tPA体外纤溶活性及半定量检测方法的确立

Establishment of a Modified Procedure for Assay of In vitro Fibrinolysis Activity and Semi-quantity of t-PA

在原有的纤维蛋白平板溶圈法 (fibrin agarose plate assay,FAPA)检测系统中引入纤溶酶原 (plasminogen) ,使该法检测组织型纤溶酶原激活剂 (tissue plasminogen activator,t- PA)的灵敏度提高 30倍以上 ,达到 0 .0 1ng(约0 .0 0 1IU )水平 ;并对不同反应时间、纤维蛋白含量、琼脂糖凝胶厚度下的溶圈结果进行对比研究。结果表明 ,37℃温育18～ 2 4h可以得到较满意结果 ;其他因素虽不同程度地影响溶圈出现时间及大小 ,但对其溶圈梯度及标准曲线无明显影响。从而确立了以 FAPA为基础的快速、灵敏、可靠的重组 t- PA体外纤溶活性半定量检测方法。

Fibrin agarose plate assay(FAPA) is a kind of procedure detecting the in vitro fibrinolysis activity of plasminogen activators(PAs) such as urokinase(UK) and tissue-type plasminogen activator(t-PA). Several important parameters in the protocol were not delimited in extant references, and, the sensitivity of the method is low. To solve the problems mentioned above, a modified FAPA was re-erected by regulating the parameters. The sensitivity of FAPA was raised by 30-folds after the introduction of plasminogen as an additional supplement. The main parameters, including the time of incubation, the concentration of fibrin in agarose gel, the thickness of the agarose plate, were investigated and standardized. The modified FAPA provides a convenient method for detection of low activity PAs and their mutants.