Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein.
AuthorsWitkowski A, Daunert S, Kindy MS, Bachas LG
JournalAnal Chem
PubMed ID8503503
Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production ... More
Rapid characterisation and identification of mycobacteria using fluorogenic enzyme tests.
AuthorsHamid ME, Chun J, Magee JG, Minnikin DE, Goodfellow M
JournalZentralbl Bakteriol
PubMed ID8061408
'Sixty representatives of selected Mycobacterium and Nocardia species were examined for their ability to cleave 79 fluorogenic synthetic enzyme substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone. The resultant data were analysed using the simple matching coefficient and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. ... More
Fluorogenic selective and differential medium for isolation of fecal streptococci.
AuthorsLittel KJ, Hartman PA
JournalAppl Environ Microbiol
PubMed ID6830220
'Of 44 fluorogenic substrates tested for their ability to differentiate species of fecal streptococci, four yielded species-differentiating reactions. The remaining substrates either yielded uniformly positive, negative, or variable strain-dependent reactions. One substrate, 4-methylumbelliferone-alpha-D-galactoside, was hydrolyzed by Streptococcus bovis and S. faecium and its biotypes. 4-Methylumbelliferone-alpha-D-galactoside and a colorimetric starch substrate ... More
Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates.
AuthorsMeng Y, High K, Antonello J, Washabaugh MW, Zhao Q
JournalAnal Biochem
PubMed ID16137635
'Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product ... More
Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus.
AuthorsFulton RE, Wong JP, Siddiqui YM, Tso MS
JournalJ Virol Methods
PubMed ID3146582
'A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of ... More
Sensitive avidin-biotin amplified fluorogenic enzyme immunoassay using biotinylated monoclonal antibodies for the identification and quantitation of virus.
AuthorsWong JP, Fulton RE, Siddiqui YM
JournalJ Virol Methods
PubMed ID1955488
'A highly sensitive amplified fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes the high affinity interaction of the vitamin biotin for the multiple binding sites on the glycoprotein avidin, was developed for the detection and identification of a model virus, Newcastle disease virus (NDV). Monoclonal antibodies (MCA) directed against the virus ... More
Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA).
AuthorsCrowther JR, Angarita L, Anderson J
JournalBiologicals
PubMed ID2178352
'Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain ... More
Patterning enzymes inside microfluidic channels via photoattachment chemistry.
AuthorsHolden MA, Jung SY, Cremer PS
JournalAnal Chem
PubMed ID15053641
'We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, ... More
Fluorogenic and chromogenic substrates used in bacterial diagnostics.
AuthorsManafi M, Kneifel W, Bascomb S
JournalMicrobiol Rev
PubMed ID1943991
'Methods based on the application of chromogenic and fluorogenic substrates enable specific and rapid detection of a variety of bacterial enzymatic activities. By using these techniques, enzymatic reactions can be examined simultaneously or individually, either directly on the isolation plate or in cell suspensions. For this purpose, various testing principles ... More
Properties of lysosomes in guinea pig heart: subcellular distribution and in vitro stability.
AuthorsWelman E, Peters TJ
JournalJ Mol Cell Cardiol
PubMed ID7679
Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus.
AuthorsTada J, Ohashi T, Nishimura N, Ozaki H, Fukushima S, Takano J, Nishibuchi M, Takeda Y
JournalMol Cell Probes
PubMed ID1480188
'A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction. This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. An advantage of this ... More
A comparison of the sensitivity and specificity of enzyme immunoassays and time-resolved fluoroimmunoassay.
AuthorsRoberts IM, Jones SL, Premier RR, Cox JC
JournalJ Immunol Methods
PubMed ID1919036
'Time-resolved fluoroimmunoassay (TR-FIA) and various enzyme immunoassays (EIA) were compared in order to determine the detection system which showed the greatest degree of sensitivity without sacrificing specificity. The system chosen for the evaluation of these assays was the detection of antibodies to human immunodeficiency virus (HIV). For EIA, horseradish peroxidase ... More
Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.
'Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3''-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc ... More
Enzyme immunoassay techniques. An overview.
AuthorsPorstmann T, Kiessig ST
JournalJ Immunol Methods
PubMed ID1613258
'In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to ... More
4-Methylumbelliferyl phosphate as a substrate for lysosomal acid phosphatase.
AuthorsRobinson D, Willcox P
JournalBiochim Biophys Acta
PubMed ID5823497
A Specific effect of external ATP on the permeability of transformed 3T3 cells.
AuthorsRozengurt E, Heppel LA
JournalBiochem Biophys Res Commun
PubMed ID1039
Lysosomal enzymes of cultured amniotic fluid cells.
AuthorsButterworth J, Sutherland GR, Broadhead DM, Bain AD
JournalClin Chim Acta
PubMed ID4694473
Determination of acid hydrolases in human platelets.
AuthorsDangelmaier CA, Holmsen H
JournalAnal Biochem
PubMed ID6247939
Amplification systems in immunoenzymatic techniques.
AuthorsAvrameas S
JournalJ Immunol Methods
PubMed ID1613256
Staining for enzymatic activity after gel electrophoresis, I.
AuthorsGabriel O, Gersten DM
JournalAnal Biochem
PubMed ID1381872
Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity.
AuthorsGore MG, Greasley PJ, Ragan CI
JournalJ Biochem Biophys Methods
PubMed ID1331221
This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. ... More
A rapid micro method for counting cells "in situ" using a fluorogenic alkaline phosphatase enzyme assay.
AuthorsHuschtscha LI, Lucibello FC, Bodmer WF
JournalIn Vitro Cell Dev Biol
PubMed ID2914812
A new method has been developed to count cells "in situ", based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay ... More
Enzymatic quantification of cell-matrix and cell-cell adhesion.
AuthorsLöster K, Horstkorte R
JournalMicron
PubMed ID10568230
Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of ... More
Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish.
AuthorsVaz-Velho M, Duarte G, Gibbs P
JournalJ Microbiol Methods
PubMed ID10699670
This study was conducted to evaluate the efficacy of the mini-VIDAS Listeria monocytogenes (LMO) system (BioMérieux Vitek, Inc., Missouri, USA) for detection of L. monocytogenes in environmental and fish samples from three Portuguese cold-smoking plants and from their fresh fish suppliers. Mini-VIDAS-LMO is a fully automated system that uses fluorescent ... More
Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.
AuthorsPastula C, Johnson I, Beechem JM, Patton WF
JournalComb Chem High Throughput Screen
PubMed ID12769677
A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. ... More
A fluorescence assay to monitor vesicle fusion and lysis.
AuthorsKendall DA, MacDonald RC
JournalJ Biol Chem
PubMed ID6815181
An assay based on the fluorescent compound 2',7'-([bis(carboxymethyl)amino]methyl) fluorescein (calcein) has been developed to investigate vesicle fusion and lysis. The assay involves encapsulating the nonfluorescent Co2+ complex of calcein in one set of vesicles and EDTA in a second set. If fusion occurs, EDTA chelates Co2+, releasing calcein which may ... More
Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles.
AuthorsGodsey JH, Matteo MR, Shen D, Tolman G, Gohlke JR
JournalJ Clin Microbiol
PubMed ID7016897
A total of 539 clinical isolates belonging to 10 species of the Enterobacteriaceae family were identified by enzyme activity profiles within 30 min of test inoculation. Each isolate was grown at 37 degrees C for 18 h on Mueller-Hinton agar and suspended to an optical density of 200 Klett units ... More
A fluorescent microplate assay for diarrheic shellfish toxins.
AuthorsVieytes MR, Fontal OI, Leira F, Baptista de Sousa JM, Botana LM
JournalAnal Biochem
PubMed ID9177752
A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of ... More
A quantitative fluorescence enzyme immunoassay for plant cytokinins.
AuthorsTrione EJ, Banowetz GM, Krygier BB, Kathrein JM, Sayavedra-Soto L
JournalAnal Biochem
PubMed ID3605594
An enzyme-linked immunosorbent assay (ELISA) which used 4-methylumbelliferyl phosphate as an enzyme substrate was used to quantify two plant cytokinins. This assay detected as little as 0.03 pmol (approximately 10 pg) of cytokinin in microplate wells coated with a cytokinin-ovalbumin conjugate. The method measured competition between free cytokinin and the ... More
Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.
AuthorsVlieger AM, Medenblik AM, van Gijlswijk RP, Tanke HJ, van der Ploeg M, Gratama JW, Raap AK
JournalAnal Biochem
PubMed ID1332534
In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or ... More
Requirement of calmodulin binding by HIV-1 gp160 for enhanced FAS-mediated apoptosis.
AuthorsMicoli KJ, Pan G, Wu Y, Williams JP, Cook WJ, McDonald JM
JournalJ Biol Chem
PubMed ID10625668
Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 ... More
Enzyme-linked immunosorbent fluorescence assay and high-pressure liquid chromatography for analysis of humoral immune responses to Coxiella burnetti proteins.
AuthorsSchmeer N, Müller HP, Baumgärtner W, Wieda J, Krauss H
JournalJ Clin Microbiol
PubMed ID3068247
A microtiter enzyme-linked immunosorbent fluorescence assay based on alkaline phosphatase conjugate and 4-methylumbelliferyl phosphate as fluorogenic substrate was developed and adapted to quantitatively analyze immunoglobulin G subclass 1 (IgG1) and IgG2 responses of vaccinated and infected cattle to proteins of Coxiella burnetii. The enzyme-linked immunosorbent fluorescence assay surpassed the conventional ... More
Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.
AuthorsObzansky DM, Rabin BR, Simons DM, Tseng SY, Severino DM, Eggelte H, Fisher M, Harbron S, Stout RW, Di Paolo MJ
JournalClin Chem
PubMed ID1893577
A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active ... More
Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase?
AuthorsPorstmann B, Porstmann T, Nugel E, Evers U
JournalJ Immunol Methods
PubMed ID3923120
Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying ... More
Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.
AuthorsGee KR, Sun WC, Bhalgat MK, Upson RH, Klaubert DH, Latham KA, Haugland RP
JournalAnal Biochem
PubMed ID10452797
Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase ... More
Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.
AuthorsCasciola-Rosen LA, Hubbard AL
JournalJ Biol Chem
PubMed ID1671861
We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was ... More
Controlling programmed cell death with a cyclophilin-cyclosporin-based chemical inducer of dimerization.
BACKGROUND: Cell death can occur either from physical damage (necrosis) or cellular suicide (apoptosis). Apoptosis is essential for the development of multicellular organisms and disregulated apoptosis underlies many human diseases. The Fas receptor (Fas) is a membrane signaling protein that mediates a death signal following its aggregation by the Fas ... More