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Homogeneous Substrate‐Labeled Fluorescent Immunoassay for Carbamazepine
Author(s) -
Li Thomas M.,
Miller Judith E.,
Ward Frederick E.,
Burd John F.
Publication year - 1982
Publication title -
epilepsia
Language(s) - Spanish
Resource type - Journals
SCImago Journal Rank - 2.687
H-Index - 191
eISSN - 1528-1167
pISSN - 0013-9580
DOI - 10.1111/j.1528-1157.1982.tb05425.x
Subject(s) - carbamazepine , immunoassay , chromatography , chemistry , high performance liquid chromatography , anticonvulsant , coefficient of variation , substrate (aquarium) , reagent , fluorescence , homogeneous , enzyme , therapeutic drug monitoring , hydrolysis , antibody , drug , pharmacology , epilepsy , biochemistry , medicine , immunology , organic chemistry , biology , ecology , physics , quantum mechanics , psychiatry , thermodynamics
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). RESUMEN La carbamazepina es una droga anticonvulsiva útil en el tratamiento de la epilepsia. Debido al estrecho rango terapeiitico su monitorización sérica es necessaria para una terapeútica adecuada sin toxicidad. Exponemos el desarrollo de un inmunoensayo fluorescen‐te en suero con sustratos marcados. Se ha sintetizado un reactivo fluorogénico (RF) de la carbamazepina, el cual, mediante hidrolisis con β‐galactosidasa, el RF no fluorescente produce un producto fluorescente. Esta hidrolisis enzimática se inhibe cuando el RF se une a anticuerpos anticarbamazepina. Esta inhibition es liberada cuando la carbamazepina compite con el RF disponible en los lugares de captatión de anticuerpos. Así pues, aumentos de los niveles de carbamazepina conducen a incrementos de los niveles de fluorescencia que pueden ser monitorizados con‐venientemente con cualquier fluorómetro convencional. Para bajos, medios y altos sueros control (4, 12, y 16 μg de carbamazepina/ml) el coeficiente “intra‐proceso” de variación para el ensayo es 5.5%, 1.6% y 2.9% respectivamente, mientras que, los correspondientes coeficientes de variation “entre‐pro‐cesos” son 3.5%, 1.9%, y 2.3%. Se estudiaron 53 muestras séricas clínicas con el SLFIA, cromatografía de gases (GC), cromatografia de líquidos de altos resultados (HPLC) y un método de immunoensayo enzimático ( r = 0.98, declinatión = 1.07, interceptión cony = 0.82) y el metodo GC ( r = 0.95, declinación = 1.01, interceptión cony = ‐0.30).