Blake D A, Skarstedt M T, Shultz J L, Wilson D P
Clin Chem. 1984 Sep;30(9):1452-6.
In this ligand-binding assay procedure, sensitivity is enhanced by successive generation of enzyme active sites via two zymogens from the blood-coagulation cascade: Factor X and prothrombin. A protease fraction from Russell's viper venom (RVV) acts upon Factor X, initiating a two-step cascade that culminates in generation of thrombin, the activity of which is monitored with a chromogenic substrate. In the model presented here, the analyte of interest, biotin, is covalently coupled to Factor X. In the presence of avidin, a biotin-binding protein, RVV cannot initiate cascade activity; however, the inhibition can be competitively overcome by addition of free biotin to the reaction mixture. In a complete system, the dose-response curve is linear from 20 to 100 nmol of biotin per liter. Such an assay offers improved sensitivity over many radioisotope-independent immunoassay methods, and may be applicable to a wide variety of analytes.
在这种配体结合分析方法中，通过来自血液凝固级联反应的两种酶原（凝血因子X和凝血酶原）连续产生酶活性位点来提高灵敏度。来自锯鳞蝰蛇毒（RVV）的蛋白酶组分作用于凝血因子X，启动一个两步级联反应，最终产生凝血酶，其活性通过显色底物进行监测。在此处呈现的模型中，目标分析物生物素与凝血因子X共价偶联。在存在抗生物素蛋白（一种生物素结合蛋白）的情况下，RVV无法启动级联活性；然而，通过向反应混合物中添加游离生物素可以竞争性地克服这种抑制作用。在完整系统中，剂量反应曲线在每升20至100纳摩尔生物素范围内呈线性。这种分析方法比许多非放射性同位素免疫分析方法具有更高的灵敏度，并且可能适用于多种分析物。
