hymecromone and 4-methylumbelliferyl-phosphate

4-MUP, 4-Methylumbelliferyl phosphate; 4-MU, 4-Methylumbelliferone; RFV, Relative Fluorescent Values; RFU, Relative Fluorescent Units; QDs, Quantum Dots; LoD, Limit of Detection.

Topics: Alkaline Phosphatase; Biocatalysis; Enzyme-Linked Immunosorbent Assay; Fluorescence; Hydrolysis; Hymecromone; Kinetics; Recombinant Proteins

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain.

Topics: Animals; Aphids; Carboxylic Ester Hydrolases; Cell Line; Hymecromone; Insecticide Resistance; Insecticides; Molecular Docking Simulation; Mutation; Naphthols; Organophosphates; Spodoptera

We introduce a method for tracking the rate and extent of delivery of liposome contents in vivo based on encapsulation of 4-methylumbelliferyl phosphate (MU-P), a profluorophore of 4-methylumbelliferone (MU). MU-P is rapidly dephosphorylated by endogenous phosphatases in vivo to form MU after leakage from the liposome. The change in fluorescence spectra when MU-P is converted to MU allows for quantification of entrapped (MU-P) and released (MU) liposome contents by fluorescence or by a sensitive high performance liquid chromatography assay. We define the "cellular availability" of an agent encapsulated in a liposome as the ratio of the amount of released agent in the tissue to the total amount of agent in the tissue; this parameter quantifies the fraction of drug available for therapy. The advantage of this method over existing technologies is the ability to decouple the signals of entrapped and released liposome contents. We validate this method by tracking the circulation and tissue distribution of MU-P loaded liposomes after intravenous administration. We use this assay to compare the cellular availability of liposomes composed of engineered phosphocholine lipids with covalently attached cholesterol, sterol-modified lipids (SML), to liposomes composed of conventional phospholipids and cholesterol. The SML liposomes have similar pharmacokinetic and biodistribution patterns as conventional phospholipid-cholesterol liposomes but a slower rate of contents delivery into the tissue. Thus, MU-P enables the tracking of the rate and extent of liposome contents release in tissues and should facilitate a better understanding of the pharmacodynamics of liposome-encapsulated drugs in animals.

Topics: Administration, Intravenous; Animals; Fluorescent Dyes; Hymecromone; Lipids; Liposomes; Liver; Mice; Serum; Spectrometry, Fluorescence; Spleen; Tissue Distribution

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

Topics: Amino Acid Sequence; Amino Acid Substitution; Amphibian Proteins; Animals; Cloning, Molecular; DNA, Complementary; Embryo, Nonmammalian; Epoxide Hydrolases; Escherichia coli; Gene Expression; Hymecromone; Kinetics; Lysophospholipids; Molecular Sequence Data; Mutation; Phosphoric Monoester Hydrolases; Sequence Alignment; Sequence Homology, Amino Acid; Solubility; Species Specificity; Substrate Specificity; Xenopus laevis

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

Topics: Alkaline Phosphatase; Animals; Antibodies, Monoclonal; Benzidines; Beverages; Caffeine; Chromatography, Liquid; Cosmetics; Fluorescent Dyes; Horseradish Peroxidase; Hymecromone; Immunoenzyme Techniques; Nitrophenols; Organophosphorus Compounds; Phenylpropionates; Sensitivity and Specificity; Tandem Mass Spectrometry

We developed a method for detecting phosphatase activities in crude tissue extracts after separation of proteins by a novel non-denaturing two-dimensional electrophoresis. In the first dimension, protein samples were separated by a MicroRotofor, a liquid-phase isoelectric focusing, in the presence or absence of urea. In the second dimension, fractionated proteins by the MicroRotofor were resolved by a native polyacrylamide gel electrophoresis in the presence of 20 mM 2-mercaptoethanol. After electrophoresis, the polyacrylamide gel was directly immersed in a reaction mixture containing 4-methylumbelliferyl phosphate (MUP), a fluorogenic substrate, and phosphatase activities were detected as fluorescent bands. In this assay, a variety of phosphatase activities were clearly detected in gel when the tissue extracts were separated by the MicroRotofor in the presence of 1.5 M urea. Furthermore, after detecting phosphatase activities in polyacrylamide gel at neutral pH, its activities at acidic pH could be detected by immersing the gel in sodium citrate buffer (pH 3.0). Therefore, this method is a quite useful technique to analyze various phosphatases by sequential reactions with MUP under different conditions after sample separation by the two-dimensional electrophoresis.

Topics: Animals; Brain; Electrophoresis, Gel, Two-Dimensional; Embryo, Nonmammalian; Enzyme Assays; Fluorescent Dyes; Hydrogen-Ion Concentration; Hymecromone; Isoelectric Focusing; Male; Phosphoric Monoester Hydrolases; Protein Denaturation; Rats; Rats, Wistar; Tissue Extracts; Urea; Zebrafish; Zebrafish Proteins

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.

Topics: Alkaline Phosphatase; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Gels; Hymecromone; Male; Nuclear Proteins; Phosphoprotein Phosphatases; Protein Phosphatase 2C; Rats; Rats, Wistar; Recombinant Proteins

This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records.. The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line.. Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor.. These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Topics: Acid Phosphatase; Animals; Candida albicans; CD4 Lymphocyte Count; Cell Adhesion; Cell Line; Child; Enzyme Inhibitors; Epithelial Cells; HIV; HIV Seronegativity; HIV Seropositivity; Humans; Hydrogen-Ion Concentration; Hymecromone; Indicators and Reagents; Mouth Mucosa; Vanadates; Viral Load

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Topics: Acid Phosphatase; Bacterial Proteins; Cations, Divalent; Clostridium perfringens; Dimerization; Enzyme Activators; Enzyme Inhibitors; Hymecromone; Kinetics; Molecular Weight; Nitrophenols; Nucleosides; Organophosphorus Compounds; Substrate Specificity

We compared the accuracy and reliability of three amplification systems for enzyme immunoassays in the detection of specific IgG antibodies for the diagnosis of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in patients from an endemic area in Rio de Janeiro, Brazil. Partially soluble antigens obtained from the promastigote forms of L. (V.) braziliensis were used. For development of the reaction, two chromogens, 1,2-orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB), and a fluorogen, 4-methylumbelliferylphosphate (MUP), were tested. The performance of each system was compared using the following parameters: accuracy, intraclass correlation coefficient (ICC), and area under the receiver operating characteristic (ROC) curve. Sensitivity was the same (97.4%) for all systems. The reliability was excellent (ICC = 98.6, 98.7, and 99.1%) and specificity was 93.7, 95.8, and 97.4% for OPD, MUP, and TMB, respectively, showing no statistical significance. Despite the absence of differences in the performance of the three systems, the use of TMB is suggested because of its operational advantages, such as low cost compared with fluorogens, easy manipulation, greater stability, and lower toxicity.

Topics: Animals; Antigens, Protozoan; Benzidines; Confidence Intervals; Humans; Hymecromone; Immunoenzyme Techniques; Immunoglobulin G; Leishmania braziliensis; Leishmaniasis, Cutaneous; Phenylenediamines; Predictive Value of Tests; Reproducibility of Results; ROC Curve; Sensitivity and Specificity

Oxidative damage to proteins, implicated amongst other in the etiology and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), results in the loss of specific biological protein function. A simple, sensitive, and cost-effective fluorimetric test to assess the antioxidant capacity of new chemical entities to protect proteins from loss of activity caused by reactive oxygen species (ROS) was developed using alkaline phosphatase (ALP) as model protein. Protein oxidation was induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and the decrease in catalytic activity of ALP to hydrolyze 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) was monitored as a marker of protein degradation. According to their capacity to protect ALP from peroxyl radical-induced activity loss, ten reference antioxidants were divided into three classes, namely efficient (pIC(50) > 5 for quercetin, chlorogenic acid, caffeic acid, mangiferin, and resveratrol), intermediate (4 < pIC(50) < or = 5 for melatonin, trolox, and ascorbic acid), and poor antioxidants (pIC(50) < 4 for glutathione and D-mannitol). Multifunctional drugs, having the ability to interact with several disease-related targets are of interest in PD. Therefore, the capacity of three catechol-O-methyltransferase (COMT) inhibitors, entacapone, nitecapone, and tolcapone to protect ALP from oxidative damage was also investigated and found to be very similar to the most potent reference antioxidants.

Topics: Alkaline Phosphatase; Amidines; Antioxidants; Catalysis; Catechol O-Methyltransferase Inhibitors; Drug Evaluation, Preclinical; Enzyme Inhibitors; Fluorometry; Hymecromone; Peroxides; Reactive Oxygen Species

Protein phosphatase type 5 (PP5) belongs to the PPP family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5 are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by hormones (i.e., glucocorticoids) or cellular stress (i.e., hypoxia, oxidative stress). Recently, studies conducted with short interfering RNA and antisense oligonucleotides indicate that PP5 plays an important role in the regulation of stress-induced signaling cascades that influence both cell growth and the onset of apoptosis. Therefore, the identification of small molecule inhibitors of PP5 is desired for use in studies to further define the biological/pathological roles of PP5. Such inhibitors may also prove useful for development into novel antitumor agents. Here we describe methods to express and purify large amounts of biologically active PP5c, an inhibitor titration-based assay to determine the amount of PP5 in solution, and a fluorescent phosphatase assay that can be used to screen chemical libraries and natural extracts for the presence of catalytic inhibitors.

Topics: 4-Nitrophenylphosphatase; Catalysis; Enzyme Inhibitors; Escherichia coli; Fluorescent Dyes; Humans; Hymecromone; Indicators and Reagents; Nuclear Proteins; Phosphoprotein Phosphatases; Spectrometry, Fluorescence

We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (10(4)-10(6)). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro.

Topics: Alkaline Phosphatase; Cell Adhesion; Fluorometry; Humans; Hymecromone; Neutrophils; Substrate Specificity

The purpose of the present study was to implement a fluorometric method for detecting and quantifying viral antigens in human meduloblastoma cells infected by two types of fixed rabies virus (CVS-MB and CVS-BHK) and a street virus using a cell-enzyme linked immunosorbent assay (cell-ELISA) technique; alkaline phosphatase was used as the antibody-marker enzyme and 4-methyl-umbelliferyl-phosphate as the fluorogenic substrate. The system was used for detecting up to 1:10,000 viral inoculums, followed by evaluating the effect of heparin on infection. Infected cultures were reliably differentiated from their respective negative controls in both assays allowing data to be analysed statistically. As reported in another study, heparin produces strong inhibition when the CVS-BHK viral strain is used for infection; it has thus been suggested that it binds to the neural cell adhesion molecule and could be blocked by using this drug. This fluorometric method is less time-consuming, has increased reproducibility and useful for quantitation of collected data and can therefore be considered as a useful tool for research.

Topics: Antibody Specificity; Antigens, Viral; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fluorometry; Heparin; Humans; Hymecromone; Neural Cell Adhesion Molecules; Rabies virus

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.

Topics: Binding Sites; Culture Media; Dose-Response Relationship, Drug; Hymecromone; Indicators and Reagents; Monoacylglycerol Lipases; Organophosphorus Compounds; Pseudomonas aeruginosa; Titrimetry

Protein phosphatase inhibition assays currently used for the detection of cyanobacterial peptide hepatotoxins in drinking water require an enrichment step using C18 cartridges to achieve lower the detection limit. This paper describes a colorimetric and fluorometric protein phosphatase inhibition method for the direct detection of microcystin-LR (MCYST-LR) in drinking water without complex clean-up steps and preconcentration procedures. In this assay three different substrates, p-nitrophenyl phosphate (p-NPP) and two fluorogenic compounds, 4-methylumbelliferyl phosphate (MUP) and 6,8-difluoro-4-methylumbelliferyl phosphate DiFMUP), were tested. The detection limits of the assay are 0.25 and 0.1 microg/l using colorimetric and fluorometric methods, respectively. These levels are well below the provisional guideline value for MCYST-LR of 1 microg/l of drinking water. The detection limit of the fluorometric method is comparable to that of the classical ELISA test. Although both the latter tests allow the detection of MCYST-LR in drinking water directly without pretreatment, the protein phosphatase inhibition assay remain less expensive and therefore more attractive for use in the routine assessment of drinking water contamination by microcystins.

Topics: Bacterial Toxins; Colorimetry; Cyanobacteria; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fluorometry; Hymecromone; Marine Toxins; Microcystins; Nitrophenols; Organophosphorus Compounds; Peptides, Cyclic; Phosphoprotein Phosphatases; Reproducibility of Results; Sensitivity and Specificity; Water

Activities of the extracellular enzymes beta-glucosidase and phosphatase and bacterial densities were investigated during the filtration process at several sites in a groundwater recharge plant at the Ruhr river (Hengsen recharge plant in Schwerte. Germany). Low numbers of microorganisms and low levels of activity in this type of habitat, compared to most surface waters, caused methodological problems when determining microbial activity. In this study, fluorigenic model substrates, which enable hydrolytic rates as low as 1 nmol (L x h)(-1) to be measured, were used to determine extracellular enzyme activities. Highest activities were determined in surface water (107 nmol (L x h)(-1) for beta-glucosidase and 252 nmol (L x h)(-1) for phosphatase). which decreased during the filtration process in the gravel prefilter and the main sand filter until the end of subsurface flow (1.6 nmol (L x h)(-1) and 6.8 nmol (L x h)(-1), respectively). Similarly, bacterial numbers decreased from 3.4 x 10(6) to 0.29 x 10(6) cells mL(-1). These data showed that microbial activity within the prefilter and the shallow layers of the sand filter had the greatest impact on water quality. In addition to its involvement in the continuous purification of surface water, the microbial community in the sand filter probably acts as a biological buffer against ephemeral increases in the loads of organic matter and nutrients in the recharge plant.

Topics: beta-Glucosidase; Colony Count, Microbial; Extracellular Space; Filtration; Fresh Water; Germany; Glucosides; Hymecromone; Models, Biological; Phosphoric Monoester Hydrolases; Silicon Dioxide; Water Supply

The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.

Topics: Acid Phosphatase; Bacterial Typing Techniques; Chromogenic Compounds; Clostridium perfringens; Culture Media; Fluorescent Dyes; Hymecromone; Nitrophenylgalactosides; Sensitivity and Specificity; Time Factors

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.

Topics: Acridines; Animals; Bivalvia; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Fluorescein; Fluorometry; Hymecromone; Indicators and Reagents; Marine Toxins; Okadaic Acid; Phosphoprotein Phosphatases; Reproducibility of Results; Shellfish; Substrate Specificity

Previously it was reported that promastigotes of virtually all pathogenic Leishmania species, except Leishmania major, release a structurally conserved soluble acid phosphatase (AcP) activity during their growth in vitro (P. S. Doyle and D. M. Dwyer, Exp. Parasitol. 77, 435-444 1993). In the current study we used a highly sensitive fluorogenic detection method to demonstrate that soluble AcPs were in fact produced by promastigotes of several different strains of L. major. These L. major AcP activities were readily immunoprecipitated with a rabbit antibody previously generated against the L. donovani AcP. Results of metabolic labeling and immunoprecipitations demonstrated that AcPs produced by the L. majors strains examined had an apparent molecular mass of approximately 77 kDa. Results of Southern hybridization analysis with an L. donovani AcP gene probe showed that the AcP gene loci were conserved in the L. major strains examined. Taken together, these results indicate that the AcP enzyme has been structurally and functionally conserved throughout the evolution of pathogenic species of Leishmania. Such conservation suggests that the AcPs play a functional role in the growth and survival of this group of important human pathogens.

Topics: Acid Phosphatase; Aniline Compounds; Animals; Antigens, Protozoan; Blotting, Southern; Conserved Sequence; DNA, Protozoan; Epitopes; Hymecromone; Indicators and Reagents; Leishmania major; Organophosphorus Compounds; Precipitin Tests; Rabbits; Solubility

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 the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

Topics: Animals; Diarrhea; Fluoresceins; Fluorescent Dyes; Fluorometry; Humans; Hymecromone; Marine Toxins; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Protein Phosphatase 2; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Shellfish Poisoning; Substrate Specificity

Amylin is a 37-amino-acid polypeptide synthesized in and secreted from pancreatic beta cells along with insulin. Its biological actions include the slowing and reduction of postmeal increases in plasma glucose concentrations. Studies of the basic amylin biology in humans have been hampered by the lack of a rapid, sensitive assay capable of measuring physiological concentrations of amylin in small volumes of plasma. We report here two sandwich-type immunoassays that use pairs of monoclonal antibodies, the fluorescent substrate 4-methylumbelliferyl phosphate, and the enzyme alkaline phosphatase. The minimum detectable concentration of amylin in 50 microL of plasma was 0.5 to 2 pmol/L, and the dynamic range was 2 to 100 pmol/L. The assays had average intraassay CVs of <10%, average interassay CVs of <15%, and good linearity on dilution and recovery of added amylin. The two assays use the same detection antibody, which binds to the carboxyl terminus of the molecule, but different capture antibodies. One of the assays measures only human amylin; the other also detects amylin-like peptides. Examples of measurements in human plasma are provided in subjects with impaired glucose tolerance and in nondiabetic controls.

Topics: Alkaline Phosphatase; Amyloid; Antibodies, Monoclonal; Fluoroimmunoassay; Glucose Tolerance Test; Humans; Hymecromone; Indicators and Reagents; Islet Amyloid Polypeptide; Peptides; Sensitivity and Specificity

A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence (r = 0.99 for both Staph. aureus and E. coli). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline (r = 0.99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.

Topics: Animals; Cattle; Colony Count, Microbial; Culture Media; Escherichia coli; Evaluation Studies as Topic; Fluorometry; Hymecromone; In Vitro Techniques; Indicators and Reagents; Leukocytes; Phagocytosis; Staphylococcus aureus; Time Factors

To improve the sensitivity of detecting biotin-labeled DNA Probes, a new fluorescent substrate of alkaline phosphatase, 4-methylum belliferylphosphate (4-mup) was studied instead of conventional BCIP-NBT. The result of dot-blot hybridization demonstrates that this new substrate can be used for the colorimetric detection of biotin-labeled probes after hybridization to immobilized nucleic acids. The sensitivity is about one order of magnitude higher than that of BCIP-NBT system, and the time required for color development is very short, only about five min. It is suggested that the Bio-SA-Bio-AP-4-MUP colorimetric detection system can be widely used in gene diagnosis.

Topics: Biotin; DNA Probes; Fluorescence; Hymecromone; Nucleic Acid Hybridization; Sensitivity and Specificity

A novel simple agar plate capable of detecting methicillin-resistant Staphylococcus aureus (MRSA) by single culture was developed. The agar plate included 1) mannitol and a pH indicator, and 4-methylumbelliferyl phosphoric acid, both for identification of Staphylococcus aureus by its ability to digest mannitol and produce phosphatase; detection of the former by change in color of pH indicator and the latter by fluorescence of 4-methylumbelliferone, 2) oxacillin at a minimum concentration just to eliminate methicillin-sensitive Staphylococcus aureus (MSSA), and 3) phenylethyl alcohol to inhibit growth of most gram negative rods. The method was simple, sensitive, time-saving, and quite effective in the identification of MRSA directly from clinical specimens. Neither false positive nor false negative results were observed by the present method.

Topics: Agar; Bacteriological Techniques; Culture Media; Fluorescence; Hydrogen-Ion Concentration; Hymecromone; Mannitol; Methicillin Resistance; Oxacillin; Staphylococcus aureus

As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.

Topics: Alkaline Phosphatase; Base Sequence; Biotin; DNA Ligases; DNA Transposable Elements; DNA, Bacterial; Electrophoresis; Fluorescent Dyes; Gene Amplification; Hymecromone; Indoles; Luminescent Measurements; Molecular Sequence Data; Mycobacterium tuberculosis; NADP; Nitroblue Tetrazolium; Nitrophenols; Organophosphorus Compounds; Tuberculosis

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Topics: Adamantane; Bacterial Proteins; Biotin; Deoxyuracil Nucleotides; Digoxigenin; DNA, Bacterial; Enzymes, Immobilized; Hymecromone; Indicators and Reagents; Listeria monocytogenes; Nitrophenols; Organophosphorus Compounds; Polymerase Chain Reaction; Sensitivity and Specificity; Streptavidin; Substrate Specificity; Thymine Nucleotides

A test to determine the epitope specificity of monoclonal antibodies (MCA) was developed for hybridoma clones producing antibodies against Newcastle disease virus (NDV). The virus was first immobilized on nitrocellulose membranes of Millititer HA plates. Dilutions of MCA were then added, singly, or simultaneously in pairs, and bound antibody was quantitated with alkaline phosphatase-labelled detector antibody and a fluorogenic substrate, 4-methylumbelliferyl phosphate (4-MUP). Fluorescence count was measured fluorometrically. Additivity indices were calculated and plotted against dilutions of paired MCA. Antibodies that recognized identical epitopes displayed non-additivity at saturating antibody dilutions, followed by partial additivity and by total additivity at low, non-saturating dilutions. In contrast, MCA that recognized distinct epitopes exhibited total additivity throughout the curve. MCA that exhibited partial additivity were interpreted as competing for overlapping shared epitopes, or, distinct epitopes in close proximity, resulting in steric hinderance.

Topics: Antibodies, Monoclonal; Antibodies, Viral; Antigens, Viral; Epitopes; Hymecromone; Immunoenzyme Techniques; Newcastle disease virus; Sensitivity and Specificity

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. The kinetic constants for the enzyme using this new substrate are described.

Topics: Animals; Cattle; Cyclohexanols; Fluorescence; Hymecromone; Kinetics; Lithium; Magnesium; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Spectrophotometry

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.

Topics: Adamantane; Alkaline Phosphatase; Base Sequence; DNA Probes; Fluorescent Dyes; Hymecromone; Membranes, Artificial; Molecular Sequence Data; Nitrophenols; Nucleic Acid Hybridization; Organophosphorus Compounds

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 detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.

Topics: Acetylglucosamine; Animals; Asialoglycoproteins; Cell Compartmentation; Endocytosis; Endosomes; Glucosamine; Glycoproteins; Hydrolases; Hymecromone; Liver; Lysosomes; Male; Pinocytosis; Rats; Rats, Inbred Strains; Serum Albumin, Bovine

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 (HRP) and alkaline phosphatase (AP) were investigated, each with a number of different substrates. HRP with its fluorogenic substrate, 3-(p-hydroxyphenyl)propionic acid (HPPA) was 1.6 times (p less than 0.01) more sensitive than with 3,3',5,5'-tetramethylbenzidine (TMB) and four times (p less than 0.001) more sensitive than with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). AP with its fluorogenic substrate, 4-methylumbelliferyl phosphate (4MeUP), was 6-7 times (p less than 0.001) more sensitive than with phenolphthalein monophosphate (PMP) and 8-13 times (p less than 0.001) more sensitive than with p-nitrophenyl phosphate (pNPP). TR-FIA with Eu3(+)-labelled anti-human IgG was equivalent in sensitivity to HRP with TMB and AP with 4MeUP.

Topics: Acquired Immunodeficiency Syndrome; Alkaline Phosphatase; Aniline Compounds; Benzidines; Benzothiazoles; Chromogenic Compounds; Fluoroimmunoassay; HIV; Horseradish Peroxidase; Humans; Hymecromone; Immunoenzyme Techniques; Organophosphorus Compounds; Phenolphthaleins; Phenylpropionates; Sensitivity and Specificity; Sulfonic Acids

We explored the biochemical basis for the disorder pseudohypophosphatasia (PsHYPT) in one patient by examining the substrate specificity and localization of alkaline phosphatase (ALP) in cultured dermal fibroblasts. Despite substantial ALP activity, in cell homogenates, toward the artificial substrate 4-methyl-umbelliferyl phosphate (4-MUP), there was a marked deficiency in ALP activity toward the natural substrates pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), indicating altered substrate specificity. Furthermore, although 4-MUP phosphatase (4-MUP-P'tase) activity was predominantly localized as an ecto-enzyme, the small amount of PLP phosphatase (PLP-P'tase) activity was intracellular. This differential localization was apparent in intact cells, since (1) brief acidification of the medium at 4 degrees C inactivated a majority of the 4-MUP-P'tase activity but only 15% of the PLP-P'tase activity (in contrast to greater than 85% inactivation of both in homogenates), (2) greater than 70% of the 4-MUP-P'tase activity but only 30% of the PLP-P'tase activity was released by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, and (3) degradation of extracellular PLP was less than 35% of that of disrupted cells. Both 4-MUP- and PLP-P'tase activities were predominantly in a lipid-anchored form that could be converted to a soluble, lipid-free form by PI-PLC digestion. Our findings suggest that the clinical and biochemical presentation of this PSHPT patient results from the production of two aberrant ALP species. One form of ALP has appropriate ectoorientation but is preferentially deficient in activity toward natural substrates; the other ALP species has appropriate substrate specificity but is sequestered from substrates by its intracellular location.

Topics: Alkaline Phosphatase; Cells, Cultured; Ethanolamines; Fibroblasts; Humans; Hymecromone; Hypophosphatasia; Isoenzymes; Kinetics; Mutation; Octoxynol; Phosphoric Monoester Hydrolases; Polyethylene Glycols; Pyridoxal Phosphate; Skin; Substrate Specificity

The major acid phosphatase of human seminal fluid was purified to homogeneity by chromatography on Sephadex G-150, and DEAE-Sephadex, and by isoelectric focusing (pI, 4.3). This purified preparation of seminal fluid acid phosphatase blocked superoxide anion production by neutrophils stimulated with fMet-Leu-Phe (fMLP) by 50%. The phosphatase also hydrolysed myo-inositol 1,4,5-trisphosphate (IP3) in vitro, an intracellular second messenger which releases Ca2+ from intracellular pools, at nearly one-third the rate at which the nonphysiologic substrate 4-methylumbelliferylphosphate (MUP) was cleaved. In contrast, two phosphoinositide lipids, namely phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate, were poor phosphatase substrates. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by phosphatase-treated cells was reduced by 69%. These results indicate that the human seminal fluid acid phosphatase may compromise the neutrophil's microbicidal response to the organism by hydrolyzing a second messenger (IP3) which is directly involved in the regulation of intracellular Ca2+ concentrations. The seminal fluid phosphatase also inhibited by 85% the ability of murine natural killer (NK) cells to inactivate target cells.

Topics: Acid Phosphatase; Animals; Chromatography; Humans; Hydrolysis; Hymecromone; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Isoelectric Focusing; Killer Cells, Natural; Male; Mice; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Semen; Superoxides

We describe a new clinical laboratory instrument, the IMx, used to automate immunoassay testing in the clinical laboratory. The IMx incorporates a novel technology called Microparticle capture Enzyme ImmunoAssay (MEIA) for assays of high-molecular-mass analytes, and fluorescence polarization immunoassay (FPIA) for hapten assays. A front-surface fluorometer is used to quantify the enzymatic generation of fluorescent product at a rate proportional to the concentration of the analyte in an MEIA, and a fluorescence polarization optical system is used to quantify results in an FPIA. The microprocessor-based instrument uses a robotic arm with two degrees of freedom and a rotating carousel to process the samples for assay. One assay can be done on each of 24 patients' specimens in 30 to 40 min with "walk-away" automation. Calibration curves are stable for at least two weeks. Instrument control involves software-labeled "command keys," a numeric keypad, and an interactive display. Results are output to a thermal printer or computer interface.

Topics: alpha-Fetoproteins; Autoanalysis; Chorionic Gonadotropin; Fluorescence Polarization; Fluorometry; Hot Temperature; Humans; Hymecromone; Immunoassay; Immunoenzyme Techniques; Optics and Photonics

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Hymecromone; Immunoglobulin G; Mice; Pollen; Predictive Value of Tests; Umbelliferones

A continuous spectrofluorimetric assay of calmodulin-dependent phosphatase is described. The assay monitors the formation of a fluorescent 4-methylumbelliferone from the dephosphorylation of 4-methylumbelliferyl phosphate and detects as little as 1 pmol of 4-methylumbelliferone. The phosphatase shows a Km of 1.3 mM for the substrate and a Vmax of 100 nmol/mg/min.

Topics: Animals; Brain; Calmodulin-Binding Proteins; Cattle; Hydrolysis; Hymecromone; Spectrometry, Fluorescence; Substrate Specificity; Time Factors; Umbelliferones

The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available.

Topics: Antibodies, Viral; Antigens, Viral; Enzyme-Linked Immunosorbent Assay; Hymecromone; Immunoenzyme Techniques; Nitrophenols; Organophosphorus Compounds; Rubella virus; Simplexvirus

A new enzyme-linked immunoassay for the determination of anti-erythrocyte antibodies is described. A fluorogenic substrate of alkaline phosphatase, the 4-MUP, was used to reveal the antigen-antibody reaction. This substrate was chosen in order to avoid interferences with haemoglobin, since it emits a light with a wavelength outside the absorption spectrum of haemoglobin. This method reaches a sensitivity in the order of 1 ng/ml. Sera from 66 Rh-immunized pregnant women and 20 control subjects have been studied. The specificity and reproducibility of the results and their good correlation with the biological and clinical parameters of the hemolytic disease of the newborn confirm the validity of the method, encouraging its routine use.

Topics: Alkaline Phosphatase; Antibodies; Coombs Test; Enzyme-Linked Immunosorbent Assay; Erythroblastosis, Fetal; Erythrocytes; Female; Fluorometry; Humans; Hymecromone; Methods; Pregnancy; Prenatal Diagnosis; Rh-Hr Blood-Group System

Lysosomal acid phosphatase (AcPase) activity was measured in individual segments of rat and rabbit nephrons employing 4-methylumbelliferyl phosphate as the substrate. Generation of reaction product was linear with incubation time up to 127 min and with tubule length. Activity was much higher in glomeruli and proximal tubules of rat than rabbit kidney. In both rat and rabbit there were higher activities in juxtamedullary than in superficial glomeruli. In rats, AcPase activity decreased from S1 to S3 segments, which parallels the known decrease in the number of lysosomes. Surprisingly, in rabbits of both sexes AcPase activity in the cortical collecting duct (CCD), which contains a limited number of lysosomes, was comparable to levels measured in the S1 and S2 segments of the proximal tubule. Similarly, in the male rat values for AcPase activity in the cortical thick ascending limb, distal convoluted tubule, CCD, and medullary collecting duct paralleled those in the S3 segment. These findings suggest that a considerable amount of AcPase in the distal nephron is either extralysosomal or that the amount of lysosomal AcPase activity per unit volume is greater in distal nephron segments compared with the proximal tubule. Different K'm values for AcPase in S1 segments and CCD were found in the rabbit, suggesting the presence of different isoenzymes.

Topics: Acid Phosphatase; Animals; Female; Fluorometry; Hymecromone; Kidney Tubules; Kinetics; Male; Nephrons; Rabbits; Rats; Rats, Inbred Strains; Sex Factors; Sodium Fluoride; Time Factors

A variety of substrates can be employed in enzyme immunoassays (EIAs) for the measurement of enzyme-labeled immunoreactants. We compared the sensitivities of a fluorescent and a colorigenic substrate in an EIA system for the measurement of Haemophilus influenza purified polyribose phosphate. After a 10-min substrate incubation, the EIA in which the fluorescent substrate was used could detect 10 pg of polyribose phosphate per ml, whereas the EIA in which the colorigenic substrate was used required the addition of 640 pg of polyribose phosphate per ml to generate a positive reading. However, the use of longer substrate incubation periods led to an increase in sensitivity of the colorigenic EIA. After an incubation period of 240 min, the sensitivity was equal to that of the EIA in which the fluorescent substrate was used. These results suggest that the ultimate limit of sensitivity of EIA systems is determined by the nature of the antigen-antibody reactions. However, the use of high-energy substrates in EIA systems can allow for the attainment of maximal sensitivity after short enzyme-substrate incubation periods.

Topics: Chromogenic Compounds; Fluorescent Dyes; Haemophilus influenzae; Hymecromone; Immunoenzyme Techniques; Pentosephosphates; Polysaccharides, Bacterial; Time Factors

Topics: Acid Phosphatase; Ascitic Fluid; Esterases; Humans; Hymecromone; Isoelectric Focusing; Isoenzymes; Macrophages; Monocytes; Pulmonary Alveoli; Umbelliferones

Enzyme immunofluorescence assays for cytomegalovirus antibodies could be read satisfactorily using a light box with ultraviolet illumination. Higher antibody titers were obtained with a fluorogenic substrate than with a color-producing substrate.

Topics: Antibodies, Viral; Cytomegalovirus; Humans; Hymecromone; Immunoenzyme Techniques; Nitrophenols; Organophosphorus Compounds; Spectrometry, Fluorescence

Three variants of the classical acyl-enzyme mechanism were compared theoretically with respect to the predicted transient kinetics of substrate hydrolysis by Escherichia coli alkaline phosphatase. In all three, acyl-enzyme hydrolysis was assumed to exist initially primarily as a noncovalent complex with the acid product, inorganic phosphate. In one mechanism, the pre-steady-state rate-controlling step was assumed to be the dissociation of acid product from its initial complex with enzyme. In the other two, pre-steady-state rate control was assigned to an enzyme isomerization occurring before or after substrate binding to free enzyme. Under concentration conditions of excess substrate and acid product, integrated rate laws were used to reject the possibility of pre-steady-state rate control by enzyme isomerization between phosphate dissociation and substrate binding. Whereas this mechanism predicts a pre-steady-state noncompetitive relationship between substrate and acid product, the stopped-flow kinetics of 4-methylumbelliferyl phosphate hydrolysis demonstrates a competitive relationship, consistent with either of the other two mechanisms. Under concentration conditions of stoichiometrically limiting substrate, computer simulations eliminated the possibility of rate control by enzyme isomerization after substrate binding. This mechanism predicts a substrate concentration dependence for the apparent first-order rate constant of substrate hydrolysis which disagrees with previously published data [Halford, S. E. (1971) Biochem. J. 125, 319--327]; the other two mechanisms are consistent with experiment. Comparison of transient kinetic theory and experiment under these two contrasting concentration conditions suggests strongly that the rate-controlling step in phosphate ester hydrolysis by E. coli alkaline phosphate is the dissociation of "sticky" acid product from its noncovalent complex with enzyme. This mechanism explains an anomaly in the stopped-flow kinetic trace, a substoichiometric pre-steady-state burst of alcohol product release.

Topics: Alkaline Phosphatase; Catalysis; Escherichia coli; Hymecromone; Kinetics; Models, Chemical; Organophosphorus Compounds; Umbelliferones