Substrate-labeled fluorescent immunoassay for phenobarbital.
AuthorsKrausz LM, Hitz JB, Buckler RT, Burd JF
JournalTher Drug Monit
PubMed ID7013165
An assay for the anticonvulsant drug phenobarbital (PB) has been developed that is based on the principles of the substrate-labeled fluorescent immunoassay. A fluorogenic enzyme substrate, galactosyl umbelliferone, was covalently linked to a derivative of PB. The labeled drug, galactosyl umbelliferone-PB (GUPB), is nonfluorescent under conditions of the assay; however, ... More
Substrate-labeled fluorescent immunoassay for measuring dibekacin concentrations in serum and plasma.
AuthorsPlace JD, Thompson SG
JournalAntimicrob Agents Chemother
PubMed ID6357071
'We have developed a homogeneous substrate-labeled fluorescent immunoassay for the measurement of dibekacin concentrations in serum and plasma. The fluorogenic enzyme substrate beta-galactosyl-umbelliferone was covalently attached to tobramycin, an aminoglycoside structurally similar to dibekacin, to prepare a fluorogenic drug reagent (FDR). The FDR is nonfluorescent under assay conditions, but fluoresces ... More
A homogeneous fluorescent immunoassay for human immunoglobulin M.
AuthorsWorah D, Yeung KK, Ward FE, Carrico RJ
JournalClin Chem
PubMed ID7014030
'We describe a homogeneous substrate-labeled fluorescent immunoassay for human IgM. In this competitive-binding method we use a fluorogenic substrate for Escherichia coli beta-galactosidase, N-(6-aminohexyl)-7-beta-galactosyl-coumarin-3-carboxamide, which is covalently coupled to IgM. The fluorescence emission intensity of the labeled IgM at 450 nm (with excitation at 400 nm) is negligible, but when ... More
Substrate-labeled fluorescent immunoassay for phenytoin in human serum.
'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 ... More
Adenovirus-facilitated nuclear translocation of adeno-associated virus type 2.
AuthorsXiao W, Warrington KH, Hearing P, Hughes J, Muzyczka N
JournalJ Virol
PubMed ID12388712
'We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from ... More
Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum.
AuthorsBurd JF, Wong RC, Feeney JE, Carrico RJ, Boguslaski RC
JournalClin Chem
PubMed ID326439
'We applied a homogeneous reactant-labeled fluorescent immunoassay to the measurement of therapeutic drug concentrations in human serum, exemplified here by gentamicin. A derivative of umbelliferyl-beta-galactoside was coupled covalently to the drug and this conjugate was found to be nonfluorescent under assay conditions. The drug/dye conjugate was a substrate for bacterial ... More
Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.
AuthorsNazarenko DA, Dertinger SD, Gasiewicz TA
JournalBiotechniques
PubMed ID11314260
'beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form ... More
Homogeneous substrate-labeled fluorescent immunoassay for carbamazepine.
AuthorsLi TM, Miller JE, Ward FE, Burd JF
JournalEpilepsia
PubMed ID7047157
'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 ... More
Homogeneous substrate-labeled fluorescent immunoassay for disopyramide in human serum: semi- and fully automated procedures.
AuthorsJohnson PK, Messenger LJ
JournalClin Chem
PubMed ID3510781
'In this immunoassay for disopyramide in serum, to form the label, disopyramide is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid, which is nonfluorescent under the conditions of the assay. Hydrolysis, catalyzed by beta-galactosidase, yields a fluorescent product. As a result of competitive protein binding reactions between drug and ... More
Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.
AuthorsLi TM, Benovic JL, Buckler RT, Burd JF
JournalClin Chem
PubMed ID7449119
'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 ... More
Serum theophylline determination by fluorescence polarization immunoassay utilizing an umbelliferone derivative as a fluorescent label.
AuthorsLi TM, Benovic JL, Burd JF
JournalAnal Biochem
PubMed ID7337204
The homogeneous substrate-labeled fluorescent immunoassay.
AuthorsBurd JF
JournalMethods Enzymol
PubMed ID6798371
A comparison of serum phenytoin determination by the substrate-labeled fluorescent immunoassay with gas chromatography, liquid chromatography, radioimmunoassay and "EMIT".
AuthorsWong RC, George R, Yeung R, Burd JF
JournalClin Chim Acta
PubMed ID6985851
Patients' sera were analyzed for phenytoin by the Substrate-Labeled Fluorescent Immunoassay (SLFIA), gas chromatography (GC), liquid chromatography (LC), Radioimmunoassay (RIA) and EMIT and the results were compared. The EMIT assay was performed using a "PACER Analyzer". All assay results compared favorably. The correlation coefficients were: SLFIA vs GC, 0.995 (n ... More
Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin.
AuthorsArmenta R, Tarnowski T, Gibbons I, Ullman EF
JournalAnal Biochem
PubMed ID3922243
A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a beta-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked beta-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ... More
Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells.
Certain serotypes of salmonellae carry virulence plasmids that greatly enhance the pathogenicity of these bacteria in experimentally infected mice. This phenotype is largely attributable to the 8-kb spv regulon. However, spv genes are not expressed while bacteria grow in vitro. We now show that spvB, which is required for virulence, ... More
Substrate-labeled fluorescent immunoassay for amikacin in human serum.
AuthorsThompson SG, Burd JF
JournalAntimicrob Agents Chemother
PubMed ID7004339
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the ... More
Mammalian Suppressor of Sec4 Modulates the Inhibitory Effect of Rab15 during Early Endocytosis.
Authors Strick David J; Francescutti Dina M; Zhao Yali; Elferink Lisa A;
JournalJ Biol Chem
PubMed ID12105226
Rab15 is a novel endocytic Rab that counters the stimulatory effect of Rab5-GTP on early endocytic trafficking. Rab15 may interfere with Rab5 function directly by sequestering Rab5 effectors or indirectly through novel sets of effector interactions. To distinguish between these possibilities, we examined the effector binding properties of Rab15. Rab15 ... More
Glucocorticoid receptor pathways are involved in the inhibition of astrocyte proliferation.
AuthorsCrossin KL, Tai MH, Krushel LA, Mauro VP, Edelman GM
JournalProc Natl Acad Sci U S A
PubMed ID9122257
In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes both in vitro and in vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAM in vitro. ... More
Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM.
To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of beta-galactosidase under the ... More
Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB.
AuthorsQanungo S, Starke DW, Pai HV, Mieyal JJ, Nieminen AL
JournalJ Biol Chem
PubMed ID17468103
In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. ... More
Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer.
Authors
JournalSci Adv
PubMed ID33514539
PAX3-FOXO1 drives miR-486-5p and represses miR-221 contributing to pathogenesis of alveolar rhabdomyosarcoma.