Frederick E. Ward

Homogeneous Substrate-Labeled Fluorescent Immunoassay for Carbamazepine

Summary: Carbamazepine is an anticonvulsant drug useful in the management of epilepsy. Because of the narrow therapeutic range, serum carbamazepine monitoring is useful for ensuring adequate drug therapy without toxicity. We report the development of a homogeneous substrate‐labeled fluorescent immunoassay for carbamazepine in human serum. A carbamazepine fluorogenic reagent (FR) has been synthesized. Upon hydrolysis by β‐galactosidase, the nonfluorescent FR produces a fluorescent product. This enzymic hydrolysis is inhibited when the FR binds with antibody against carbamazepine. The inhibition is relieved when carbamazepine competes with FR for available antibody binding sites. Thus, increasing levels of carbamazepine result in increasing levels of fluorescence that can be conveniently monitored with any conventional fluorometer. For low, medium, and high control sera (4, 12, and 16 μg carbamazepine/ml), the within‐run coefficient of variation for the assay is 5.5%, 1.6%, and 2.9%, respectively, while the respective between‐run coefficients of variation are 3.5%, 1.9%, and 2.3%. Fifty‐three clinical serum samples were assayed by the SLFIA, gas chromatography (GC), high pressure liquid chromatography (HPLC), and an enzyme immunoassay method. The SLFIA method compares favorably with the HPLC technique (r= 0.97, slope = 1.10, v‐intercept = 1.21), the enzyme immunoassay (r= 0.98, slope = 1.07, y‐intercept = 0.82), and the GC method (r= 0.95, slope = 1.01, y‐intercept = ‐0.30).