Substrate-labeled fluorescent immunoassay for phenytoin in human serum
| Autor: | John F. Burd, Robert J. Carrico, Robert T. Buckler, Robert C. Boguslaski, R C Wong, J Thoma |
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| Rok vydání: | 1979 |
| Předmět: |
Phenytoin
chemistry.chemical_classification Chromatography medicine.diagnostic_test Chemistry Metabolite digestive oral and skin physiology Biochemistry (medical) Clinical Biochemistry Substrate (chemistry) Fluorescence nervous system diseases stomatognathic diseases chemistry.chemical_compound Hydrolysis Enzyme Immunoassay otorhinolaryngologic diseases medicine heterocyclic compounds medicine.drug Conjugate |
| Zdroj: | Clinical Chemistry. 25:686-691 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1093/clinchem/25.5.686 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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