Wong R C, Burd J F, Carrico R J, Buckler R T, Thoma J, Boguslaski R C
Clin Chem. 1979 May;25(5):686-91.
A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.
一种均相底物标记荧光免疫分析法已应用于测定人血清中苯妥英的浓度。我们将一种荧光酶底物半乳糖基伞形酮共价连接到苯妥英的一种衍生物上。在分析条件下，这种药物 - 底物共轭物无荧光，但在细菌β - 半乳糖苷酶催化水解后会变得有荧光。当苯妥英抗体与药物 - 底物共轭物结合时，它作为酶底物是无活性的。向竞争性结合反应中加入苯妥英可解除失活，产生的荧光与苯妥英浓度成正比。我们通过将用该技术获得的苯妥英值与通过气相色谱法和酶免疫分析法（EMIT）获得的值进行比较，验证了荧光免疫分析法。这三种方法相关性良好。苯妥英的主要代谢物5 - （对羟基苯基） - 5 - 苯基乙内酰脲以及血清中预期浓度的其他药物对该分析无影响。荧光免疫分析法操作快速简便，每次检测仅需2微升血清样本。
