Yuan A S, Gilbert J D
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
J Pharm Biomed Anal. 1996 May;14(7):773-81. doi: 10.1016/0731-7085(95)01718-6.
A solid-phase immunoassay with detection based on time-resolved fluorescence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to the wells of microtitration strips. At the end of the first incubation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with the biotin already bound to the solid-phase antibody. After a washing step, the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence from each sample is inversely proportional to the concentration of the drug in the sample. The assay demonstrates good accuracy, reproducibility and specificity at serum concentrations down to 0.5 ng ml-1. However, the useful concentration range of TR-FIA is much narrower than that obtained by double antibody radioimmunoassay (RIA).
已开发出一种基于时间分辨荧光（TR-FIA）检测的固相免疫测定法，用于测定人血清中的赖诺普利和依那普利拉。免疫原是通过二氟二硝基苯的两步反应将赖诺普利与牛血清白蛋白偶联制备的。使用了对赖诺普利和依那普利拉均具有特异性的抗血清。该测定法基于竞争性免疫测定原理，其中药物与生物素标记的药物竞争有限量的通过羊抗兔球蛋白结合到微量滴定条孔中的一抗。在第一次孵育结束时，未结合的生物素标记的药物被洗去。在第二步中，铕标记的链霉亲和素（对生物素具有特异性）与已经结合到固相抗体上的生物素反应。经过洗涤步骤后，加入增强溶液使铕离子从标记的链霉亲和素中解离到溶液中。每个样品的荧光与样品中药物的浓度成反比。该测定法在低至0.5 ng/ml的血清浓度下显示出良好的准确性、重现性和特异性。然而，TR-FIA的有效浓度范围比双抗体放射免疫测定法（RIA）获得的范围窄得多。
