A2McAb 与 UK 的亲和常数为8.43×10~8M^(-1)。2B_8McAb 亦为IgG_(?)亚类,特异地识别 HMW-UK 及其氨基端1～135氨基酸片段(ATF),不抑制活性,EIA 测定2B_8McAb 与UK 的亲和常数为2.8×10~9M^(-1)。2B_8McAb 可用于导向溶栓的研究以及 UK 与其受体间反应的研究。

Two hybridoma cell lines,which secret anti—urokinase monoclonal antibodies 2B8、3A2,were obtained by fusing the murine myeloma cell line SP2/0 with the spleen cells from Balb/C mouse immunized with high—molecular weight urokinase(HMW—UK).These antibodies were purified with SPA affinity chromatography or with Water's 650 FPLC.3A2 McAb is an IgG1 that binds selectively to HMW—UK and to the low—molecular weight urokinase LMW—UK),not to the 17000 Mr aminoterminal fragment of the A chain(ATF).3A2 McAb also inhibits the activity of UK.This strongly suggests that 3A2 McAb recognizes B chain of UK.The Kaff of 3A2 McAb is 3.43×10~8M^(-1).2B8 McAb recognizes B chain of UK.2B8 McAb cannot inhibit the activity of UK.The kaff of 2B8 McAb is 2.08×10~9M(-1).2B8McAb may be a useful tool for antibody—targeting thrombolysis and for the study of UK receptor physiology.