Automation of the homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum: application to the determination of phenytoin and phenobarbital in serum.

A substrate labeled fluorescent immunoassay procedure for serum theophylline has been automated. The method uses the Ames (SLFIA) substrate-labeled fluorescent immunoassay reagents and Technicon AutoAnalyzer equipment. Forty-seven sera analyzed for theophylline by the automated Ames SLFIA procedure and by the Syva EMIT method yielded the regression equation y (Ames) = 0.92x (Syva) + 0.47, SE, sy.x = 1.67, and correlation coefficient r = 0.934. Recoveries ranged from 94.6 to 100% by the automated SLFIA method. Within-run mean and SD for a sample in and above the therapeutic range was 14.5 +/- 0.37 and 22.7 +/- 1.0 mg/L, respectively. Day-to-day reproducibility was 13.4 +/- 0.92 and 24.7 +/- 1.71 mg/L for a sample in and above the therapeutic range, respectively. The automated SLFIA procedure analyzes serum for theophylline at the rate of 40 per hour and gives results comparable to the Syva EMIT method. The same equipment was also used for the SLFIA determination of phenytoin and phenobarbital and yielded reliable results. The manifold and equipment were the same. The only change was the substitution of the Ames phenytoin and phenobarbital reagents for the theophylline reagent.