Kulisek E S, Holm S E, Johnston K H
Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans 70112.
Anal Biochem. 1989 Feb 15;177(1):78-84. doi: 10.1016/0003-2697(89)90017-1.
A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.
已开发出一种直接固相显色测定法，用于检测由结合在硝酸纤维素上的纤溶酶原激活剂相互作用产生的纤溶酶（EC 3.4.21.7），该方法使用纤溶酶特异性三肽底物H-D-缬氨酰-亮氨酰-赖氨酸-对硝基苯胺。纤溶酶裂解赖氨酸-对硝基苯胺所释放的对硝基苯胺被衍生化为重氮盐，随后原位偶联到N-1-萘基乙二胺，在纤溶酶原激活剂位点形成强烈红色的重氮氨基化合物。该方法不仅用于测定粗细菌上清液中的链球菌纤溶酶原激活剂链激酶，还用于检测单个细菌菌落分泌的链激酶。此外，该固相测定法用于鉴定对链激酶具有特异性的单克隆抗体，这些抗体可抑制链激酶对人纤溶酶原的激活。该方法还允许对纤溶酶原激活剂进行同时的免疫学和生化鉴定，从而进行明确的比较观察。该测定法是定量的，对纳克量的激活剂敏感，与可溶性测定法得到的结果相当。该方法也可能适用于检测其他纤溶酶原激活剂，如组织纤溶酶原激活剂、尿激酶和葡萄球菌激酶，还适用于检测可裂解用对硝基苯胺衍生化的其他底物的固定化蛋白酶。该测定法中使用的试剂价格便宜且易于制备。
