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At least 235 records · Page 13Linked to original sources
PubMed1995

A newly developed immunofluorescence assay for simultaneous detection of antibodies to human immunodeficiency virus type 1 and type 2.

Immunofluorescence assays (IFA) that simultaneously distinguish between antibodies against closely related human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) infections have not been readily available. Serum specimens from 95 HIV-1-infected, 26 HIV-2-infected and 3 HIV-1/HIV-2 dually infected individuals and 106 seronegative blood donors were evaluated for the ability to serologically discriminate HIV-1 and HIV-2 infections by means of IFA employing three types of cells whose morphology varied within one field of microscopy. Mixtures of HIV-1-infected, HIV-2-infected and uninfected cells were used in the present study. In consequence, all serum specimens from individuals infected with HIV were confirmed to contain antibodies to HIV-1 and/or HIV-2. None of the sera from the blood donors were positive. Serum specimens from HIV-1-infected or HIV-2-infected individuals were diagnosed as single infection with HIV-1 (85/95) and HIV-2 (22/26), respectively, by this new assay. Although another 14 (10/95 and 4/26) were shown to be seropositive for both HIV-1-infected and HIV-2-infected cells, these results suggest that this assay is potentially simple and useful for screening and confirming both HIV-1 and HIV-2 infections simultaneously.

Cell Line↗
PubMed2006

Detection of recombinant Alt a1 in a two-site, IgM based, sandwich ELISA opens up possibilities of developing alternative assays for the allergen.

Alternaria alternata is well known to induce IgE-mediated asthma in humans. Alt a1, a 29 kD glycoprotein doublet composed of 14.5 and 16 kD subunits, is the major allergen of this mould. Detection of Alt a1 relies on a two-site sandwich ELISA using the same IgG subclass immunoglobulin as primary and secondary antibody. In this study, we have compared two IgM monoclonal antibodies against recombinant and native Alt a1 in detecting the allergen in a two-site sandwich ELISA. Although both IgM clones detected the native and the recombinant allergen by SDS-PAGE immunoblotting and by the antibody-capture ELISA, only the IgM against recombinant Alt a1 was able to detect the corresponding, and not the native allergen, in a two-site sandwich ELISA. The IgM against native Alt a1 was unable to detect either allergen by this method. A combination of the two IgM clones and with a commercially available IgG failed to detect both allergens. However, atopic human IgE detected both forms of the allergen with the two IgM clones as primary antibody. This is the first time to demonstrate detection of Alt a1 in a two-site, IgM based, sandwich ELISA opening up possibilities for exploring novel detection methods, based on this approach.

Allergens↗
PubMed2005

High-throughput screening for inhibitors of the e3 ubiquitin ligase APC.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.

Drug Evaluation, Preclinical↗
PubMed2006

Development of assays for nuclear receptor modulators using fluorescently tagged proteins.

This chapter describes a method for designing cell-based assays to screen for nuclear receptor modulators. The basic strategy consists in following the movement of the receptors from the cytoplasm into the nucleus in response to ligand binding or analogous activating events. The receptors are tagged with green fluorescent protein for automated, fluorescent detection. In the case of constitutively nuclear receptors, they are engineered for cytoplasmic retention in the absence of an activating signal by fusing them to specific regions of the glucocorticoid receptor, which is found predominantly in the cytoplasm of cultured cells. The resulting chimeras respond to ligands or receptor modulators by translocating into the nucleus. This movement is monitored easily by automated fluorescent microscopy and serves as the basis for screening libraries. Finally, secondary assays built into the cell system can differentiate between modulators that stimulate, inhibit, or do not affect the transcriptional activity of the receptor under study. This approach has been validated for both the estrogen receptor and the retinoic acid receptor and should be applicable to any member of the superfamily, facilitating the identification of new ligands and selective receptor modulators.

Amino Acid Sequence↗
PubMed1999

A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles.

This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another.

Animals↗
PubMed1990

Improved fluorometric high-performance liquid chromatographic assay for (-)-carbovir in rat blood and urine.

Carbovir is a carbocyclic guanosine analogue with potent in vitro activity against the human immunodeficiency virus. All of the activity resides in the (-)-enantiomer. An ion-paired liquid chromatographic assay for (-)-carbovir was developed on a Spherisorb C8 column with fluorescence detection (275 nm excitation, 345 nm emission). Guanosine nucleosides are fluorescent at a pH less than 2.5, and fluorescence detection resulted in a four-fold improvement in the limit of quantitation (0.039 microgram/ml) compared to the previously developed assay with ultraviolet detection. Standard curves were processed with an internal standard at (-)-carbovir concentrations of 0.039-40 micrograms/ml in whole rat blood with a solid-phase extraction technique. Total variability was less than 16% at all concentrations and less than 10% at concentrations greater than 0.3 microgram/ml. Within-day variability was less than 7.5% at concentrations greater than 0.3 microgram/ml. Urine was analyzed directly after dilution and an diethyl ether wash to remove impurities. The total coefficients of variation were less than 10% from 0.5-20 micrograms/ml in urine. The concentrations of (-)-carbovir in rat blood were detectable for as long as 8 h after intravenous and oral doses of 20 and 60 mg/kg, respectively.

Analysis of Variance↗
PubMed2003

Combination of chiral capillary electrochromatography with electrospray ionization mass spectrometry: method development and assay of warfarin enantiomers in human plasma.

The hyphenation of chiral capillary electrochromatography (CEC) with electrospray ionization mass spectrometry (ESI-MS) is very challenging but promising due to the fact that it combines sensitivity with high specificity and selectivity. In this work, CEC capillaries packed with (3R,4S)-Whelk-O1 chiral stationary phase were used for simultaneous enantioseparation of (+/-)-warfarin and its internal standard, (+/-)-coumachlor. Furthermore, both the chiral CEC separation and MS detection parameters were examined in detail. First, the influence of different column fabrication was investigated. Second, enantioseparation was optimized by varying CEC parameters, including acetonitrile concentration, buffer pH, and ionic strength. Under the optimum chiral CEC conditions, ESI-MS parameters such as sheath liquid pH and composition, sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizer pressure, and fragmentor voltage were investigated to achieve maximum MS signals of the separated enantiomers. Finally, using solid-phase extraction as sample preparation method, (+/-)-warfarin spiked in 100-microL human plasma samples were analyzed. The calibration curves showed good linearity for both (R)-warfarin (R = 0.9979) and (S)-warfarin (R = 0.9978) enantiomers. The experimental limit of detection was approximately 25 ng/mL for both enantiomers. Even though the data are still preliminary, we can state with confidence that chiral CEC-ESI-MS has the potential to establish itself as a very powerful technique for the determination of enantiomeric ratios in human body fluid.

Buffers↗
PubMed1982

Nuclear oestrogen receptors in rat liver. Development of assay conditions, characterization and the translocation of oestrogens in vivo.

A method for the determination of specific oestrogen-receptor binding sites in rat liver nuclei is described. Nuclear receptors showed a high affinity for oestradiol (Kd approximately 3 x 10(-9)M), a low capacity, and a distinct specificity for substances with known oestrogenic and anti-oestrogenic activity. No sex differences were seen in the concentrations of nuclear receptors from either vehicle- or ethynyloestradiol-pretreated rats. Only a limited number of binding sites could be extracted with 0.4 M-KCl. The remaining sites, which were solubilized by sonication and treatment with deoxyribonuclease I, sedimented at 3-4 S. Of four oestrogens tested (oestradiol, ethynyloestradiol, diethylstilboestrol, tri-p-anisylchloroethylene), ethynyloestradiol was the most effective translocation agent in vivo, nuclear uptake occurring at doses below 1 microgram/rat; changes in salt extractability of nuclear receptors occurred at doses lower than those required to achieve absolute increases in nuclear receptor concentrations.

Animals↗
PubMed1990

Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3',5'-cyclic monophosphate-synthesizing enzyme.

A method is described for the separation of cytidine 3',5'-cyclic monophosphate (cyclic CMP) from cytidine tri-, di- and mono-phosphates and from cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate-3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine monophosphate, compounds previously shown to be the result of putative cytidylate cyclase activity. This separation, involving elution of a novel bilayer column of QAE-Sephadex and alumina with 0.03 M-HCl, has been incorporated into an assay protocol to determine the enzyme-catalysed conversion of radiolabelled CTP to cyclic CMP. By this assay, cytidylate cyclase activity has been shown to be present in rat lung, spleen, ovary, testes, brain, stomach, liver, heart and kidney preparations; the activity was of a similar order in each tissue and had a sharp pH optimum of 7.0-7.5. The liver preparation had a Vmax. of 1.2 nmol of cyclic CMP formed/min per mg, and a Km of 220 microM-CTP, and although active in the absence of added cations, it was stimulated by Fe2+ and Mn2+ ions. In several of the tissues examined, the cytidylate cyclase activity was inversely proportional to age of the animals.

Aging↗
PubMed1999

Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents.

A series of cationic nucleic acid carriers was evaluated for their ability to deliver pLuc plasmid DNA or a 2'-O-methyl-oligoribonucleoside phosphorothioate, ON-705. Oligonucleotide delivery and its antisense function were assayed by a recently developed assay based on alternative splicing of modified luciferase pre-mRNA (Kang et al., 1998). This assay scores only the nuclear and sequence-specific antisense activity of the oligonucleotides. The results show that the efficiencies of delivery of plasmid DNA and oligonucleotides by the tested carriers, with the exception of Exgene and Lipofectamine, differed markedly. The efficiency of the delivery of ON-705 oligonucleotide was reduced by 70%-90% for all carriers, except Effectene, in culture media containing 8% fetal bovine serum. Interestingly, the efficiency of delivery of the ON-705-Effectene complex increased with serum concentrations of up to 30%.

Alternative Splicing↗
PubMed2004

Axon mediated interneuron migration.

Mammalian forebrain development requires extensive cell migration for cells to reach their appropriate location in the adult brain. Defects in this migration result in human malformations and neurologic deficits. Thus, understanding the mechanisms underlying normal cell migration during development is essential to understanding the pathogenesis of human malformations. Radial glia are known to support radial cell migration, while axons have been proposed as substrate for some non-radially migrating cells. Herein we have directly tested the hypothesis that axons can support non-radial cell migration. One population of cells known to migrate non-radially is the inhibitory interneurons that move from the ganglionic eminence to the cerebral cortex. We first show that early born GABAergic cells colocalize with TAG-1-positive (TAG-1+) axons, while later born cells colocalize with intermediate weight neurofilament-positive, TAG-1-negative (TAG-1-) processes, suggesting temporal differences in substrate specificities. We next developed an in vitro assay that allows us to observe cell migration on axons in culture. Using this assay we find that early born medial ganglionic eminence-derived interneurons migrate preferentially on TAG-1+ axons, while later born cells only migrate on neurofilament-positive/TAG-1- processes. These data provide the first direct evidence that ganglionic eminence cells migrate on axons and that there is an age-dependent substrate preference. Furthermore, the assay developed and characterized herein provides a robust method to further study the molecular substrates and guidance cues of axonophilic cell migration in neural development.

Aging↗
PubMed1992

Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light. II. Results of testing three sunscreen ingredients.

Three sunscreen ingredients, derivatives of benzylidene camphor, were tested for photomutagenic potential. These were benzenesulfonic acid, 4-[(4,7,7,-trimethyl-3-oxo-bicyclo [2.2.1] hept-2-ylidene) methyl] (Mexoryl SL), 4-(2-oxo 3-bornylidenemethyl) phenyl trimethylammonium methyl sulphate (Mexoryl SO) and 3,3'-(1,4-phenylenedimethylidyne) bis [7,7-dimethyl-2-oxo-bicyclo [2.2.1] heptane-1-methanesulfonic acid] (Mexoryl SX). Two complementary assay systems were used, one involving the induction of reverse mutations in Escherichia coli strain WP2, the other measuring the induction of chromosome damage in Chinese hamster ovary (CHO) cells. Irradiation with UVA and/or UVB was provided by an Osram Ultra-Vitalux sunlamp. None of the three sunscreens, tested either to the limit of solubility or toxicity, gave any indication of photomutagenicity in either assay, under conditions in which the positive control compound, 8-methoxypsoralen, was extremely photomutagenic. It is concluded that Mexoryls SL, SO and SX can be exposed to UV light without producing photomutagenicity measurable using a bacterial reverse mutation or a mammalian chromosome aberration assay.

Animals↗
PubMed2000

Serodiagnosis of Bartonella bacilliformis infection by indirect fluorescence antibody assay: test development and application to a population in an area of bartonellosis endemicity.

Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B. bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting B. bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where B. bacilliformis is endemic and where the point prevalence of infection is 45%.

Antibodies, Bacterial↗
PubMed1994

Mitochondrial F1-ATPase activity of canine myocardium: effects of hypoxia and stimulation.

Recent studies have suggested that modifications in mitochondrial F1-adenosinetriphosphatase (ATPase) activity may play an important role in the regulation of myocardial oxidative phosphorylation. The goal of the present study was to develop and characterize an assay of F1-ATPase activity that could be performed repeatedly on an intact heart under various physiological states. With the use of submitochondrial particles prepared from biopsy samples of canine myocardium, we found reproducible F1-ATPase activity when normalized to the activity of the intramitochondrial enzyme citrate synthase. The oligomycin-sensitive component of the ATPase activity was found to be mainly F1-ATPase. F1-ATPase activity of normal myocardium increased by incubation in high salt-pH buffer, suggesting baseline inhibition. Five minutes after global ischemia, F1-ATPase activity decreased to 60% of baseline. Hypoxia for 10 min resulted in no significant change in F1-ATPase activity. With phenylephrine infusion, myocardial oxygen consumption more than doubled, whereas F1-ATPase activity increased by approximately 30%. Both returned to baseline levels after discontinuation of the drug. With the use of an assay developed to measure F1-ATPase activity of intact myocardium, changes of the enzyme activity were found during both ischemia and at increased work loads. These data suggest that alterations of F1-ATPase activity may contribute to the regulation of myocardial oxidative phosphorylation.

Adenosine Triphosphatases↗