Clinical Chemistry
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Clinical Chemistry 27: 22-26, 1981;
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Clinical Chemistry, Vol 27, 22-26, Copyright © 1981 by American Association for Clinical Chemistry

Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum

TM Li, JL Benovic, RT Buckler and JF Burd

A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta- galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta- galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.




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Copyright © 1981 by the American Association for Clinical Chemistry.