TIME-RESOLVED FLUOROIMMUNOASSAY FOR THE DETERMINATION OF LISINOPRIL AND ENALAPRILAT IN HUMAN SERUM

A solid-phase immunoassay with detection based on time-resolved fluore scence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by couplin g lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and e nalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a l imited quantity of primary antibody bound via sheep anti-rabbit globul in to the wells of microtitration strips. At the end of the first incu bation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with t he biotin already bound to the solid-phase antibody. After a washing s tep. the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence fro m each sample is inversely proportional to the concentration of the dr ug in the sample. The assay demonstrates good accuracy, reproducibilit y and specificity at serum concentrations down to 0.5 ng ml(-1). Howev er. the useful concentration range of TR-FIA is much narrower than tha t obtained by double antibody radioimmunoassay (RIA).