一种灵敏的体外溶栓活性检测方法——薄层聚丙烯酰胺纤维蛋白胶板法的建立

Establishment of a sensitive method, thin polyacrylamide fibrin plate assay for detection of fibrinolysis activity in vitro

目的 建立一种用于体外溶栓活性精确定量分析和高通量溶栓药物筛选鉴定的方法。方法 使用聚丙烯酰胺代替琼脂糖作载体 ,用平板电泳制胶模具代替玻璃或塑料平皿制作可控制厚度的、均匀的薄层聚丙烯酰胺纤维蛋白胶板 ,用染色剂指示纤维蛋白降解情况。结果 最适条件为胶板胶含 0 5mg·mL-1纤维蛋白原和 10 %丙烯酰胺 ,加样后 37℃保湿温育 8～ 12h ,用考马斯亮蓝染色 ,观察纤维蛋白降解后产生的透明圈。本方法最低可检测出 2 5ng蚓激酶的活性 ;蚓激酶浓度在 0 0 1～ 5mg·mL-1时 ,浓度对数与其所产生的透明圈两垂直直径算术平均值平方的对数成良好的线性关系 (r =0 9997) ;重复性试验RSD为 3 83% (n =5 )。

OBJECTIVE: To establish a method for detecting and analyzing fibrinolysis activity in vitro and high throughput screening fibrinolysis drugs. METHODS: Replaced agar with polyacrylamide and glass or plastics plate with casting gel instrument of plate electrophoresis, the controllable thickness and homogeneous thin fibrin plate were fabricated. The fibrino lusis of fibrin was indicated through protein staining. RESULTS: The optimum condition was ascertained that the plate contained 0.5 mg&middotmL-1 fibrin and 10% acrylamide. The thin polyacrylamide plate was stained with Coomassie brilliant blue after being incubated for 8-12 h at 37°C in wet. Fibrinolysis activity of 25 ng lumbrokinase was detected in minimum. Within a range of 0.01-5 mg&middotmL -1 of lumbrokinase, a good linearity(r = 0.999 7) was obtained between the logarithm of lumbrokinase concentration and the logarithm of diameter's square of transparent spots. The RSD was 3.83% (n = 5) in reproducibility test. CONCLUSION: The thin polyacrylamide fibrin plate assay was suitable for detecting fibrinolysis activity in vitro.