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Sample records for assaying quantitative

  1. Immune chromatography: a quantitative radioimmunological assay

    International Nuclear Information System (INIS)

    Davis, J.W.; Demetriades, M.; Bowen, J.M.

    1984-01-01

    Immune chromatography, a radioimmunological binding assay, employs paper chromatography to separate immune complexes from free antigen and antibodies. During chromatography free antigen and antibodies become distributed throughout the paper, while immune complexes remain near the bottoms of the strips. The chromatographic differences can be made quantitative by using either iodinated antigens or antibodies. Under these conditions nanogram quantities of antigen can be detected or antibodies in sera diluted several 1000-fold. The immune chromatography assay can also be performed as an indirect assay, since the paper strips are cut from nitrocellulose paper. In this case the immune components are absorbed by the paper during chromatography. Antigen is then detected with an iodinated second antibody. The indirect immune chromatography assay is particularly useful for identifying different sera that react with the same antigen. Reaction with the first serum before chromatography reduces the amount of antigen available to the second serum following chromatography. In addition to characterizing the immune chromatography procedure, we discuss the possible applications of chromatography assays for the quantitation of other types of molecular binding interactions. (Auth.)

  2. Rapid quantitative assay for chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

    Neumann, J.R.; Morency, C.A.; Russian, K.O.

    1987-01-01

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ([ 3 H] or [ 14 C]acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM [ 14 C]acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 37 0 C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques

  3. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  4. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Science.gov (United States)

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  5. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  6. Quantitative Fissile Assay In Used Fuel Using LSDS System

    Science.gov (United States)

    Lee, YongDeok; Jeon, Ju Young; Park, Chang-Je

    2017-09-01

    A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241) was done at Korea Atomic Energy Research Institute (KAERI), using lead slowing down spectrometer (LSDS). The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with 2% uncertainty for Pu239. By applying the covering (neutron absorber), the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  7. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

    Science.gov (United States)

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

    2015-03-30

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

  8. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    Science.gov (United States)

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  9. Assay Validation For Quantitation of Sn 2+ In Radiopharmaceutical Kits

    International Nuclear Information System (INIS)

    Muthalib, A; Ramli, Martalena; Herlina; Sarmini, Endang; Suharmadi; Besari, Canti

    1998-01-01

    An assay validation for quantitation of Sn2+ in radiopharmaceutical kits based on indirect iodometric titration is described. The method is based on the oxidation of sn2+ using a known excess of iodine and the excess unreacted iodine titrated with thiosulphate. Typical analytical parameters considered in this assay validation are precision, accuracy, selectivity or specificity, range, and linearity. The precision of the analytical method is quit good represented by coefficient of variance in range of 1.0% to 6.9 %, for 10 runs of analysis except one analysis shows the coefficient of 10.2 %. The method has an accuracy of 95.6 % - 99 % as percent recoveries at theoretical Sn2+ amounts of 463 μg to 2318μg

  10. [Clinical evaluation of a novel HBsAg quantitative assay].

    Science.gov (United States)

    Takagi, Kazumi; Tanaka, Yasuhito; Naganuma, Hatsue; Hiramatsu, Kumiko; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi

    2007-07-01

    The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations in HBV-infected individuals remains unclear. The aim of this study was to evaluate a novel fully automated Chemiluminescence Enzyme Immunoassay (Sysmex HBsAg quantitative assay) by comparative measurements of the reference serum samples versus two independent commercial assays (Lumipulse f or Architect HBsAg QT). Furthermore, clinical usefulness was assessed for monitoring of the serum HBsAg levels during antiviral therapy. A dilution test using 5 reference-serum samples showed linear correlation curve in range from 0.03 to 2,360 IU/ml. The HBsAg was measured in total of 400 serum samples and 99.8% had consistent results between Sysmex and Lumipulse f. Additionally, a positive linear correlation was observed between Sysmex and Architect. To compare the Architect and Sysmex, both methods were applied to quantify the HBsAg in serum samples with different HBV genotypes/subgenotypes, as well as in serum contained HBV vaccine escape mutants (126S, 145R). Correlation between the methods was observed in results for escape mutants and common genotypes (A, B, C) in Japan. Observed during lamivudine therapy, an increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for all HBV genetic variants common in Japan. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response to lamivudine treatment and diagnosis of the breakthrough hepatitis.

  11. Quantitative relationship between the local lymph node assay and human skin sensitization assays.

    Science.gov (United States)

    Schneider, K; Akkan, Z

    2004-06-01

    The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to

  12. Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

    Science.gov (United States)

    Ackerman, S B; Kelley, E A

    1983-03-01

    The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

  13. A new trend to determine biochemical parameters by quantitative FRET assays.

    Science.gov (United States)

    Liao, Jia-yu; Song, Yang; Liu, Yan

    2015-12-01

    Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

  14. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

    2017-10-23

    Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

  15. Quantitative CrAssphage PCR Assays for Human Fecal ...

    Science.gov (United States)

    Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

  16. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld

    2011-01-01

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency...

  17. A multiparametric assay for quantitative nerve regeneration evaluation

    OpenAIRE

    Weyn, Barbara; Van Remoortere, M; Nuydens, R; Meert, Theo; Van de Wouwer, G

    2005-01-01

    We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet t...

  18. A multiparametric assay for quantitative nerve regeneration evaluation.

    Science.gov (United States)

    Weyn, B; van Remoortere, M; Nuydens, R; Meert, T; van de Wouwer, G

    2005-08-01

    We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.

  19. Gold nanoparticle immunochromatographic assay for quantitative detection of urinary RBP

    Directory of Open Access Journals (Sweden)

    XU Kuan

    2013-04-01

    Full Text Available A rapid quantitative detection of urinary RBP was established by using nano-gold immunochromatography (sandwich method and trisodium citrate reduction method and a rapid immunochromatographic test strip was developed. Theimmunochromatographic test strip can quantitatively detect RBP within 15 minutes. The detection limit was 150ng/mL and detection range was from 150 to 5000 ng/mL. There were no cross-reactions with others kidney disease markers,such as urinary albumin (ALB,transferrin protein (TRF,β2-microglobulin (β2-MG,urinary fiber connecting protein (FN,and lysozyme (LZM. The results indicate that it is a quick and simple method with strong specificity,high sensitivity,and wide detection range. The rapid detection method will have extensive clinical applications in the early diagnosis of proximal tubular damage,kidney disease,diabetic nephropathy,and process monitoring.

  20. Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, J P; Vladutiu, A O; Moreno, D M; Cohen, S A; Camara, D S [State University of New York, Buffalo (USA). School of Medicine

    1982-11-26

    The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay. The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself. Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins. The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide. Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

  1. Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study

    NARCIS (Netherlands)

    Hammond, Emma L.; Sayer, David; Nolan, David; Walker, Ulrich A.; Ronde, Anthony de; Montaner, Julio S. G.; Cote, Helene C. F.; Gahan, Michelle E.; Cherry, Catherine L.; Wesselingh, Steven L.; Reiss, Peter; Mallal, Simon

    2003-01-01

    Background: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. Objectives: A collaborative study was undertaken to evaluate intra-assay precision and between

  2. Smartphone based visual and quantitative assays on upconversional paper sensor.

    Science.gov (United States)

    Mei, Qingsong; Jing, Huarong; Li, You; Yisibashaer, Wuerzha; Chen, Jian; Nan Li, Bing; Zhang, Yong

    2016-01-15

    The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    Science.gov (United States)

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Gamma aminobutyric acid radioreceptor assay: a confirmatory quantitative assay for toxaphene in environmental and biological samples

    International Nuclear Information System (INIS)

    Saleh, M.A.; Blancato, J.N.

    1993-01-01

    Toxaphene is a complex mixture of polychlorinated monoterpenes, and was found to be acutely and chronically toxic to aquatic and wild life and posed a carcinogenic risk to humans before its ban in 1982. However, it is still found in the environment due to its relative persistence with an estimated half life time of about 10 years in soils. Toxaphenes neurotoxicity is attributed to a few isomers with a mode of action through binding to the chloride channel of the gamma-aminobutyric acid (GABA) receptor ionophore complex. [ 35 S] tertiary butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABA receptor for the development of radioreceptor assay technique. The GABA receptor was prepared by a sequence of ultra centrifugation and dialysis of mammalian (rats, cows, catfish and goats) brain homogenates. The receptor is then labeled with [ 35 S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with the sample containing the analytes. The assay is fast, sensitive and requires very little or no sample preparation prior to the analysis. (Author)

  5. A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

    Science.gov (United States)

    Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

    2017-10-01

    In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

  6. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

    Science.gov (United States)

    Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung

    2013-03-01

    The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Microfluorometric mithramycin assay for quantitating the effects of immunotoxicants on lymphocyte activation

    International Nuclear Information System (INIS)

    Quattrone, A.J.; Ranney, D.F.

    1981-01-01

    A semiautomated, microfluorometric assay has been developed for the detection of toxicant-induced changes in lymphocyte DNA content at standard intervals after mitogen activation. DNA is quantitated by solubilizing the cells and determining the fluorescence enhancement that results from formation of the highly specific mithramycin:DNA adduct. The limit of detection is 0.21 μg (30,000 resting cell equivalents) per microliter well. Correlation with the less sensitive, nonautomatable, diphenylamine DNA assay give a correlation coefficient r = 0.91. Prototype substances representative of true immunotoxicants (prostaglandin E 2 ) and common interfering substances (thymidine at 14 M) have been tested. The latter substance produces false positive results in the standard [ 3 H] thymidine assay. The mithramycin assay does not inappropriately detect this interfering substance. It has the characteristics of a highly specific, accurate technique of screening and quantitating immunotoxic drugs, agents, and mediators in patient sera and other complex biological fluids

  8. Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

    International Nuclear Information System (INIS)

    Kasprzyk, P.G.; Cuttitta, F.; Avis, I.; Nakanishi, Y.; Treston, A.; Wong, H.; Walsh, J.H.; Mulshine, J.L.

    1988-01-01

    A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones

  9. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    Science.gov (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  10. High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

    Science.gov (United States)

    Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

    2002-01-01

    A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

  11. Quantitative assay for the measurement of immune responses directed against the human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Davies, M; Sutcliffe, R G [Glasgow Univ. (UK)

    1982-02-12

    A quantitative in vitro immune assay based on the classical chromium release assay has been developed to detect immune responses directed against alien antigens expressed by the developing foetus and present on the maternal-facing surface of the human placenta. A plasma membrane fraction from the surface of the placenta was prepared and the vesicles thus formed were radiolabelled with /sup 51/Cr. The /sup 51/Cr-labelled vesicles, by various criteria, were found to be suitable for use as targets in a release assay. Further, by means of experimentally immunised animals, the target membranes were shown to be capable of detecting both cellular and humoral anti-placental activity.

  12. A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

    Science.gov (United States)

    Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

    2012-01-01

    ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

  13. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  14. A novel assay to quantitate MASP-2/ficolin-3 complexes in serum

    DEFF Research Database (Denmark)

    Csuka, Dorottya; Munthe-Fog, Lea; Skjoedt, Mikkel-Ole

    2013-01-01

    in the circulation. The significance of lectin pathway complexes in the circulation is unknown. Thus, we established an assay for the measurement of circulating MASP-2/ficolin-3 complexes. A quantitative sandwich ELISA was developed for the measurement of the MASP-2/ficolin-3 complexes in serum based on monoclonal...

  15. A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

    Science.gov (United States)

    Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

    1984-10-01

    A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

  16. [Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

    Science.gov (United States)

    Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

    2005-07-01

    To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

  17. A multiplex branched DNA assay for parallel quantitative gene expression profiling.

    Science.gov (United States)

    Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

    2006-05-01

    We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

  18. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    International Nuclear Information System (INIS)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

  19. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    Science.gov (United States)

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. © 2015 Society for Laboratory Automation and Screening.

  20. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  1. Small unilamellar vesicles as reagents: a chemically defined, quantitative assay for lectins

    Energy Technology Data Exchange (ETDEWEB)

    Rando, R.R.

    1981-01-01

    Samll unilamellar vesicles containing synthetic glycolipids can be prepared. These vesicles are aggregated by the appropriate lectin (Orr et al., 1979; Rando and Bangerter, 1979; Slama and Rando, 1980). It is shown here that extent of aggregation of these vesicles as measured by light scattering at 360 nm, is, under certain conditions, linear with amount of lectin added. This forms the basis of a rapid and simple quantitative assay for lectins using the modified vesicles as a defined chemical substrate. The assay is sensitive to lectin concentrations in the low ..mu..g range. The assay is applied here to studies on concanavalin A, Ricinus communis agglutinin and the ..cap alpha..-fucosyl binding lectin from Ulex europaeus (Type I).

  2. A quantitative assay measuring the function of lipase maturation factor 1

    Science.gov (United States)

    Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós

    2009-01-01

    Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043

  3. A new sensitive and quantitative HTLV-I-mediated cell fusion assay in T cells

    International Nuclear Information System (INIS)

    Pare, Marie-Eve; Gauthier, Sonia; Landry, Sebastien; Sun Jiangfeng; Legault, Eric; Leclerc, Denis; Tanaka, Yuetsu; Marriott, Susan J.; Tremblay, Michel J.; Barbeau, Benoit

    2005-01-01

    Similar to several other viruses, human T cell leukemia virus type I (HTLV-I) induces the formation of multinucleated giant cells (also known as syncytium) when amplified in tissue culture. These syncytia result from the fusion of infected cells with uninfected cells. Due to the intrinsic difficulty of infecting cells with cell-free HTLV-I virions, syncytium formation has become an important tool in the study of HTLV-I infection and transmission. Since most HTLV-I-based cell fusion assays rely on the use of non-T cells, the aim of this study was to optimize a new HTLV-I-induced cell fusion assay in which HTLV-I-infected T cell lines are co-cultured with T cells that have been transfected with an HTLV-I long terminal repeat (LTR) luciferase reporter construct. We demonstrate that co-culture of various HTLV-I-infected T cells with different transfected T cell lines resulted in induction of luciferase activity. Cell-to-cell contact and expression of the viral gp46 envelope protein was crucial for this induction while other cell surface proteins (including HSC70) did not have a significant effect. This quantitative assay was shown to be very sensitive. In this assay, the cell fusion-mediated activation of NF-κB and the HTLV-I LTR occurred through previously described Tax-dependent signaling pathways. This assay also showed that cell fusion could activate Tax-inducible cellular promoters. These results thus demonstrate that this new quantitative HTLV-I-dependent cell fusion assay is versatile, highly sensitive, and can provide an important tool to investigate cellular promoter activation and intrinsic signaling cascades that modulate cellular gene expression

  4. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M

    1994-01-01

    variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer......Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal...... antibodies recognizing the non-ligand binding part of this receptor, and it detects both free and occupied uPAR, in contrast to ligand-binding assays used previously. In a variant of the assay, the occupied fraction of uPAR is selectively detected with a uPA antibody. To be used as a standard, a soluble...

  5. Development and validation of a quantitative PCR assay for Ichthyophonus spp.

    Science.gov (United States)

    White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

    2013-04-29

    Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

  6. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

    Directory of Open Access Journals (Sweden)

    Sylvie Faucher

    Full Text Available BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127 and common gamma (CD132 chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127 is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

  7. Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

    Directory of Open Access Journals (Sweden)

    Hanae Takatsuki

    Full Text Available The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50 is reached approximately 10(10/g brain (values varies 10(8.79-10.63/g. A genetic case (GSS-P102L yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

  8. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  9. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

    Directory of Open Access Journals (Sweden)

    Adam R Brown

    Full Text Available Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin, were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.

  10. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  11. Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

    Science.gov (United States)

    Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

    2017-06-01

    Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

  12. Development of a Quantitative PCR Assay for Thermophilic Spore-Forming Geobacillus stearothermophilus in Canned Food.

    Science.gov (United States)

    Nakano, Miyo

    2015-01-01

    The thermophilic spore forming bacteria Geobacillus stearothermophilus is recognized as a major cause of spoilage in canned food. A quantitative real-time PCR assay was developed to specifically detect and quantify the species G. stearothermophilus in samples from canned food. The selected primer pairs amplified a 163-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 12.5 fg of pure culture DNA, corresponding to DNA extracted from approximately 0.7 CFU/mL of G. stearothermophilus. Analysis showed that the bacterial species G. stearothermophilus was not detected in any canned food sample. Our approach presented here will be useful for tracking or quantifying species G. stearotethermophilus in canned food and ingredients.

  13. Phytochrome quantitation in crude extracts of Avena by enzyme-linked immunosorbent assay with monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Y; Cordonnier, M M; Pratt, L H

    1983-01-01

    An enzyme-linked immunosorbent assay (ELISA), which uses both rabbit polyclonal and mouse monoclonal antibodies to phytochrome, has been adapted for quantitation of phytochrome in crude plant extracts. The assay has a detection limit of about 100 pg phytochrome and can be completed within 10 h. Quantitation of phytochrome in crude extracts of etiolated oat seedlings by ELISA gave values that agreed well with those obtained by spectrophotometric assay. When etiolated oat seedlings were irradiated continuously for 24 h, the amount of phytochrome detected by ELISA and by spectrophotometric assay decreased by more than 1000-fold and about 100-fold, respectively. This discrepancy indicates that phytochrome in light-treated plants may be antigenically distinct from that found in fully etiolated plants. When these light-grown oat seedlings were kept in darkness for 48 h, phytochrome content detected by ELISA increased by 50-fold in crude extracts of green oat shoots, but only about 12-fold in extracts of herbicide-treated oat shoots. Phytochrome reaccumulation in green oat shoots was initially more rapid in the more mature cells of the primary leaf tip than near the basal part of the shoot. The inhibitory effect of Norflurazon on phytochrome accumulation was much more evident near the leaf tip than the shoot base. A 5-min red irradiation of oat seedlings at the end of a 48-h dark period resulted in a subsequent, massive decrease in phytochrome content in crude extracts from both green and Norflurazon-bleached oat shoots. These observations eliminate the possibility that substantial accumulation of chromophore-free phytochrome was being detected and indicate that Norflurazon has a substantial effect on phytochrome accumulation during a prolonged dark period. 25 references, 9 figures, 3 tables.

  14. Analysis of Nanobody-Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.

    Science.gov (United States)

    Früholz, Simone; Pimpl, Peter

    2017-01-01

    Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.

  15. Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

    Science.gov (United States)

    Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

    2017-01-01

    Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

  16. Quantitative genotoxicity assays for analysis of medicinal plants: A systematic review.

    Science.gov (United States)

    Sponchiado, Graziela; Adam, Mônica Lucia; Silva, Caroline Dadalt; Soley, Bruna Silva; de Mello-Sampayo, Cristina; Cabrini, Daniela Almeida; Correr, Cassyano Januário; Otuki, Michel Fleith

    2016-02-03

    Medicinal plants are known to contain numerous biologically active compounds, and although they have proven pharmacological properties, they can cause harm, including DNA damage. Review the literature to evaluate the genotoxicity risk of medicinal plants, explore the genotoxicity assays most used and compare these to the current legal requirements. A quantitative systematic review of the literature, using the keywords "medicinal plants", "genotoxicity" and "mutagenicity", was undertakenQ to identify the types of assays most used to assess genotoxicity, and to evaluate the genotoxicity potential of medicinal plant extracts. The database searches retrieved 2289 records, 458 of which met the inclusion criteria. Evaluation of the selected articles showed a total of 24 different assays used for an assessment of medicinal plant extract genotoxicity. More than a quarter of those studies (28.4%) reported positive results for genotoxicity. This review demonstrates that a range of genotoxicity assay methods are used to evaluate the genotoxicity potential of medicinal plant extracts. The most used methods are those recommended by regulatory agencies. However, based on the current findings, in order to conduct a thorough study concerning the possible genotoxic effects of a medicinal plant, we indicate that it is important always to include bacterial and mammalian tests, with at least one in vivo assay. Also, these tests should be capable of detecting outcomes that include mutation induction, clastogenic and aneugenic effects, and structural chromosome abnormalities. In addition, the considerable rate of positive results detected in this analysis further supports the relevance of assessing the genotoxicity potential of medicinal plants. Copyright © 2016. Published by Elsevier Ireland Ltd.

  17. A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

    Science.gov (United States)

    Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

    2013-12-27

    Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

  18. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    ) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect r = 0.99) over...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  19. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    Science.gov (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

  20. Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

    Science.gov (United States)

    Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

    2015-02-06

    Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

  1. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

  2. A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples.

    Science.gov (United States)

    Jiang, Bo; Li, Guanghe; Xing, Yi; Zhang, Dayi; Jia, Jianli; Cui, Zhisong; Luan, Xiao; Tang, Hui

    2017-10-01

    Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013. The chromium(VI) contaminated soils were pretreated by water extraction, and directly exposed to the bioreporter in two phases: aqueous soil extraction (water phase) and soil supernatant (solid phase). The results indicated that both extractable and soil particle fixed chromium(VI) were bioavailable to the bioreporter, and the solid-phase contact bioreporter assay provided a more precise evaluation of soil genotoxicity. For crude oil contaminated seawater, the response of the bioreporter clearly illustrated the spatial and time change in genotoxicity surrounding the spill site, suggesting that the crude oil degradation process decreased the genotoxic risk to ecosystem. In addition, the performance of the bioreporter was simulated by a modified cross-regulation gene expression model, which quantitatively described the DNA damage response of the E. coli bioreporter. Accordingly, the bioluminescent response of the bioreporter was calculated as the mitomycin C equivalent, enabling quantitative comparison of genotoxicities between different environmental samples. This bioreporter assay provides a rapid and sensitive screening tool for direct genotoxicity assessment of environmental samples. Copyright © 2017. Published by Elsevier Ltd.

  3. A Quantitative Point-of-Need Assay for the Assessment of Vitamin D3 Deficiency.

    Science.gov (United States)

    Vemulapati, S; Rey, E; O'Dell, D; Mehta, S; Erickson, D

    2017-10-26

    Vitamin D is necessary for the healthy growth and development of bone and muscle. Vitamin D deficiency, which is present in 42% of the US population, is often undiagnosed as symptoms may not manifest for several years and long-term deficiency has been linked to osteoporosis, diabetes and cancer. Currently the majority of vitamin D testing is performed in large-scale commercial laboratories which have high operational costs and long times-to-result. Development of a low-cost point-of-need assay could be transformative to deficiency analysis in limited-resource settings. The best biomarker of vitamin D status, 25hydroxyvitamin D 3 (25(OH)D 3 ), however, is particularly challenging to measure in such a format due to complexities involved in sample preparation, including the need to separate the marker from its binding protein. Here we present a rapid diagnostic test for the accurate, quantitative assessment of 25(OH)D 3 in finger-stick blood. The assay is accompanied by a smartphone-assisted portable imaging device that can autonomously perform the necessary image processing. To achieve accurate quantification of 25(OH)D 3 , we also demonstrate a novel elution buffer that separates 25(OH)D 3 from its binding protein in situ, eliminating the need for sample preparation. In human trials, the accuracy of our platform is 90.5%.

  4. A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

    Directory of Open Access Journals (Sweden)

    Symonds Meegan L

    2002-07-01

    Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

  5. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

    Science.gov (United States)

    Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

    2018-01-01

    Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

  6. Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

    Science.gov (United States)

    Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

    2018-01-01

    Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

  7. Characterization of a method for quantitating food consumption for mutation assays in Drosophila

    International Nuclear Information System (INIS)

    Thompson, E.D.; Reeder, B.A.; Bruce, R.D.

    1991-01-01

    Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period. Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity

  8. Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

    Directory of Open Access Journals (Sweden)

    Roberta Fedriga

    2001-01-01

    Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

  9. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    Science.gov (United States)

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  10. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD......) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...

  11. Quantitation of biotinylated compounds by a solid-phase assay using a /sup 125/I-labelled biotin derivative

    Energy Technology Data Exchange (ETDEWEB)

    Smith, P J; Warren, R M; Holt, C von

    1987-05-11

    The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with /sup 125/I. This analogue has been used in conjunction with immobilized streptavidin in an assay which detects as little as 1 fmol biotin or biotinylated molecules in solution. The determination of biotinylated insulin in a tissue extract and the quantitation of a transcription assay are given as examples. (Auth.). 12 refs.; 4 figs.; 3 tabs.

  12. Quantitative assay for the number of leukemic spleen colony forming unit in radiation-induced murine myeloid leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Nara, N [Tokyo Medical and Dental Univ. (Japan). School of Medicine; Bessho, M

    1981-11-01

    In mice with myelogenous leukemia, leukemic spleen colony forming units were assayed quantitatively. When 5 x 10/sup 3/ - 2 x 10/sup 4/ leukemic cells were transplanted to other mice of the same strain, a rectilinear relationship (p < 0.01) was found between the number of the cells transplanted and that of the colonies formed on the surface of the spleen. From these results, the authors considered that myelogenous leukemia in mice is an adequate model for acute myelogenous leukemia in human adults, and that the quantitative assay of the leukemic colony forming units can be used for sensitivity tests of antileukemic agents.

  13. A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

    Science.gov (United States)

    Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

    2017-01-01

    A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

  14. Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

    International Nuclear Information System (INIS)

    Schwarz, S.; Ellen, R.P.; Grove, D.A.

    1987-01-01

    There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of 3 H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence

  15. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN

    Directory of Open Access Journals (Sweden)

    Hans Bigalke

    2015-11-01

    Full Text Available The historical method for the detection of botulinum neurotoxin (BoNT is represented by the mouse bioassay (MBA measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.

  16. Development of stable isotope dilution assays for the quantitation of Amadori compounds in foods.

    Science.gov (United States)

    Meitinger, Michael; Hartmann, Sandra; Schieberle, Peter

    2014-06-04

    During thermal processing of foods, reducing carbohydrates and amino acids may form 1-amino-1-desoxyketoses named Amadori rearrangement products after the Italian chemist Mario Amadori. Although these compounds are transient intermediates of the Maillard reaction, they are often used as suitable markers to measure the extent of a thermal food processing, such as for spray-dried milk or dried fruits. Several methods are already available in the literature for their quantitation, but measurements are often done with external calibration without addressing losses during the workup procedure. To cope with this challenge, stable isotope dilution assays in combination with LC-MS/MS were developed for the glucose-derived Amadori products of the seven amino acids valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and histidine using the respective synthesized [(13)C6]-labeled isotopologues as internal standards. The quantitation of the analytes added to a model matrix showed a very good sensitivity with the lowest limits of detection for the Amadori compound of phenylalanine of 0.1 μg/kg starch and 0.2 μg/kg oil, respectively. Also, the standard deviation measured in, for example, wheat beer was only ±2% for this analyte. Application of the method to several foods showed the highest concentrations of the Amadori product of valine in unroasted cocoa (342 mg/kg) as well as in dried bell pepper (3460 mg/kg). In agreement with literature data, drying of foods led to the formation of Amadori products, whereas they were degraded during roasting of, for example, coffee or cocoa. The study presents for the first time results on concentrations of the Amadori compounds of tyrosine and histidine in foods.

  17. Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

    International Nuclear Information System (INIS)

    Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo

    2011-01-01

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA) based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA) based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R 2= 0.838, P 2= 0.067, P=0.316 by IRMA, and R 2= 0.101, P=0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA based method in chronic hepatitis B patients

  18. Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo [Seoul National Univ. Seoul (Korea, Republic of)

    2011-03-15

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA) based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA) based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R{sup 2=}0.838, P<0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R{sup 2=}0.067, P=0.316 by IRMA, and R{sup 2=}0.101, P=0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA based method in chronic hepatitis B patients.

  19. Quantitation of Rotundone in Grapefruit (Citrus paradisi) Peel and Juice by Stable Isotope Dilution Assay.

    Science.gov (United States)

    Nakanishi, Akira; Fukushima, Yusuke; Miyazawa, Norio; Yoshikawa, Keisuke; Maeda, Tomoko; Kurobayashi, Yoshiko

    2017-06-21

    Aroma extract dilution analyses of the aromas of peels and juices of white and pink grapefruits revealed that rotundone, responsible for peppery, spicy, and woody odors, was detected for the first time at high flavor dilution factors of 256-1024. In both juices, rotundone was detected at the highest flavor dilution factor of 1024. Rotundone in grapefruits was quantitated by a stable isotope dilution assay with a newly synthesized deuterium-labeled internal standard, rotundone-d 2,3 : its levels were 2180 and 1920 ng/kg in white and pink grapefruit peels and 29.6 and 49.8 ng/kg in white and pink grapefruit juices, respectively. On the basis of these results, sensory analysis was performed to assess the effects of rotundone on a white grapefruit juice aroma reconstitute. This sensory analysis revealed that rotundone does not impart a woody odor or affect any of the existing attributes, but increases various attributes, thus confirming that rotundone is indispensable for the aroma of grapefruit juice.

  20. Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

    Science.gov (United States)

    The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

  1. Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships (MCBIOS)

    Science.gov (United States)

    Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships Jie Liu1,2, Richard Judson1, Matthew T. Martin1, Huixiao Hong3, Imran Shah1 1National Center for Computational Toxicology (NCCT), US EPA, RTP, NC...

  2. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    Science.gov (United States)

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  3. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer......A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD...

  4. A comparison of sperm agglutination and immobilization assays with a quantitative ELISA for anti-sperm antibody in serum.

    Science.gov (United States)

    Lynch, D M; Leali, B A; Howe, S E

    1986-08-01

    An enzyme-linked immunosorbent assay (ELISA) that quantitates antisperm antibody in serum was compared with standard sperm agglutination and immobilization assays with the use of sera from 40 normal and 292 subfertile individuals. Quantitation of the assay was accomplished by standardizing assay parameters, including the incorporation of a standard reference curve, the number of whole target sperm, the optimal dilution of serum, the selection of microtiter plate, and the time and temperatures involved in the adsorption and incubation phases. With this method, the level of antisperm antibody binding to target sperm in 40 normal fertile individuals was found to be 2.3 (+/- 1.1 standard deviation [SD]) fg immunoglobulin (Ig)/sperm. An increased mean level of 7.4 +/- 3.7 fg Ig/sperm was determined in 84 infertile patients with positive agglutination and/or immobilization tests. In 208 individuals with negative agglutination and immobilization tests the mean concentration of antisperm antibody was 2.5 +/- 1.3 fg Ig/sperm. Postvasectomy patients assayed by this method had a mean Ig binding value of 7.1 +/- 2.4 fg Ig/sperm. The infertile group with positive agglutination and/or immobilization tests had a significantly higher mean antisperm antibody level than the normal fertile group, according to the Student's t-test for independent samples (P less than 0.001). This indirect serum-based assay reproducibly quantitates antisperm antibody binding to whole target sperm, suggests the normal and abnormal levels of antisperm antibody, and correlates with standard functional assays.

  5. Quantitative Assay of Pu-239 and Pu-240 by Neutron Transmission Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, E

    1971-04-15

    A method for quantitative assay of 239Pu and 240Pu has been tested at the reactor R1 in Stockholm. The method makes use of a fast chopper to measure the neutron transmission through a sample around the main resonances of these two isotopes - at 0.296 eV in 239Pu and at 1.056 eV in 240Pu. The transmission data measured are then combined with the known resonance cross sections to give the content of the isotopes. The method is nondestructive, i.e., one can use fuel pins as samples, even highly irradiated ones. A time-of-flight spectrometer of moderate capacity, like our fast chopper, is sufficient as the resonances are located at low energy. Altogether five samples have been used in the tests of the method. The results have been compared with mass spectrometer values. This comparison came out quite well for 239Pu whereas the chopper results for 240Pu were more than 10 per cent higher than the mass spectrometer results. This large deviation might be due to errors in the resonance cross section for 240Pu used in the analysis of the transmission data from the chopper. The best possible accuracy for a 15-hour run with our equipment is +- 1 per cent for 239Pu and +- 2 per cent for 240Pu, obtained for thick samples - about 3 x 1020 atoms per cm2 for each isotope. The accuracy corresponds to 68 per cent confidence level and does not include any contribution from the uncertainty in the resonance cross section

  6. Clinical performance of a new hepatitis B surface antigen quantitative assay with automatic dilution

    Directory of Open Access Journals (Sweden)

    Ta-Wei Liu

    2015-01-01

    Full Text Available Hepatitis B virus surface antigen (HBsAg levels reflect disease status and can predict the clinical response to antiviral treatment; however, the emergence of HBsAg mutant strains has become a challenge. The Abbott HBsAg quantification assay provides enhanced detection of HBsAg and HBsAg mutants. We aimed to evaluate the performance of the Abbott HBsAg quantification assay with automatic sample dilutions (shortened as automatic Architect assay, compared with the Abbott HBsAg quantification assay with manual sample dilutions (shortened as manual Architect assay and the Roche HBsAg quantification assay with automatic sample dilutions (shortened as Elecsys. A total of 130 sera samples obtained from 87 hepatitis B virus (HBV-infected patients were collected to assess the correlation between the automatic and manual Architect assays. Among the 87 patients, 41 provided 42 sera samples to confirm the linearity and reproducibility of the automatic Architect assay, and find out the correlation among the Elecsys and two Architect assays. The coefficients of variation (0.44–9.53% and R2 = 0.996–1, which were both determined using values obtained from the automatic Architect assay, showed good reproducibility and linearity. Results of the two Architect assays demonstrated a feasible correlation (n = 130 samples; R = 0.898, p  0.93 in all cases. In conclusion, the correlation between the automatic and manual dilution Architect assays was feasible, particularly in the HBeAg-negative and low DNA groups. With lower labor costs and less human error than the manual version, the Abbott automatic dilution Architect assay provided a good clinical performance with regard to the HBsAg levels.

  7. A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

    Science.gov (United States)

    Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

    2018-05-02

    Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

  8. Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

    Science.gov (United States)

    2013-01-01

    Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

  9. Quantitative PCR Assays for Detecting Loach Minnow (Rhinichthys cobitis and Spikedace (Meda fulgida in the Southwestern United States.

    Directory of Open Access Journals (Sweden)

    Joseph C Dysthe

    Full Text Available Loach minnow (Rhinichthys cobitis and spikedace (Meda fulgida are legally protected with the status of Endangered under the U.S. Endangered Species Act and are endemic to the Gila River basin of Arizona and New Mexico. Efficient and sensitive methods for monitoring these species' distributions are critical for prioritizing conservation efforts. We developed quantitative PCR assays for detecting loach minnow and spikedace DNA in environmental samples. Each assay reliably detected low concentrations of target DNA without detection of non-target species, including other cyprinid fishes with which they co-occur.

  10. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

    Science.gov (United States)

    Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

    2014-01-01

    Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

  11. Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

    Science.gov (United States)

    Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

    2017-05-29

    Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

    Science.gov (United States)

    Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

    2010-12-01

    A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

  13. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

    DEFF Research Database (Denmark)

    Kruse, N.; Persson, S.; Alcolea, D.

    2015-01-01

    (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when...... the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings...... assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program -Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits...

  14. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

    Science.gov (United States)

    Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

    2011-05-01

    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

  16. Quantitative radiological characterization of waste. Integration of gamma spectrometry and passive/active neutron assay

    Energy Technology Data Exchange (ETDEWEB)

    Simone, Gianluca; Mauro, Egidio; Gagliardi, Filippo; Gorello, Edoardo [Nucleco S.p.A., Rome (Italy)

    2016-06-15

    The radiological characterization of drums through Non-Destructive Assay (NDA) techniques commonly relies on gamma spectrometry. This paper introduces the procedure developed in Nucleco for the NDA radiological characterization of drums when the presence of Special Nuclear Material (SNM) is expected/observed. The procedure is based on the integration of a gamma spectrometry in SGS mode (Segmented Gamma Scanner) and a passive/active neutron assay. The application of this procedure is discussed on a real case of drums. The extension of the integration procedure to other gamma spectrometry systems is also discussed.

  17. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  18. A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

    Science.gov (United States)

    Glenney, Gavin W; Barbash, Patricia A; Coll, John A

    2016-03-01

    Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

  19. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

    Directory of Open Access Journals (Sweden)

    Zsolt Czimmerer

    Full Text Available Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

  20. Development of a quantitative PCR assay for measurement of trichechid herpesvirus 1 load in the Florida manatee ( Trichechus manatus latirostris).

    Science.gov (United States)

    Ferrante, Jason A; Cortés-Hinojosa, Galaxia; Archer, Linda L; Wellehan, James F X

    2017-07-01

    Trichechid herpesvirus 1 (TrHV-1) is currently the only known herpesvirus in any sirenian. We hypothesized that stress may lead to recrudescence of TrHV-1 in manatees, thus making TrHV-1 a potential biomarker of stress. We optimized and validated a TrHV-1 real-time quantitative probe hybridization PCR (qPCR) assay that was used to quantify TrHV-1 in manatee peripheral blood mononuclear cells (PBMCs). Average baseline TrHV-1 loads in a clinically healthy wild Florida manatee ( Trichechus manatus latirostris) population ( n = 42) were 40.9 ± SD 21.2 copies/100 ng DNA; 19 of 42 manatees were positive. TrHV-1 loads were significantly different between the 2 field seasons ( p < 0.025). This optimized and validated qPCR assay may be used as a tool for further research into TrHV-1 in Florida manatees.

  1. Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.

    Science.gov (United States)

    Pape, B E; Cary, P L; Clay, L C; Godolphin, W

    1983-01-01

    Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.

  2. QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers

    Science.gov (United States)

    Brener, Oleksandr; Dunkelmann, Tina; Gremer, Lothar; van Groen, Thomas; Mirecka, Ewa A.; Kadish, Inga; Willuweit, Antje; Kutzsche, Janine; Jürgens, Dagmar; Rudolph, Stephan; Tusche, Markus; Bongen, Patrick; Pietruszka, Jörg; Oesterhelt, Filipp; Langen, Karl-Josef; Demuth, Hans-Ulrich; Janssen, Arnold; Hoyer, Wolfgang; Funke, Susanne A.; Nagel-Steger, Luitgard; Willbold, Dieter

    2015-01-01

    Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease. PMID:26394756

  3. Quantitative dynamic nuclear polarization‐NMR on blood plasma for assays of drug metabolism

    DEFF Research Database (Denmark)

    Lerche, Mathilde Hauge; Meier, Sebastian; Jensen, Pernille Rose

    2011-01-01

    ‐scan 13C DNP‐NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP‐NMR data with an established analytical method (liquid chromatography‐mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP...

  4. Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

    International Nuclear Information System (INIS)

    Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

    1989-01-01

    A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells

  5. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  6. Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

    Science.gov (United States)

    Ferrante, Jason; Hunter, Margaret; Wellehan, James F.X.

    2018-01-01

    Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (Puse as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

  7. Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

    Science.gov (United States)

    Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

    2015-07-01

    Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    Science.gov (United States)

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue

    Directory of Open Access Journals (Sweden)

    Bodil Hakonen

    2014-01-01

    Full Text Available The lack of predictable in vitro methods to analyze antimicrobial activity could play a role in the development of resistance to antibiotics. Current used methods analyze planktonic cells but for the method to be clinically relevant, biofilm in in vivo like conditions ought to be studied. Hence, our group has developed a qualitative and quantitative method with in vivo like 3D tissue for prediction of antimicrobial activity in reality. Devices (wound dressings were applied on top of Pseudomonas aeruginosa inoculated Muller-Hinton (MH agar or 3D synthetic soft tissues (SST and incubated for 24 hours. The antibacterial activity was then analyzed visually and by viable counts. On MH agar two out of three silver containing devices showed zone of inhibitions (ZOI and on SST, ZOI were detected for all three. Corroborating results were found upon evaluating the bacterial load in SST and shown to be silver concentration dependent. In conclusion, a novel method was developed combining visual rapid screening and quantitative evaluation of the antimicrobial activity in both tissue and devices. It uses tissue allowing biofilm formation thus mimicking reality closely. These conditions are essential in order to predict antimicrobial activity of medical devices in the task to prevent device related infections.

  10. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

    Cox, Annie M.; Goodwin, Kelly D.

    2013-01-01

    Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

  11. Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

    Science.gov (United States)

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

  12. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

    International Nuclear Information System (INIS)

    Pierson, Theodore C.; Sanchez, Melissa D.; Puffer, Bridget A.; Ahmed, Asim A.; Geiss, Brian J.; Valentine, Laura E.; Altamura, Louis A.; Diamond, Michael S.; Doms, Robert W.

    2006-01-01

    West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody

  13. Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

    Science.gov (United States)

    Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

    2014-01-01

    A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

  14. Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

    Directory of Open Access Journals (Sweden)

    Maria Frølund

    Full Text Available A novel multiplex quantitative real-time polymerase chain reaction (qPCR for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

  15. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

    Science.gov (United States)

    Cox, Annie M; Goodwin, Kelly D

    2013-08-15

    The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

  16. Quantitative analysis of the chromatin of lymphocytes: an assay on comparative structuralism.

    Science.gov (United States)

    Meyer, F

    1980-01-01

    With 26 letters we can form all the words we use, and with a few words it is possible to form an infinite number of different meaningful sentences. In our case, the letters will be a few simple neighborhood image transformations and area measurements. The paper shows how, by iterating these transformations, it is possible to obtain a good quantitative description of the nuclear structure of Feulgen-stained lymphocytes (CLL and normal). The fact that we restricted ourselves to a small number of image transformations made it possible to construct an image analysis system (TAS) able to do these transformations very quickly. We will see, successively, how to segment the nucleus itself, the chromatin, and the interchromatinic channels, how openings and closings lead to size and spatial distribution curves, and how skeletons may be used for measuring the lengths of interchromatinic channels.

  17. Statistical inference for the within-device precision of quantitative measurements in assay validation.

    Science.gov (United States)

    Liu, Jen-Pei; Lu, Li-Tien; Liao, C T

    2009-09-01

    Intermediate precision is one of the most important characteristics for evaluation of precision in assay validation. The current methods for evaluation of within-device precision recommended by the Clinical Laboratory Standard Institute (CLSI) guideline EP5-A2 are based on the point estimator. On the other hand, in addition to point estimators, confidence intervals can provide a range for the within-device precision with a probability statement. Therefore, we suggest a confidence interval approach for assessment of the within-device precision. Furthermore, under the two-stage nested random-effects model recommended by the approved CLSI guideline EP5-A2, in addition to the current Satterthwaite's approximation and the modified large sample (MLS) methods, we apply the technique of generalized pivotal quantities (GPQ) to derive the confidence interval for the within-device precision. The data from the approved CLSI guideline EP5-A2 illustrate the applications of the confidence interval approach and comparison of results between the three methods. Results of a simulation study on the coverage probability and expected length of the three methods are reported. The proposed method of the GPQ-based confidence intervals is also extended to consider the between-laboratories variation for precision assessment.

  18. Hessian-based quantitative image analysis of host-pathogen confrontation assays.

    Science.gov (United States)

    Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

    2018-03-01

    Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  19. Quantitative assay for the colonization ability of heterogeneous bacteria on controlled nanopillar structures

    International Nuclear Information System (INIS)

    Jin, Lin; Guo, Wen; Zhang, Yali; Xue, Peihong; Gao, Hainan; Zhao, Ming; Zheng, Chen; Han, Dong

    2015-01-01

    The colonization ability of bacteria on biomaterial surfaces is influenced by the morphology of the bacteria and the nanotopography of the biomaterial. However, interactions between the bacterial morphology and nanotopography of biomaterials have not yet been completely elucidated. In this article, we quantitatively characterized the bacterial morphology to illuminate the integrated effects of polyethylene terephthalate (PET) nanopillar arrays on the colonization of bacteria cells with different shapes. Our results demonstrated that the interaction between interpillar spacing and the diameter of the bacterial cells impacted the number of bacterial cells that adhered to different PET substrates. The interpillar spacing of nanopillar arrays promotes bacterial adhesion in a definite range (<50 nm). However, further increasing the interpillar spacing inhibited the adhesion of bacteria to the nanopillar arrays. Moreover, the interpillar spacing also influenced the morphologies of adherent bacterial cells on the PET nanopillar arrays, which consequently facilitated bacterial adhesion to the nanopillar arrays. Our findings enhance the understanding of interactions between controlled nanotopography and bacterial colonization and provide an appropriate parameter for the design of antibacterial materials with nanotopography. (paper)

  20. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    Science.gov (United States)

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  1. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... probes. The limits of detection ware found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid...

  2. Magnetic particle separation technique: a reliable and simple tool for RIA/IRMA and quantitative PCR assay

    International Nuclear Information System (INIS)

    Shen Rongsen; Shen Decun

    1998-01-01

    Five types of magnetic particles without or with aldehyde, amino and carboxyl functional groups, respectively were used to immobilize first or second antibody by three models, i. e. physical adsorption, chemical coupling and immuno-affinity, forming four types of magnetic particle antibodies. The second antibody immobilized on polyacrolein magnetic particles through aldehyde functional groups and the first antibodies immobilized on carboxylic polystyrene magnetic particles through carboxyl functional groups were recommended to apply to RIAs and/or IRMAs. Streptavidin immobilized on commercial magnetic particles through amino functional groups was successfully applied to separating specific PCR product for quantification of human cytomegalovirus. In the paper typical data on reliability of these magnetic particle ligands were reported and simplicity of the magnetic particle separation technique was discussed. The results showed that the technique was a reliable and simple tool for RIA/IRMA and quantitative PCR assay. (author)

  3. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    Science.gov (United States)

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

  4. A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

    Science.gov (United States)

    Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

    2015-01-01

    A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

  5. Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    Jelena V Jovanovic

    2011-10-01

    Full Text Available Molecular detection of minimal residual disease (MRD has become established to assess remission status and guide therapy in patients with PML-RARA+ acute promyelocytic leukemia (APL. However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their relative rarity (<1% of APL we identified 6 cases (PLZF-RARA, n=5; STAT5b-RARA, n=1, established the respective breakpoint junction regions and designed real-time quantitative polymerase chain reaction (RQ-PCR assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17-associated APL, affording assay sensitivities of ~1 in 104-105. Serial samples were available from 2 PLZF-RARA APL patients. One showed persistent PCR positivity, predicting subsequent relapse, and remains in CR2, ~11 years post-autograft. The other, achieved molecular remission (CRm with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RQ-PCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly-defined subsets of acute leukemia.

  6. Development of Real-Time Quantitative Polymerase Chain Reaction Assays to Track Treatment Response in Retinoid Resistant Acute Promyelocytic Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Jovanovic, Jelena V. [Cancer Genetics Laboratory, Department of Medical and Molecular Genetics, King’s College London School of Medicine, London (United Kingdom); Rennie, Kristian [GSTS Pathology, Guy’s Hospital, London (United Kingdom); Culligan, Dominic [Department of Haematology, Aberdeen Royal Infirmary, Aberdeen (United Kingdom); Peniket, Andrew [Department of Haematology, John Radcliffe Hospital, Oxford (United Kingdom); Lennard, Anne [Department of Haematology, Royal Victoria Infirmary, Newcastle (United Kingdom); Harrison, Justin [Department of Haematology, Hemel Hempstead Hospital, Hemel Hempstead (United Kingdom); Vyas, Paresh [Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford (United Kingdom); Grimwade, David, E-mail: david.grimwade@genetics.kcl.ac.uk [Cancer Genetics Laboratory, Department of Medical and Molecular Genetics, King’s College London School of Medicine, London (United Kingdom)

    2011-10-25

    Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 10{sup 4}−10{sup 5}. Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

  7. Development of Real-Time Quantitative Polymerase Chain Reaction Assays to Track Treatment Response in Retinoid Resistant Acute Promyelocytic Leukemia

    International Nuclear Information System (INIS)

    Jovanovic, Jelena V.; Rennie, Kristian; Culligan, Dominic; Peniket, Andrew; Lennard, Anne; Harrison, Justin; Vyas, Paresh; Grimwade, David

    2011-01-01

    Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 10 4 −10 5 . Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

  8. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Directory of Open Access Journals (Sweden)

    Xingmei Xie

    Full Text Available Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR. Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY, five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377, one X/Y-common STR (X22, and two autosomal STRs (D13S305 and D21S11. Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

  9. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    Science.gov (United States)

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.

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    Markus Kopp

    Full Text Available Because of minimal data available on folate analysis in dried matrix spots (DMSs, we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs and dried plasma spots (DPSs as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.

  11. Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

    Science.gov (United States)

    Wilbur, Ami E; Ford, Susan E; Gauthier, Julie D; Gomez-Chiarri, Marta

    2012-12-27

    The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

  12. Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm

    International Nuclear Information System (INIS)

    Olive, D.L.; Weinberg, J.B.; Haney, A.F.

    1987-01-01

    The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with 111 indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of 111 indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of 111 indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract

  13. Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production.

    Science.gov (United States)

    Azam, Philippe; Peiffer, Jean-Luc; Ourlin, Jean-Claude; Bonnet, Pierre-Antoine; Tissier, Marie-Hélène; Vian, Laurence; Fabre, Isabelle

    2005-01-15

    The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells. At the mRNA level, no change in the IL-2 gene expression level was detected by real-time PCR analysis. At the protein level, various experimental conditions were checked in order to improve the irritant versus sensitizer discrimination with a restricted set of prototypic compounds. In particular, the use of phytohemagglutinin A (PHA) in an ex vivo cell culture step showed an improvement of both signal and discrimination. In these optimised conditions, a panel of irritants and potency-graded sensitizers was used to assess the performance of the modified method. IFN-gamma production was used as a positive control. For each compound, a dose-response was performed and stimulation indexes (SI) were determined. Effective concentrations (EC) for each sensitizers were then extracted and compared to the literature data of the regular LLNA. The IL-2-based LLNA showed similar performances at both qualitative and quantitative levels compared to regular LLNA.

  14. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    Science.gov (United States)

    Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  15. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    Directory of Open Access Journals (Sweden)

    Carmen Rueda-Martínez

    Full Text Available Bicuspid aortic valve (BAV is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40% incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR assays. A total of 51 adult (180-240 days old and 56 old (300-440 days old animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30, or to the affected strain of hamsters with TAV (n = 45 or BAV (n = 32. The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  16. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Feroze Ahmed Ganaie

    2015-01-01

    Full Text Available Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment] in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.

  17. Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay

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    Daniel Hubert Darius

    2009-01-01

    Full Text Available Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR for accurate quantitation and screening of HBV. Materials and Methods: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV antibody and human immunodeficiency virus (HIV antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay′s capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001. Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

  18. Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography-Mass Spectrometry

    Science.gov (United States)

    Xu, Xia; Veenstra, Timothy D.

    2012-01-01

    The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) have…

  19. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    Science.gov (United States)

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

  20. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  1. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    International Nuclear Information System (INIS)

    Fichorova, Raina N.; Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F.

    2015-01-01

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  2. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

    Science.gov (United States)

    Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

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    Isabel Hostettler

    2014-12-01

    Full Text Available Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48–72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

  4. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  5. Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified common bean (Phaseolus vulgaris).

    Science.gov (United States)

    Treml, Diana; Venturelli, Gustavo L; Brod, Fábio C A; Faria, Josias C; Arisi, Ana C M

    2014-12-10

    A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

  6. Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

    Science.gov (United States)

    Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

    2016-01-01

    To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

  7. Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

    2015-06-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

  8. Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

    2015-01-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

  9. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    International Nuclear Information System (INIS)

    Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

    2010-01-01

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V max ) and the michaelis constant (k m ) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  10. Quantitative comparison of in house irma/ria methods using reagents supplied in bulk with assays based on commercial kits

    International Nuclear Information System (INIS)

    Sajid, K.M.; Jafri, S.R.A.

    1997-01-01

    Due to high cost of commercial kit assays, local trials were started to establish low cost immunoassay techniques at MINAR, Multan. First available alternate of commercial kits was the use of matched reagents supplied in bulk by NETRIA through INMOL, Lahore under IAEA assistance. As quality is crucial in RIA estimations this laboratory collected passive quality control data of 50, 51 and 52 in-house assay batches of T3,T4 and TSH to compare with the same number of last Amerlex RIAs(successive lots). A qualitative comparison based on computerized data analysis shows linear correlation between the results of two assay systems with low and acceptable precision in T3 and T4 assays. In TSH assays both systems show high imprecision although Amerlex RIA system is relatively more precise than in-house TSH- IRMA. T3 and T4 assays in both the systems show wide working ranges covering all clinical regions. In TSH, working ranges of both the techniques do not cover all clinical regions. In-house TSH assay excludes below 5.5 mulU/ml, whereas Amerlex excludes levels below 4.5 mulL/ml. This may reduce the clinical efficacy of these tests. Amerlex-M T3 and T4 assays show high negative drift with relatively less between variation, whereas in-house assays show low positive drift with high between batch variation. (author)

  11. Embolus location affects the sensitivity of a rapid quantitative D-dimer assay in the diagnosis of pulmonary embolism

    NARCIS (Netherlands)

    de Monyé, Wouter; Sanson, Bernd-Jan; Mac Gillavry, Melvin R.; Pattynama, Peter M. T.; Büller, Harry R.; van den Berg-Huysmans, Annette A.; Huisman, Menno V.

    2002-01-01

    D-dimer blood tests have been suggested to rule out pulmonary embolism. Despite evidence of the safety of withholding anticoagulant treatment in patients with suspected pulmonary embolism and a normal D-dimer assay result, clinicians remain reluctant to use a D-dimer assay as a sole diagnostic test.

  12. Comparison of real-time and quantitative polymerase chain reaction assays in detection of cytomegalovirus DNA in clinical specimens

    International Nuclear Information System (INIS)

    Gokahmetoglu, S.; Deniz, E.

    2007-01-01

    To compare the real-time (RT) and qualitative (Q) polymerase chain reaction (PCR) assays for detection of Cytomegalovirus (CMV) DNA. The study took place in the Department of Microbiology, Erciyes University, Kayseri and in Iontek Laboratory, Istanbul, Turkey, from August to December 2006. One hundred and seven clinical specimens from 67 patients were included in the study. Cytomegalovirus DNA was investigated using RT-PCR kit (Fluorion Iontek, Turkey) and Q-PCR kit (Fluorion Iontek, Turkey). Deoxyribonucleic acid sequencing was applied to the samples that yielded discrepant results in both assays. Mac Nema's Chi Square test was used for statistical analysis. Of the specimens, 27 were found positive with both assays: 9 with only RT-PCR, and 11 with only Q-PCR assay. Both assays were found negative in 60 of the specimens. There was a good agreement between the 2 assays in 87(81.3%) of the specimens. There was no statistical significant difference between the assays (p>0.05). Two of the 11 samples that RT-PCR negative Q-PCR positive, and 3 of 9 samples that RT-PCR positive Q-PCR negative were found to be CMV DNA positive by DNA sequencing. A good level of concordance between RT-PCR and Q-PCR assays for CMV DNA detection has been found. (author)

  13. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  14. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

    An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

  15. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    Science.gov (United States)

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  16. Branching enzyme assay: selective quantitation of the alpha 1,6-linked glucosyl residues involved in the branching points.

    Science.gov (United States)

    Krisman, C R; Tolmasky, D S; Raffo, S

    1985-06-01

    Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.

  17. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  18. A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

    Science.gov (United States)

    Li, Ye; Cassone, Vincent M

    2015-09-01

    A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

    Science.gov (United States)

    Huang, Weidong; Reinholz, Monica; Weidler, Jodi; Yolanda, Lie; Paquet, Agnes; Whitcomb, Jeannette; Lingle, Wilma; Jenkins, Robert B; Chen, Beiyun; Larson, Jeffrey S; Tan, Yuping; Sherwood, Thomas; Bates, Michael; Perez, Edith A

    2010-08-01

    The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

  20. STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

    Science.gov (United States)

    Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

    2012-01-01

    To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

  1. Performance of the new Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: Conversion to international units and comparison with the Roche COBAS amplicor HCV monitor, version 2.0, assay

    NARCIS (Netherlands)

    Beld, Marcel; Sentjens, Roel; Rebers, Sjoerd; Weegink, Christine; Weel, Jan; Sol, Cees; Boom, René

    2002-01-01

    We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0

  2. Thyroglobulin (Tg) recovery testing with quantitative Tg antibody measurement for determining interference in serum Tg assays in differentiated thyroid carcinoma

    NARCIS (Netherlands)

    Persoon, ACM; Links, TP; Wilde, J; Sluiter, WJ; Wolffenbuttel, BHR; van den Ouweland, JMW

    Background: Thyroglobulin (Tg) measurements are complicated by interference from Tg autoantibodies (TgAbs) or heterophilic antibodies (HAMAs). We used a new automated immunochemiluminometric assay (ICMA) with Tg recovery (TgR) on the Nichols Advantage (R) platform to reassess the clinical utility of

  3. Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

    Directory of Open Access Journals (Sweden)

    Touseef Hussain

    2018-05-01

    Full Text Available Phytophthora infestans (mont de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

  4. Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens.

    Science.gov (United States)

    Eischeid, Anne C; Kim, Bang-hyun; Kasko, Sasha M

    2013-06-19

    Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 10⁶ parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.

  5. Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

    International Nuclear Information System (INIS)

    Dold, K.M.; Greenlee, W.F.

    1990-01-01

    A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells

  6. Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

    Science.gov (United States)

    Cecchinato, Mattia; Lupini, Caterina; Munoz Pogoreltseva, Olga Svetlana; Listorti, Valeria; Mondin, Alessandra; Drigo, Michele; Catelli, Elena

    2013-01-01

    In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10(-0.41) median infectious dose/ml and 10(1.15) median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were 0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

  7. Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

    OpenAIRE

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A.; Saul, Allan; Gerke, Christiane

    2014-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Int...

  8. Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation.

    Science.gov (United States)

    Schwendimann, Livia; Kauf, Peter; Fieseler, Lars; Gantenbein-Demarchi, Corinne; Miescher Schwenninger, Susanne

    2015-08-01

    To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Science.gov (United States)

    Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

  10. Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I

    2001-01-01

    We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...... used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...

  11. High performance liquid chromatographic-mass spectrometric assay for the quantitation of BMS-204352 in dog K(3)EDTA plasma.

    Science.gov (United States)

    Yao, Ming; Mantha, Subbarao; Shah, Vinod R; Vachharajani, Nimish N; Arnold, Mark E; Pursley, Janice M; Srinivas, Nuggehally R

    2002-05-01

    A high performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of BMS-204352 in dog K(3)EDTA plasma. A 0.5 mL aliquot of control plasma was spiked with BMS-204352 and internal standard (IS) and buffered with 1 mL of 5 mM ammonium acetate. The mixture was then extracted with 3 mL of toluene. After separation and evaporation of the organic phase to dryness using nitrogen at 40 degrees C, the residue was reconstituted in the mobile phase and 25 microL of the sample were injected onto a Hypersil C(18) column (2 x 50 mm; 3 microm) at a flow rate of 0.5 mL/min. The mobile phase was consisted of two solvent mixtures (A and B). Solvent A was composed of 5 mM ammonium acetate and 0.1% triethylamine in 75:25 v/v water:methanol, pH adjusted to 5.5 with glacial acetic acid, and solvent B was 5 mM ammonium acetate in methanol. A linear gradient system was used to elute the analytes. The mass spectrometer was programmed to admit the de-protonated molecules at m/z 352.7 (IS) and m/z 357.9 (BMS-204352). Standard curves of BMS-204352 were linear (r(2) > or = 0.998) over the concentration range of 0.5-1000 ng/mL. The mean predicted quality control (QC) concentrations deviated less than 5.1% from the corresponding nominal values (ie 4, 80, 400 and 2000 ng/mL); the within- and between-assay precision of the assay were within 5.5% relative standard deviation. Stability of BMS-204352 was confirmed after at least three freeze/thaw cycles and BMS-204532 was stable in dog plasma when stored frozen at or below -20 degrees C for at least 16 weeks in spiked QC samples and for at least 4 1/2 weeks for in vivo study samples. BMS-204352 and IS were stable in the injection solvent at room temperature for at least 24 h. The assay was applied to delineate the pharmacokinetic disposition of BMS-204352 in dogs following a single intravenous dose administration. In conclusion, the assay is accurate, precise, specific, sensitive and

  12. Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

    Science.gov (United States)

    Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

    2007-01-01

    Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can

  13. Individuals with autism have higher 8-Iso-PGF2α levels than controls, but no correlation with quantitative assay of Paraoxonase 1 serum levels.

    Science.gov (United States)

    Pop, Bianca; Niculae, Alexandru-Ștefan; Pop, Tudor Lucian; Răchișan, Andreea Liana

    2017-12-01

    Autism spectrum disorder (ASD) represents a very large set of neurodevelopmental issues with diverse clinical outcomes. Various hypotheses have been put forth for the etiology of autism spectrum disorder, including issues pertaining to oxidative stress. In this study, we conducted measurements of serum 8-Iso-Prostaglanding F2 α (8-iso-PGF2α, which is the results of non-enzimatically mediated polyunsaturated fatty acid oxidation) in a population of individuals with autism and a control group of age and sex matched controls. A quantitative assay of Paraoxonase 1 (PON1) was conducted. Data regarding comorbidities, structural MRI scans, medication, intelligence quotient (IQ) and Childhood Autism Rating Scale scores (CARS) were also included in our study. Our results show that patients diagnosed with autism have higher levels of 8-iso-PGF2α than their neurotypical counterparts. Levels of this particular metabolite, however, do not correlate with quantitative serum levels of Paraoxonase 1, which has been shown to be altered in individuals with autism. Neither 8-iso-PGF2α nor quantitative levels of PON1 provide any meaningful correlation with clinical or neuroimaging data in this study group. Future research should focus on providing data regarding PON 1 phenotype, in addition to standard quantitative measurements, in relation to 8-iso-PGF2α as well as other clinical and structural brain findings.

  14. Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

    Science.gov (United States)

    Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

    2012-01-01

    During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

  15. A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element.

    Science.gov (United States)

    Haudenshield, James S; Song, Jeong Y; Hartman, Glen L

    2017-01-01

    Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5'-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction.

  16. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

    Directory of Open Access Journals (Sweden)

    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  17. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

    Science.gov (United States)

    Durant, Jean-Francois; Irenge, Leonid M; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean-Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean-Luc

    2012-12-07

    Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  18. Endogenous Reference Genes and Their Quantitative Real-Time PCR Assays for Genetically Modified Bread Wheat (Triticum aestivum L.) Detection.

    Science.gov (United States)

    Yang, Litao; Quan, Sheng; Zhang, Dabing

    2017-01-01

    Endogenous reference genes (ERG) and their derivate analytical methods are standard requirements for analysis of genetically modified organisms (GMOs). Development and validation of suitable ERGs is the primary step for establishing assays that monitoring the genetically modified (GM) contents in food/feed samples. Herein, we give a review of the ERGs currently used for GM wheat analysis, such as ACC1, PKABA1, ALMT1, and Waxy-D1, as well as their performances in GM wheat analysis. Also, we discussed one model for developing and validating one ideal RG for one plant species based on our previous research work.

  19. Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

    Science.gov (United States)

    Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

    2013-04-10

    Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

  20. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

    Science.gov (United States)

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.

    Science.gov (United States)

    Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H

    2017-02-05

    Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (neratinib. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  3. A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

    Science.gov (United States)

    Cho, Min Seok; Lee, Jang Ha; Her, Nam Han; Kim, Changkug; Seol, Young-Joo; Hahn, Jang Ho; Baeg, Ji Hyoun; Kim, Hong Gi; Park, Dong Suk

    2012-06-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

  4. Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

    Science.gov (United States)

    Malapelle, Umberto; Mayo-de-Las-Casas, Clara; Molina-Vila, Miguel A; Rosell, Rafael; Savic, Spasenija; Bihl, Michel; Bubendorf, Lukas; Salto-Tellez, Manuel; de Biase, Dario; Tallini, Giovanni; Hwang, David H; Sholl, Lynette M; Luthra, Rajyalakshmi; Weynand, Birgit; Vander Borght, Sara; Missiaglia, Edoardo; Bongiovanni, Massimo; Stieber, Daniel; Vielh, Philippe; Schmitt, Fernando; Rappa, Alessandra; Barberis, Massimo; Pepe, Francesco; Pisapia, Pasquale; Serra, Nicola; Vigliar, Elena; Bellevicine, Claudio; Fassan, Matteo; Rugge, Massimo; de Andrea, Carlos E; Lozano, Maria D; Basolo, Fulvio; Fontanini, Gabriella; Nikiforov, Yuri E; Kamel-Reid, Suzanne; da Cunha Santos, Gilda; Nikiforova, Marina N; Roy-Chowdhuri, Sinchita; Troncone, Giancarlo

    2017-08-01

    Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational

  5. Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

    Science.gov (United States)

    Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

    2001-01-01

    Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

  6. Quantitative determination of trace hydrogen peroxide in the presence of sulfide using the Amplex Red/horseradish peroxidase assay.

    Science.gov (United States)

    Wang, Nannan; Miller, Christopher J; Wang, Peng; Waite, T David

    2017-04-22

    The Amplex Red/horseradish peroxidase (AR/HRP) assay for H 2 O 2 is one of the most sensitive and simple approaches for H 2 O 2 quantification, which is effected by measuring the highly-fluorescent resorufin formed from oxidation of AR by the oxidizing intermediates generated by reaction of HRP and H 2 O 2 . The direct reactions of S(-II) with both H 2 O 2 and resorufin are too slow to be of relevance on analytical timescales, however, the reaction between S(-II) and the HRP/H 2 O 2 oxidizing intermediates is rapid enough to compete with the desired reaction of these oxidizing intermediates with AR, suppressing formation of the resorufin analyte. As this mode of interference can be considered simply a competition between the AR reagent and S(-II) for the intermediate oxidizing species, a simple equation is derived in this work enabling one to correct for this interference and obtain a good estimate of the true H 2 O 2 concentration after measuring the apparent H 2 O 2 concentration and the S(-II) concentration. This mode of interference is general to any compound that can act as a HRP substrate even if not directly reactive with H 2 O 2 . As such, the approach described is widely applicable to many potential reducing interferents and opens up the use of the AR/HRP assay to a much wider range of conditions, as well as demonstrating the utility of explicitly considering the mechanism of the analytical process. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Comparison of Alcian blue and total carbohydrate assays for quantitation of transparent exopolymer particles (TEP) in biofouling studies.

    Science.gov (United States)

    Li, Xu; Skillman, Lucy; Li, Dan; Ela, Wendell P

    2018-04-15

    Transparent exopolymer particles (TEP) and their precursors are gel-like acidic polysaccharide particles. Both TEP precursors and TEP have been identified as causal factors in fouling of desalination and water treatment systems. For comparison between studies, it is important to accurately measure the amount and fouling capacity of both components. However, the accuracy and recovery of the currently used Alcian blue based TEP measurement of different surrogates and different size fractions are not well understood. In this study, we compared Alcian blue based TEP measurements with a total carbohydrate assay method. Three surrogates; xanthan gum, pectin and alginic acid; were evaluated at different salinities. Total carbohydrate concentrations of particulates (>0.4 μm) and their precursors (10 kDa) varied depending on water salinity and method of recovery. As xanthan gum is the most frequently used surrogate in fouling studies, TEP concentration is expressed as xanthan gum equivalents (mg XG eq /L) in this study. At a salinity of 35 mg/L sea salt, total carbohydrate assays showed a much higher particulate TEP fraction for alginic acid (38%) compared to xanthan gum (9%) and pectin (12%). The concentrations of particulate TEP therefore may only represent ∼10% of the total mass; while precursor TEP represents ∼80% of the total TEP. This highlights the importance of reporting both particulate and precursor TEP for membrane biofouling studies. The calculated concentrations of TEP and their precursors in seawater samples are also highly dependent on type of surrogate and resulting calibration factor. A linear correlation between TEP recovery and calibration factor was demonstrated in this study for all three surrogates. The relative importance and accuracy of measurement method, particulate size, surrogate type, and recovery are described in detail in this study. Copyright © 2017. Published by Elsevier Ltd.

  8. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

    Science.gov (United States)

    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

    2016-01-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

  9. Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

    Science.gov (United States)

    Su, Y-L; Feng, J; Li, Y-W; Bai, J-S; Li, A-X

    2016-02-01

    Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish. © 2015 John Wiley & Sons Ltd.

  10. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays.

    Science.gov (United States)

    Hahn, Cassidy M; Iwanowicz, Luke R; Cornman, Robert S; Mazik, Patricia M; Blazer, Vicki S

    2016-12-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest. Published by Elsevier Inc.

  11. Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip.

    Science.gov (United States)

    Pang, Bo; Ding, Xiong; Wang, Guoping; Zhao, Chao; Xu, Yanan; Fu, Kaiyue; Sun, Jingjing; Song, Xiuling; Wu, Wenshuai; Liu, Yushen; Song, Qi; Hu, Jiumei; Li, Juan; Mu, Ying

    2017-12-27

    Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 10 3 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria. Additionally, as a result of the use of a mixed dye and SPC chip, the quantitative result could be easily acquired, avoiding the requirement of sophisticated instruments and tedious operation. Also, CMD-LAMP was rapid and cost-effective. Conclusively, CMD-LAMP has great potential in realizing the on-site quantitative analysis of VP for food safety.

  12. Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

    Science.gov (United States)

    Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

    2007-02-01

    Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

  13. Rapid and simple half-quantitative measurement alpha-fetoprotein by poly(dimethylsiloxane) microfluidic chip immunochromatographic assay

    Science.gov (United States)

    Tong, Chao; Jin, Qinghui; Zhao, Jianlong

    2008-03-01

    In this article, a kind of microfluidic method based on MEMS technology combined with gold immunochromatographic assay (GICA) is developed and discussed. Compared to the traditional GICA, this method supplies us convenient, multi-channel, in-parallel, low cost and similar efficiency approach in the fields of alpha-fetopro-tei (AFP)detection. Firstly, we improved the adhesion between the model material SU-8 and Silicon wafer, optimized approaches of the fabrication of the SU-8 model systematically, and fabricate the PDMS micro fluid chip with good reproduction successfully. Secondly, Surface modification and antibody immobilization methods with the GICA on the PDMS micro fluid analysis chip are studied, we choose the PDMS material and transfer GICA to the PDMS micro fluid chip successfully after researching the antibody immobilization efficiency of different materials utilized in fabrication of the micro fluid chip. In order to improve the reaction efficiency of the immobilized antibody, we studied the characteristics of micro fluid without the gas drive, and the fluid velocity control in our design; we also design structure of grove to strengthen the ability of immobilizing the antibody. The stimulation of the structure shows that it achieves great improvement and experiments prove the design is feasible.

  14. sxtA-Based Quantitative Molecular Assay To Identify Saxitoxin-Producing Harmful Algal Blooms in Marine Waters ▿ †

    Science.gov (United States)

    Murray, Shauna A.; Wiese, Maria; Stüken, Anke; Brett, Steve; Kellmann, Ralf; Hallegraeff, Gustaaf; Neilan, Brett A.

    2011-01-01

    The recent identification of genes involved in the production of the potent neurotoxin and keystone metabolite saxitoxin (STX) in marine eukaryotic phytoplankton has allowed us for the first time to develop molecular genetic methods to investigate the chemical ecology of harmful algal blooms in situ. We present a novel method for detecting and quantifying the potential for STX production in marine environmental samples. Our assay detects a domain of the gene sxtA that encodes a unique enzyme putatively involved in the sxt pathway in marine dinoflagellates, sxtA4. A product of the correct size was recovered from nine strains of four species of STX-producing Alexandrium and Gymnodinium catenatum and was not detected in the non-STX-producing Alexandrium species, other dinoflagellate cultures, or an environmental sample that did not contain known STX-producing species. However, sxtA4 was also detected in the non-STX-producing strain of Alexandrium tamarense, Tasmanian ribotype. We investigated the copy number of sxtA4 in three strains of Alexandrium catenella and found it to be relatively constant among strains. Using our novel method, we detected and quantified sxtA4 in three environmental blooms of Alexandrium catenella that led to STX uptake in oysters. We conclude that this method shows promise as an accurate, fast, and cost-effective means of quantifying the potential for STX production in marine samples and will be useful for biological oceanographic research and harmful algal bloom monitoring. PMID:21841034

  15. sxtA-based quantitative molecular assay to identify saxitoxin-producing harmful algal blooms in marine waters.

    Science.gov (United States)

    Murray, Shauna A; Wiese, Maria; Stüken, Anke; Brett, Steve; Kellmann, Ralf; Hallegraeff, Gustaaf; Neilan, Brett A

    2011-10-01

    The recent identification of genes involved in the production of the potent neurotoxin and keystone metabolite saxitoxin (STX) in marine eukaryotic phytoplankton has allowed us for the first time to develop molecular genetic methods to investigate the chemical ecology of harmful algal blooms in situ. We present a novel method for detecting and quantifying the potential for STX production in marine environmental samples. Our assay detects a domain of the gene sxtA that encodes a unique enzyme putatively involved in the sxt pathway in marine dinoflagellates, sxtA4. A product of the correct size was recovered from nine strains of four species of STX-producing Alexandrium and Gymnodinium catenatum and was not detected in the non-STX-producing Alexandrium species, other dinoflagellate cultures, or an environmental sample that did not contain known STX-producing species. However, sxtA4 was also detected in the non-STX-producing strain of Alexandrium tamarense, Tasmanian ribotype. We investigated the copy number of sxtA4 in three strains of Alexandrium catenella and found it to be relatively constant among strains. Using our novel method, we detected and quantified sxtA4 in three environmental blooms of Alexandrium catenella that led to STX uptake in oysters. We conclude that this method shows promise as an accurate, fast, and cost-effective means of quantifying the potential for STX production in marine samples and will be useful for biological oceanographic research and harmful algal bloom monitoring.

  16. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  17. Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

    Science.gov (United States)

    Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

    2017-07-01

    Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  18. Quantitation of (R)- and (S)-linalool in beer using solid phase microextraction (SPME) in combination with a stable isotope dilution assay (SIDA).

    Science.gov (United States)

    Steinhaus, Martin; Fritsch, Helge T; Schieberle, Peter

    2003-11-19

    A stable isotope dilution assay (SIDA) was developed for the quantitation of both linalool enantiomers using synthesized [2H(2)]R/S-linalool as the internal standard. For enrichment of the target compound from beer, a solid phase microextraction method (SPME) was developed. In comparison to the more time-consuming extraction/distillation cleanup of the beer samples, the results obtained by SPME/SIDA were very similar, even under nonequilibration conditions. Analysis of five different types of beer showed significant differences in the linalool concentrations, which were clearly correlated with the intensity of the hoppy aroma note as evaluated by a sensory panel. In addition, significant differences in the R/S ratios were measured in the beers. The SPME/SIDA yielded exact data independently from headspace sampling parameters, such as exposure time or ionic strength of the solution.

  19. Portable and quantitative point-of-care monitoring of Escherichia coli O157:H7 using a personal glucose meter based on immunochromatographic assay.

    Science.gov (United States)

    Huang, Haoran; Zhao, Guangying; Dou, Wenchao

    2018-06-01

    Here we innovate a portable and quantitative immunochromatographic assay (ICA) with a personal glucose meter (PGM) as readout for the detection of Escherichia coli O157:H7 (E. coli O157:H7). The carboxyl group coated Fe 3 O 4 nanoparticles (MNPs) were synthesized by a one pot method and used as carriers of invertase and monoclonal antibody against E. coli O157:H7. Initially, the invertase and antibody double functionalized MNPs (Invertase-MNPs-IgG) conjugates were prepared and used as label probe in this assay system. Before laminating onto the baking card, the absorbent pad was soaked in sucrose solution and desiccated. MNPs produced brown band at the detection zone of the ICA when acting as direct labels. As they were also coupled with invertase, the invertase catalyzed the hydrolysis of sucrose on the absorbent pad into glucose, which was detected by the PGM. To increase the sensitivity, antibody functionalized MNPs were used to enrich E. coli O157:H7 from sample solution. The innovative aspect of this approach lies in the visualization and quantification of E. coli O157:H7 through Invertase-MNPs-IgG and the detection of glucose concentration using PGM. Although the feasibility is demonstrated using E. coli O157:H7 as a model analyte, this approach can be easily developed to be a universal analysis system and applied to detection of a wide variety of foodborne pathogens and protein biomarkers. This study proposed a qualitative and quantitative analysis device for the clinic diagnostics and food safety analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

    Science.gov (United States)

    Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

    2017-05-17

    Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

  1. Development of a quantitation method to assay both lyoniresinol enantiomers in wines, spirits, and oak wood by liquid chromatography-high resolution mass spectrometry.

    Science.gov (United States)

    Cretin, Blandine N; Dubourdieu, Denis; Marchal, Axel

    2016-05-01

    Wine taste balance evolves during oak aging by the release of volatile and non-volatile compounds from wood. Among them, an enantiomer of lyoniresinol, (+)-lyoniresinol, has been shown to exhibit bitterness. To evaluate the impact of (+)-lyoniresinol on wine taste, a two-step quantitation method was developed and validated. First, (±)-lyoniresinol was assayed in wines, spirits, and oak wood macerates by C-18 liquid chromatography-high resolution mass spectrometry (LC-HRMS). Then, the lyoniresinol enantiomeric ratio was determined by chiral LC-HRMS in order to calculate the (+)-lyoniresinol content. In red and white wines, the average concentrations of (+)-lyoniresinol were 1.9 and 0.8 mg/L, respectively. The enantiomer proportions were not affected by bottle aging, and lyoniresinol appeared to remain stable over time. The sensory study of (+)-lyoniresinol established its perception threshold at 0.46 mg/L in wine. All the commercial wines quantitated were above this perception threshold, demonstrating its impact on wine taste by an increase in bitterness. In spirits, (+)-lyoniresinol ranged from 2.0 to 10.0 mg/L and was found to be released continuously during oak aging. Finally, neither botanical origin nor toasting was found to significantly affect the (+)-lyoniresinol content of oak wood. Graphical abstract From oak wood to wine: evaluation of the influence of (+)-lyoniresinol on the bitterness of wines and spirits.

  2. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

    Science.gov (United States)

    Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

    2015-08-26

    Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

  3. Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control.

    Science.gov (United States)

    Hartleif, Steffen; Göhring, Katharina; Goelz, Rangmar; Jahn, Gerhard; Hamprecht, Klaus

    2016-11-01

    For cytomegalovirus screening of breastfeeding mothers of preterm infants under risk, we present a rapid, quantitative real-time PCR protocol using the hybridization format of the viral gB target region. For quantification, we used an external gB fragment cloned into a vector system. For standardization, we created an internal control-plasmid by site-directed mutagenesis with an exchange of 9 nucleotides. Spiked with internal control, patient wildtype amplicons could be discriminated from internal controls by hybridization probes using two-channel fluorescence detection. Potential bias of formerly reported false nucleotide sequence data of gB-hybridization probes was excluded. Using this approach, we could demonstrate excellent analytical performance and high reproducibility of HCMV detection during lactation. This assay shows very good correlation with a commercial quantitative HCMV DNA PCR and may help to identify rapidly HCMV shedding mothers of very low birth weight preterm infants to prevent HCMV transmission. On the other hand, negative DNA amplification results allow feeding of milk samples of seropositive mothers to their preterm infants under risk (<30 weeks of gestational age, <1000g birth weight) during the onset and late stage of HCMV shedding during lactation. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Chemical applicability domain of the local lymph node assay (LLNA) for skin sensitisation potency. Part 4. Quantitative correlation of LLNA potency with human potency.

    Science.gov (United States)

    Roberts, David W; Api, Anne Marie

    2018-07-01

    Prediction of skin sensitisation potential and potency by non-animal methods is the target of many active research programmes. Although the aim is to predict sensitisation potential and potency in humans, data from the murine local lymph node assay (LLNA) constitute much the largest source of quantitative data on in vivo skin sensitisation. The LLNA has been the preferred in vivo method for identification of skin sensitising chemicals and as such is potentially valuable as a benchmark for assessment of non-animal approaches. However, in common with all predictive test methods, the LLNA is subject to false positives and false negatives with an overall level of accuracy said variously to be approximately 80% or 90%. It is also necessary to consider the extent to which, for true positives, LLNA potency correlates with human potency. In this paper LLNA potency and human potency are compared so as to express quantitatively the correlation between them, and reasons for non-agreement between LLNA and human potency are analysed. This leads to a better definition of the applicability domain of the LLNA, within which LLNA data can be used confidently to predict human potency and as a benchmark to assess the performance of non-animal approaches. Copyright © 2018. Published by Elsevier Inc.

  5. Alchemy: A web 2.0 real-time quality assurance platform for human immunodeficiency Virus, hepatitis C Virus, and BK Virus quantitation assays

    Directory of Open Access Journals (Sweden)

    Emmanuel Agosto-Arroyo

    2017-01-01

    Full Text Available Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT. Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs contain all the data elements necessary to do accurate quality assurance (QA reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV quantitation, hepatitis C virus (HCV quantitation, and BK virus (BKV quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

  6. Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

    Science.gov (United States)

    Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

    2017-01-01

    The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

  7. Detection of gene copy number aberrations in mantle cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value.

    Science.gov (United States)

    Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Ruminy, Philippe; Cornic, Marie; Penther, Dominique; Bertrand, Philippe; Lanic, Hélène; Cassuto, Ophélie; Humbrecht, Catherine; Lemasle, Emilie; Wautier, Agathe; Bastard, Christian; Tilly, Hervé

    2009-09-01

    The t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL). Additional genetic alterations occur in the majority of cases. This study aimed to design a polymerase chain reaction (PCR) assay to determine the incidence and relevance of recurrent gene copy number aberrations in this disease. Forty-two MCL cases with frozen- or paraffin-embedded (FFPE) tissues were selected. Three different quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) assays were designed to simultaneously analyse eight genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, MDM2), to analyse the 9p21 locus (CDKN2A/CDKN2B) and FFPE tissues. Gains of MYC, CDK2, CDKN1B, and MDM2 were observed in 10% of cases. Losses of RB1, CDKN2A, ATM or TP53 were observed in 38%, 31%, 24% and 10% of cases, respectively. Analysis of the 9p21 locus indicated that, in most cases, tumours displayed a complete inactivation of p14(ARF)/p15I(NK4B)/p16I(NK4A). CDKN2A and MYC aberrations were associated with a high MCL international prognostic index (MIPI). CDK2/MDM2 gains and CDKN2A/TP53 losses correlated with an unfavourable outcome. PCR experiments with frozen and FFPE-tissues indicated that our approach is valid in a routine diagnostic setting, providing a powerful tool that could be used for patient stratification in combination with MIPI in future clinical trials.

  8. Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

    Science.gov (United States)

    Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

    2015-10-01

    Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

    Science.gov (United States)

    Pitetti, Raymond D; Laus, Stella; Wadowsky, Robert M

    2003-08-01

    Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all

  10. Quantitative characterization of chitosan in the skin by Fourier-transform infrared spectroscopic imaging and ninhydrin assay: application in transdermal sciences.

    Science.gov (United States)

    Nawaz, A; Wong, T W

    2016-07-01

    The chitosan has been used as the primary excipient in transdermal particulate dosage form design. Its distribution pattern across the epidermis and dermis is not easily accessible through chemical assay and limited to radiolabelled molecules via quantitative autoradiography. This study explored Fourier-transform infrared spectroscopy imaging technique with built-in microscope as the means to examine chitosan molecular distribution over epidermis and dermis with the aid of histology operation. Fourier-transform infrared spectroscopy skin imaging was conducted using chitosan of varying molecular weights, deacetylation degrees, particle sizes and zeta potentials, obtained via microwave ligation of polymer chains at solution state. Both skin permeation and retention characteristics of chitosan increased with the use of smaller chitosan molecules with reduced acetyl content and size, and increased positive charge density. The ratio of epidermal to dermal chitosan content decreased with the use of these chitosan molecules as their accumulation in dermis (3.90% to 18.22%) was raised to a greater extent than epidermis (0.62% to 1.92%). A larger dermal chitosan accumulation nonetheless did not promote the transdermal polymer passage more than the epidermal chitosan. A small increase in epidermal chitosan content apparently could fluidize the stratum corneum and was more essential to dictate molecular permeation into dermis and systemic circulation. The histology technique aided Fourier-transform infrared spectroscopy imaging approach introduces a new dimension to the mechanistic aspect of chitosan in transdermal delivery. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  11. Quantitation using a stable isotope dilution assay (SIDA) and thresholds of taste-active pyroglutamyl decapeptide ethyl esters (PGDPEs) in sake.

    Science.gov (United States)

    Hashizume, Katsumi; Ito, Toshiko; Igarashi, Shinya

    2017-03-01

    A stable isotope dilution assay (SIDA) for two taste-active pyroglutamyl decapeptide ethyl esters (PGDPE1; (pGlu)LFGPNVNPWCOOC 2 H 5 , PGDPE2; (pGlu)LFNPSTNPWCOOC 2 H 5 ) in sake was developed using deuterated isotopes and high-resolution mass spectrometry. Recognition thresholds of PGDPEs in sake were estimated as 3.8 μg/L for PGDPE1 and 8.1 μg/L for PGDPE2, evaluated using 11 student panelists aged in their twenties. Quantitated concentrations in 18 commercial sake samples ranged from 0 to 27 μg/L for PGDPE1 and from 0 to 202 μg/L for PGDPE2. The maximum levels of PGDPE1 and PGDPE2 in the sake samples were approximately 8 and 25 times higher than the estimated recognition thresholds, respectively. The results indicated that PGDPEs may play significant sensory roles in the sake. The level of PGDPEs in unpasteurized sake samples decreased during storage for 50 days at 6 °C, suggesting PGDPEs may be enzymatically decomposed.

  12. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

    Science.gov (United States)

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

    2014-10-03

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

    Science.gov (United States)

    Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

  14. Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.

    Science.gov (United States)

    Zarski, Lila M; High, Emily A; Nelli, Rahul K; Bolin, Steven R; Williams, Kurt J; Hussey, Gisela

    2017-10-01

    Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 10 6 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 10 6 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells

  15. Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer.

    Science.gov (United States)

    Gray, Richard G; Quirke, Philip; Handley, Kelly; Lopatin, Margarita; Magill, Laura; Baehner, Frederick L; Beaumont, Claire; Clark-Langone, Kim M; Yoshizawa, Carl N; Lee, Mark; Watson, Drew; Shak, Steven; Kerr, David J

    2011-12-10

    We developed quantitative gene expression assays to assess recurrence risk and benefits from chemotherapy in patients with stage II colon cancer. We sought validation by using RNA extracted from fixed paraffin-embedded primary colon tumor blocks from 1,436 patients with stage II colon cancer in the QUASAR (Quick and Simple and Reliable) study of adjuvant fluoropyrimidine chemotherapy versus surgery alone. A recurrence score (RS) and a treatment score (TS) were calculated from gene expression levels of 13 cancer-related genes (n = 7 recurrence genes and n = 6 treatment benefit genes) and from five reference genes with prespecified algorithms. Cox proportional hazards regression models and log-rank methods were used to analyze the relationship between the RS and risk of recurrence in patients treated with surgery alone and between TS and benefits of chemotherapy. Risk of recurrence was significantly associated with RS (hazard ratio [HR] per interquartile range, 1.38; 95% CI, 1.11 to 1.74; P = .004). Recurrence risks at 3 years were 12%, 18%, and 22% for predefined low, intermediate, and high recurrence risk groups, respectively. T stage (HR, 1.94; P < .001) and mismatch repair (MMR) status (HR, 0.31; P < .001) were the strongest histopathologic prognostic factors. The continuous RS was associated with risk of recurrence (P = .006) beyond these and other covariates. There was no trend for increased benefit from chemotherapy at higher TS (P = .95). The continuous 12-gene RS has been validated in a prospective study for assessment of recurrence risk in patients with stage II colon cancer after surgery and provides prognostic value that complements T stage and MMR. The TS was not predictive of chemotherapy benefit.

  16. 125I-Fibrin deposition in contact sensitivity reactions in the mouse. Sensitivity of the assay for quantitating reactions after active or passive sensitization

    International Nuclear Information System (INIS)

    Mekori, Y.A.; Dvorak, H.F.; Galli, S.J.

    1986-01-01

    The clotting associated with delayed hypersensitivity (DH) responses in the mouse by sensitizing the animals to the contactant oxazolone (Ox), and then administering 125 I-guinea pig fibrinogen i.v. 10 to 30 min before antigen challenge 5 days later. Early (4 to 8 hr) contact sensitivity (CS) responses in immunized mice were barely detectable by three conventional measures of CS, but the total 125 I-cpm in ears challenged with hapten was 3.6 to 4.5 x that in control ears challenged with vehicle alone; moreover, the amount of urea-insoluble cpm (cross-linked 125 I-fibrin-associated cpm) in the reactions to Ox was 6.5-fold to 8.2-fold that present in the control reactions. In 24 hr reactions that were near peak intensity by measurements of ear swelling, ear weight ratios, and ratios of 125 I-5-iodo-2-deoxyuridine-labeled leukocyte infiltration, the cpm in antigen-challenged ears exceeded that in control ears by 13-fold to 53-fold. In addition, antigen-challenged ears contained 27 to 300 x the urea-insoluble cpm present in control ears. 125 I-Fibrin deposition was not a specific characteristic of CS reactions, because a small amount of urea-insoluble reactivity was also detected in some reactions to Ox in native mice. Nevertheless, the assay was exquisitely sensitive and readily detected quantitative differences between the immunologically specific and nonspecific reactions at very early intervals after challenge or with suboptimal doses of antigen

  17. Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan® assay

    NARCIS (Netherlands)

    Czajkowski, R.L.; Wolf, van der J.M.

    2012-01-01

    A Serratia plymuthica-specific TaqMan® assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various

  18. Multicenter evaluation of the new Abbott Realtime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    M. Schutten (Martin); D. Peters (D.); N. Back (Nicole); A.W. van den Beld (Annewieke); B. Beuselinck (B.); V. Foulongne (V.); A.M. Geretti (Anna Maria); L. Pandiani (L.); M. Tiemann; H.G.M. Niesters (Bert)

    2007-01-01

    textabstractThe analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche

  19. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, Martin; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  20. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, M.; Peters, D.; Back, N. K. T.; Beld, M.; Beuselinck, K.; Foulongne, V.; Geretti, A.-M.; Pandiani, L.; Tiemann, C.; Niesters, H. G. M.

    2007-01-01

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  1. Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan(A (R)) assay

    NARCIS (Netherlands)

    Czajkowski, R.L.; Van der Wolf, J.M.

    2012-01-01

    A Serratia plymuthica-specific TaqManA (R) assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to

  2. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV....... Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan...

  3. Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

    Science.gov (United States)

    Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

    2017-01-01

    Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

  4. Binding assays for the quantitative detection of P. brevis polyether neurotoxins in biological samples and antibodies as therapeutic aids for polyether marine intoxication. Annual report, 1 December 1987-30 November 1988

    Energy Technology Data Exchange (ETDEWEB)

    Baden, D.G.

    1988-12-15

    The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) can be detected using two separate types of specific binding reaction. Using tritiated PbTx-3 as a specific probe for binding to voltage-dependent sodium channels in rat brain synaptosomes or to specific polyclonal antibodies, binding equilibria and displacement by unlabeled brevetoxins were compared. Labeled toxin can be displaced in a competitive manner by any of the other 5 naturally-occurring toxins; the quantitative displacement ability of each appears to reflect individual potency in fish bioassay. A comparison of ED50 in Radioimmunoassay and ED50 in synaptosome binding assay indicates that the former assay is useful for detection of toxins which possess the structural backbone of PbTx-3, the immunizing hapten. Thus, the two assays have quantitative applicability; the sodium channel with respect to potency and the antibodies with respect to structure. Microtiter plate assays utilizing each specific brevetoxin binding component and enzyme-linked toxin hapten have been successful and indicate a general applicability of colorimetric prototypes. There, is however, considerable manipulation required to decrease non-specific binding of the hydrophobic toxin-enzyme complex to the plates. Preliminary studies aimed at producing monoclonal antibodies have been explored using brevetoxins linked to keyhole limpet hemocyanin.

  5. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

    Science.gov (United States)

    Tien, Wei-Ping; Lim, Gareth; Yeo, Gladys; Chiang, Suzanna Nicole; Chong, Chee-Seng; Ng, Lee-Ching; Hapuarachchi, Hapuarachchige Chanditha

    2017-09-19

    The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

  7. Evaluation and validation of a real-time PCR assay for detection and quantitation of human adenovirus 14 from clinical samples.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available In 2007, the Centers for Disease Control and Prevention (CDC reported that Human adenovirus type 14 (HAdV-14 infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2 to 10(7 copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.

  8. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Science.gov (United States)

    Jamal, Syed M; Belsham, Graham J

    2015-01-01

    Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor

  9. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Directory of Open Access Journals (Sweden)

    Syed M Jamal

    Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for

  10. A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids.

    Science.gov (United States)

    Syed, Muzeeb; Srinivas, Nuggehally R

    2016-05-01

    Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the analysis of MPA alone or with MPAG/AcMPAG in various biological specimens including plasma/serum, urine, ultrafiltrate, saliva, PBMC, dried blood spots, tissue extract, tumor biopsies and vitreous humor. Based on the need for experimental work, a proper choice of the assay and internal standard may be made using the choices in the literature. While the chemical methods involving high-performance liquid chromatography (HPLC) or LC coupled with triple quadrupole mass spectrometry (LC-MS/MS) are popular, enzymatic assays, in spite of their higher bias, have been used for the routine drug monitoring of MPA. The objectives of the present review are: (a) to provide a focused systematic compilation of the HPLC or LC-MS/MS methods for MPA, MPAG and/or AcMPAG published in the last decade (2005 to current) to enable visual comparison of the methods; (b) to compare and contrast a few enzymatic assays with those of the chemical methods; and (c) to discuss relevant issues/limitations and perspectives on select assays under various subheadings. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

    Science.gov (United States)

    Abud, J E; Luque, E H; Ramos, J G; Rodriguez, H A

    2017-07-01

    GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Effects of formic acid hydrolysis on the quantitative analysis of radiation-induced DNA base damage products assayed by gas chromatography/mass spectrometry

    International Nuclear Information System (INIS)

    Swarts, S.G.; Smith, G.S.; Miao, L.; Wheeler, K.T.

    1996-01-01

    Gas chromatography/mass spectrometry (GC/ MS-SIM) is an excellent technique for performing both qualitative and quantitative analysis of DNA base damage products that are formed by exposure to ionizing radiation or by the interaction of intracellular DNA with activated oxygen species. This technique commonly uses a hot formic acid hydrolysis step to degrade the DNA to individual free bases. However, due to the harsh nature of this degradation procedure, the quantitation of DNA base damage products may be adversely affected. Consequently, we examined the effects of various formic acid hydrolysis procedures on the quantitation of a number of DNA base damage products and identified several factors that can influence this quantitation. These factors included (1) the inherent acid stabilities of both the lesions and the internal standards; (2) the hydrolysis temperature; (3) the source and grade of the formic acid; and (4) the sample mass during hydrolysis. Our data also suggested that the N, O-bis (trimethylsilyl)trifluoroacetamide (BSTFA) derivatization efficiency can be adversely affected, presumably by trace contaminants either in the formic acid or from the acid-activated surface of the glass derivatization vials. Where adverse effects were noted, modifications were explored in an attempt to improve the quantitation of these DNA lesions. Although experimental steps could be taken to minimize the influence of these factors on the quantitation of some base damage products, no single procedure solved the quantitation problem for all base lesions. However, a significant improvement in the quantitation was achieved if the relative molecular response factor (RMRF) values for these lesions were generated with authentic DNA base damage products that had been treated exactly like the experimental samples. (orig.)

  13. Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS

    Directory of Open Access Journals (Sweden)

    Gerola Paolo D

    2009-12-01

    Full Text Available Abstract Background The β-glucuronidase (GUS gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.

  14. Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

    Science.gov (United States)

    Tran, Nini; Zielke, Ryszard A; Vining, Oliver B; Azevedo, Mark D; Armstrong, Donald J; Banowetz, Gary M; McPhail, Kerry L; Sikora, Aleksandra E

    2013-09-01

    Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

  15. A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Srinivas, Nuggehally R

    2016-01-01

    Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the an...

  16. Liquid chromatography-tandem mass spectrometric assay for the quantitative determination of the tyrosine kinase inhibitor quizartinib in mouse plasma using salting-out liquid-liquid extraction

    NARCIS (Netherlands)

    Retmana, Irene A; Wang, Jing; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2017-01-01

    A bioanalytical assay for quizartinib -a potent, and selective FLT3 tyrosine kinase inhibitor- in mouse plasma was developed and validated. Salting-out assisted liquid-liquid extraction (SALLE), using acetonitrile and magnesium sulfate, was selected as sample pretreatment with deuterated quizartinib

  17. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  18. Binary Classification of a Large Collection of Environmental Chemicals from Estrogen Receptor Assays by Quantitative Structure-Activity Relationship and Machine Learning Methods

    Science.gov (United States)

    ABSTRACT: There are thousands of environmental chemicals subject to regulatory decisions for endocrine disrupting potential. A promising approach to manage this large universe of untested chemicals is to use a prioritization filter that combines in vitro assays with in silico QSA...

  19. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin–DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L.; Powers, Alvin C.; Liu, Guozheng

    2016-01-01

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ~95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  20. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark® for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

    Directory of Open Access Journals (Sweden)

    Jeffrey S. Larson

    2010-01-01

    Full Text Available We report here the results of the analytical validation of assays that measure HER2 total protein (H2T and HER2 homodimer (H2D expression in Formalin Fixed Paraffin Embedded (FFPE breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC (HercepTest. The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC or on indirect assessments of gene amplification (FISH.

  1. Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA.

    Science.gov (United States)

    Qurollo, Barbara A; Archer, Nikole R; Schreeg, Megan E; Marr, Henry S; Birkenheuer, Adam J; Haney, Kaitlin N; Thomas, Brittany S; Breitschwerdt, Edward B

    2017-03-07

    Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp. Using 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR. The LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA q

  2. Solid phase radioimmunoassay for quantitation of IgM rheumatoid factor (RF). Comparison with agglutination techniques and radioimmunoprecipitation polyethylene glycol assay (RIPEGA)

    Energy Technology Data Exchange (ETDEWEB)

    Herrmann, D.; Jaeger, L.; Hein, G.; Henzgen, M.; Fiebig, H.; Schlenvoigt, G.; Vogelsang, H. (Friedrich-Schiller-Universitaet, Jena (German Democratic Republic). Bereich Medizin; Karl-Marx-Universitaet, Leipzig (German Democratic Republic). Sektion Biowissenschaften)

    1985-01-01

    A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor using a monoclonal anti-..mu..-chain antibody is described. Human IgG did not interfere with the detection of IgM RF by this method. The small nonspecific binding of nonRF IgM to the human IgG coated tubes utilized in the assay must be corrected for by assaying samples in parallel bovine serum albumin coated control tubes only in cases of deviation of IgM from normal range. 69 coded and randomly arranged sera from patients with rheumatoid arthritis (RA), nonrheumatic joint diseases and healthy adult control subjects were investigated by this method, agglutination techniques as well as RIPEGA. A good correlation between solid-phase radioimmunoassay and agglutination techniques was found. Patients with seropositive RA had significantly higher concentrations of IgM RF than seronegative RA patients or control subjects.

  3. Potentiometric titration in a low volume of solution for rapid assay of uranium. Application to quantitative electro-reduction of uranium(VI)

    International Nuclear Information System (INIS)

    Sahoo, P.; Ananthanarayanan, R.; Murali, N.; Mallika, C.; Falix Lawrence; Kamachi Mudali, U.

    2012-01-01

    A simple, inexpensive PC based potentiometric titration technique for the assay of uranium using low volumes of sample aliquot (25-100 μL) along with all reagents (total volume of solution being less than 2.5 mL) is presented. The technique involves modification of the well known Davies and Gray Method recommended for assay of uranium(VI) in nuclear materials by introducing an innovative potentiometric titration device with a mini cell developed in-house. After appropriate chemical conditioning the titration is completed within a couple of minutes with display of online titration plot showing the progress of titration. The first derivative plot generated immediately after titration provides information of end point. The main advantage of using this technique is to carry out titration with minimum volumes of sample and reagents generating minimum volume of wastes after titration. The validity of the technique was evaluated using standard certified samples. This technique was applied for assay of uranium in a typical sample collected from fuel reprocessing laboratory. Further, the present technique was deployed in investigating the optimum conditions for efficient in situ production of U(IV). The precision in the estimation of uranium is highly satisfactory (RSD less than 1.0%). (author)

  4. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    Science.gov (United States)

    Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  5. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    Directory of Open Access Journals (Sweden)

    Xin Zhang

    Full Text Available Fusarium oxysporum f. sp. cubense (Foc, the causal agent of Fusarium wilt (Panama disease, is one of the most devastating diseases of banana (Musa spp.. The Foc tropical race 4 (TR4 is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05. Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  6. Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

    Science.gov (United States)

    Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

    2016-06-01

    Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA.

  7. A Sensitive and Robust Ultra HPLC Assay with Tandem Mass Spectrometric Detection for the Quantitation of the PARP Inhibitor Olaparib (AZD2281 in Human Plasma for Pharmacokinetic Application

    Directory of Open Access Journals (Sweden)

    Jeffrey Roth

    2014-06-01

    Full Text Available Olaparib (AZD2281 is an orally active PARP-1 inhibitor, primarily effective against cancers with BRCA1/2 mutations. It is currently in Phase III development and has previously been investigated in numerous clinical trials, both as a single agent and in combination with chemotherapy. Despite this widespread testing, there is only one published method that provides assay details and stability studies for olaparib alone. A more sensitive uHPLC-MS/MS method for the quantification of olaparib in human plasma was developed, increasing the range of quantification at both ends (0.5–50,000 ng/mL compared to previously published methods (10–5,000 ng/mL. The wider range encompasses CMAX levels produced by typical olaparib doses and permits better pharmacokinetic modeling of olaparib elimination. This assay also utilizes a shorter analytical runtime, allowing for more rapid quantification and reduced use of reagents. A liquid-liquid extraction was followed by chromatographic separation on a Waters UPLC® BEH C18 column (2.1 × 50 mm, 1.7 µm and mass spectrometric detection. The mass transitions m/z 435.4→281.1 and m/z 443.2→281.1 were used for olaparib and the internal standard [2H8]-olaparib, respectively. The assay proved to be accurate (<9% deviation and precise (CV < 11%. Stability studies showed that olaparib is stable at room temperature for 24 h. in whole blood, at 4 °C for 24 h post-extraction, at −80 °C in plasma for at least 19 months, and through three freeze-thaw cycles. This method proved to be robust for measuring olaparib levels in clinical samples from a Phase I trial.

  8. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  9. Development and evaluation of the quantitative real-time PCR assay in detection and typing of herpes simplex virus in swab specimens from patients with genital herpes.

    Science.gov (United States)

    Liu, Junlian; Yi, Yong; Chen, Wei; Si, Shaoyan; Yin, Mengmeng; Jin, Hua; Liu, Jianjun; Zhou, Jinlian; Zhang, Jianzhong

    2015-01-01

    Genital herpes (GH), which is caused mainly by herpes simplex virus (HSV)-2 and HSV-1, remains a worldwide problem. Laboratory confirmation of GH is important, particularly as there are other conditions which present similarly to GH, while atypical presentations of GH also occur. Currently, virus culture is the classical method for diagnosis of GH, but it is time consuming and with low sensitivity. A major advance for diagnosis of GH is to use Real-time polymerase chain reaction (PCR). In this study, to evaluate the significance of the real-time PCR method in diagnosis and typing of genital HSV, the primers and probes targeted at HSV-1 DNA polymerase gene and HSV-2 glycoprotein D gene fraction were designed and applied to amplify DNA from HSV-1 or HSV-2 by employing the real-time PCR technique. Then the PCR reaction system was optimized and evaluated. HSV in swab specimens from patients with genital herpes was detected by real-time PCR. The real-time PCR assay showed good specificity for detection and typing of HSV, with good linear range (5×10(2)~5×10(8) copies/ml, r=0.997), a sensitivity of 5×10(2) copies/ml, and good reproducibility (intra-assay coefficients of variation 2.29% and inter-assay coefficients of variation 4.76%). 186 swab specimens were tested for HSV by real-time PCR, and the positive rate was 23.7% (44/186). Among the 44 positive specimens, 8 (18.2%) were positive for HSV-1 with a viral load of 8.5546×10(6) copies/ml and 36 (81.2%) were positive for HSV-2 with a viral load of 1.9861×10(6) copies/ml. It is concluded that the real-time PCR is a specific, sensitive and rapid method for the detection and typing of HSV, which can be widely used in clinical diagnosis of GH.

  10. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

    2011-08-01

    Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

  11. Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

    Science.gov (United States)

    Aigrain, Louise; Gu, Yong; Quail, Michael A

    2016-06-13

    The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

  12. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  13. The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

    Science.gov (United States)

    Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

    2015-10-01

    The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative 10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

  14. Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

    Science.gov (United States)

    Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

    2014-01-15

    Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Quantitative fluorescence-polymerase chain reaction assay for the detection of the duplication of the Charcot Marie Tooth disease type 1A critical region.

    Science.gov (United States)

    De Toffol, Simona; Bellone, Emilia; Dulcetti, Francesca; Ruggeri, Anna Maria; Maggio, Pietro Paolo; Pulimeno, Maria Rosaria; Mandich, Paola; Maggi, Federico; Simoni, Giuseppe; Grati, Francesca Romana

    2010-04-01

    Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

  16. [Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

    Science.gov (United States)

    Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

    2014-05-01

    Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  17. Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum.

    Science.gov (United States)

    Lee, Hyunju; Lim, Soo Young; Kim, Kyung Hyo

    2017-10-01

    The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05-0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. © 2017 The Korean Academy of Medical Sciences.

  18. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for first-line screening.

    Science.gov (United States)

    Fenollar, Florence; Laouira, Sonia; Lepidi, Hubert; Rolain, Jean-Marc; Raoult, Didier

    2008-09-01

    Whipple disease (WD) is a chronic infectious disease caused by Tropheryma whipplei. WD DNA has been found in stool and saliva specimens from patients and asymptomatic carriers. A total of 4418 samples that were sent to our center for determination of WD were tested by a T. whipplei-specific quantitative polymerase chain reaction (PCR) based on repetitive sequences. Definite WD was diagnosed in 71 patients, including 55 patients with classic WD (defined by positive results of periodic acid-Schiff staining and/or specific immunohistochemistry of small-bowel biopsy specimens) and 16 patients with localized WD (including patients with endocarditis, neurologic infection, and uveitis). Of the persons without WD, 2.3% had stool specimens positive for T. whipplei by PCR and 0.2% had saliva specimens positive for T. whipplei by PCR. Diagnosis of WD was likely in patients with positive results of both PCR of saliva specimens and PCR of stool specimens (positive predictive value, 95.2%). When the bacterial load was >10(4) colony-forming units per g of stool, the positive predictive value was 100%. A negative result of PCR of a saliva or stool specimen had a negative predictive value of 99.2% for classic WD. For localized WD, positive results of both PCR of saliva specimens and PCR of stool specimens had a sensitivity of 58% (compared with 94% for classic WD). The positive predictive value of testing of blood, cerebrospinal fluid, and urine specimens was 100% for each, and the positive predictive value for testing of duodenal biopsy specimens was 97.5%. T. whipplei-specific quantitative PCR of saliva and stool specimens should be performed as first-line noninvasive screening for WD. When the results for both types of specimens are positive, diagnosis of classic WD should be highly suspected, especially if a high bacterial load is detected. Because PCR of saliva and stool specimens lacks sensitivity for determination of localized WD, invasive samples should be tested on the

  19. Quantitative GC-MS assay of citric acid from humans and db/db mice blood serum to assist the diagnosis of diabetic nephropathy.

    Science.gov (United States)

    Gao, Haoxue; Yu, Xiaoyi; Sun, Runbin; Yang, Na; He, Jun; Tao, Mingxue; Gu, Huilin; Yan, Caixia; Aa, Jiye; Wang, Guangji

    2018-03-01

    The early diagnosis of diabetic nephropathy (DN) is rather challenging. Our previous study suggested that citric acid is a potential marker for the early diagnosis of diabetic nephropathy in db/db mice. For the first time, in this study, a surrogate analyte of 13 C 6 -citric acid was employed to generate calibration curves for the quantitative measurement of the endogenous citric acid in the sera of db/db mice and diabetic nephropathy patients by GC/MS after the analytes were extracted, methoximated and trimethylsilylated. The constant response factor of 13 C 6 -citric acid versus citric acid over the linear range indicated the identical ionization efficiency of these two compounds. The full validation assessments suggested that the method is sensitive, specific, reliable, reproducible and has acceptable parameters. Statistical analysis revealed cut-off citric acid concentrations of 29.24 μg/mL with a 95% confidence interval between 32.75 and 39.16 μg/mL in the diabetic nephropathy patients and 16.74 and 22.57 μg/mL in the normal controls. The areas under the receiver operating characteristic curves indicated accuracies of over 90% for the diagnoses of early diabetic nephropathy in both humans and db/db mice, which suggests that the serum citric acid level is potentially a biomarker that could assist in the diagnosis of diabetic nephropathy. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay.

    Science.gov (United States)

    Liang, Huipeng; Li, Wenfang; Luo, Qingchun; Liu, Chaolan; Wu, Zhengyun; Zhang, Wenxue

    2015-10-01

    The community structure of bacteria in aged and aging pit mud, which was judged according to their sensory and physicochemical characteristics, was analysed using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR). The phyla Firmicutes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected and the fermentative Firmicutes was predominant in both types of pit mud in the PCR-DGGE analysis. Among Firmicutes, Clostridiales was dominant in aged pit mud while Bacillales and Lactobacillales were dominant in aging pit mud. The diversity of bacterial communities in aged pit mud was higher than that in aging pit mud. In the qPCR analysis the abundance of Clostridium IV in aged pit mud was higher than that in aging pit mud and there were significant differences in the quantity of Clostridium IV between aged and aging pit mud of the same cellar (P mud. The differences in the quantity of Clostridium IV might be involved in the distinction that the aged pit mud has a strong aroma while the aging pit mud does not. © 2014 Society of Chemical Industry.

  1. Development of a Quantitative TaqMan(trademark)-PCR Assay and Feasibility of Atmosphoric Collection for Coccidioides Immits for Ecological Studies

    International Nuclear Information System (INIS)

    Daniels, J.I.; Wilson, W.J.; DeSantis, T.Z.; Gouveia, F.J.; Anderson, G.L.; Shinn, J.H.; Pletcher, R.; Johnson, S.M.; Pappagianis, D.

    2002-01-01

    identified as a select (biological) agent in the federal Anti-Terrorist and Effective Death Penalty Act. Successful demonstration of these tools in this study will place this multidisciplinary team in a credible position to proceed with additional research designed to determine the climatic signals and ecological triggers that would be associated with the presence of this microorganism environmentally and that would correlate with subsequent outbreaks of Valley Fever clinically. Results from such future research would then provide the information needed for environmental intervention of the disease occurrence, well before clinical cases appear. The technology and modeling developed for such a study also could be used for determining the ecology of other environmentally linked, medically important infectious diseases that occur naturally or that might be introduced deliberately into environmental media indoors or outside. The following approach was taken to achieve the technological objectives of this study. First, the protocols for the TaqMan(trademark)-PCR assay were enhanced to achieve the superior specificity and sensitivity required for quantifying, from a DNA signature, those C. immitis spores that are present in calibration samples (consisting of known quantities of pure culture inoculated onto air-filter concentrate, and then removed, and the DNA extracted) and those that are present within the calibration range on air filters obtained from the field and handled similarly. Second, the feasibility of using advanced nuclepore air-filter media to collect the spores from ambient air in C immitis endemic areas in the Central Valley of California was evaluated. These membranes permit the physics of high-volume air sampling to be used to filter larger amounts of air than previously possible for detecting airborne microorganisms. Thus, the higher volume of air flow improves the likelihood of capturing on the filter any C. immitis spores resuspended from nearby soil, where

  2. Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

    Directory of Open Access Journals (Sweden)

    Mojtaba Tahmoorespur

    2016-04-01

    Full Text Available Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor, using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR. It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2 was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio

  3. Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides.

    Science.gov (United States)

    Yamano, Tetsuo; Shimizu, Mitsuru; Noda, Tsutomu

    2005-07-01

    We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (microg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r=0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r=0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA.

  4. Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides

    International Nuclear Information System (INIS)

    Yamano, Tetsuo; Shimizu, Mitsuru; Noda, Tsutomu

    2005-01-01

    We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (μg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r = 0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r = 0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA

  5. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  6. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  7. In-planta detection and monitorization of endophytic colonization by a Beauveria bassiana strain using a new-developed nested and quantitative PCR-based assay and confocal laser scanning microscopy.

    Science.gov (United States)

    Landa, B B; López-Díaz, C; Jiménez-Fernández, D; Montes-Borrego, M; Muñoz-Ledesma, F J; Ortiz-Urquiza, A; Quesada-Moraga, E

    2013-10-01

    Beauveria bassiana strain 04/01-Tip obtained from larvae of the opium poppy stem gall Iraella luteipes endophytically colonizes opium poppy plants and protect it against this pest. Development of a specific, rapid and sensitive technique that allows accurately determining the process and factors leading to the establishment of this strain in opium poppy plants would be essential to achieve its efficient control in a large field scale. For that purpose in the present study, species-specific primers that can be used in conventional or quantitative PCR protocols were developed for specifically identification and detection of B. bassiana in plant tissues. The combination of the designed BB.fw/BB.rv primer set with the universal ITS1-F/ITS4 primer set in a two-step nested-PCR approach, has allowed the amplification of up to 10fg of B. bassiana. This represented an increase in sensitivity of 10000- and 1000-fold of detection than when using the BB.fw/BB.rv primers in a single or single-tube semi-nested PCR approaches, respectively. The BB.fw and BB.rv primer set were subsequently optimized to be used in real time quantitative PCR assays and allowed to accurately quantify B. bassiana DNA in different plant DNA backgrounds (leaves and seeds) without losing accuracy and efficiency. The qPCR protocol was used to monitor the endophytic colonization of opium poppy leaves byB. bassiana after inoculation with the strain EABb 04/01-Tip, detecting as low as 26fg of target DNA in leaves and a decrease in fungal biomass over time. PCR quantification data were supported in parallel with CLMS by the monitoring of spatial and temporal patterns of leaf and stem colonization using a GFP-tagged transformant of the B. bassiana EABb 04/01-Tip strain, which enabled to demonstrate that B. bassiana effectively colonizes aerial tissues of opium poppy plants mainly through intercellular spaces and even leaf trichomes. A decline in endophytic colonization was also observed by the last sampling

  8. Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

    Science.gov (United States)

    Jovanovic, J V; Ivey, A; Vannucchi, A M; Lippert, E; Oppliger Leibundgut, E; Cassinat, B; Pallisgaard, N; Maroc, N; Hermouet, S; Nickless, G; Guglielmelli, P; van der Reijden, B A; Jansen, J H; Alpermann, T; Schnittger, S; Bench, A; Tobal, K; Wilkins, B; Cuthill, K; McLornan, D; Yeoman, K; Akiki, S; Bryon, J; Jeffries, S; Jones, A; Percy, M J; Schwemmers, S; Gruender, A; Kelley, T W; Reading, S; Pancrazzi, A; McMullin, M F; Pahl, H L; Cross, N C P; Harrison, C N; Prchal, J T; Chomienne, C; Kiladjian, J J; Barbui, T; Grimwade, D

    2013-10-01

    Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

  9. Receptor assay

    Energy Technology Data Exchange (ETDEWEB)

    Kato, K; Ibayashi, H [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine

    1975-05-01

    This paper summarized present status and problems of analysis of hormone receptor and a few considerations on clinical significance of receptor abnormalities. It was pointed that in future clinical field quantitative and qualitative analysis of receptor did not remain only in the etiological discussion, but that it was an epoch-making field of investigation which contained the possiblity of artificial change of sensitivity of living body on drugs and the development connected directly with treatment of various diseases.

  10. Application of a Receptor-Binding Capture Quantitative Reverse Transcription-PCR Assay To Concentrate Human Norovirus from Sewage and To Study the Distribution and Stability of the Virus

    Science.gov (United States)

    Yang, David; Pan, Liangwen; Mandrell, Robert

    2012-01-01

    Water is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as average CT values of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 103 genomic copies per liter (gc/liter) and 4.3 × 104 gc/liter, respectively. No GI and sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage. PMID:22101044

  11. Field evaluation of an open and polyvalent universal HIV-1/SIVcpz/SIVgor quantitative RT-PCR assay for HIV-1 viral load monitoring in comparison to Abbott RealTime HIV-1 in Cameroon.

    Science.gov (United States)

    Guichet, Emilande; Aghokeng, Avelin; Eymard-Duvernay, Sabrina; Vidal, Nicole; Ayouba, Ahidjo; Mpoudi Ngole, Eitel; Delaporte, Eric; Ciaffi, Laura; Peeters, Martine

    2016-11-01

    With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log 10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log 10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples.

    Science.gov (United States)

    Schalasta, Gunnar; Börner, Anna; Speicher, Andrea; Enders, Martin

    2018-03-28

    Proper management of patients with chronic hepatitis B virus (HBV) infection requires monitoring of plasma or serum HBV DNA levels using a highly sensitive nucleic acid amplification test. Because commercially available assays differ in performance, we compared herein the performance of the Hologic Aptima HBV Quant assay (Aptima) to that of the Roche Cobas TaqMan HBV test for use with the high pure system (HPS/CTM). Assay performance was assessed using HBV reference panels as well as plasma and serum samples from chronically HBV-infected patients. Method correlation, analytical sensitivity, precision/reproducibility, linearity, bias and influence of genotype were evaluated. Data analysis was performed using linear regression, Deming correlation analysis and Bland-Altman analysis. Agreement between the assays for the two reference panels was good, with a difference in assay values vs. target 0.98). The two assays had similar bias and precision across the different genotypes tested at low viral loads (25-1000 IU/mL). Aptima has a performance comparable with that of HPS/CTM, making it suitable for use for HBV infection monitoring. Aptima runs on a fully automated platform (the Panther system) and therefore offers a significantly improved workflow compared with HPS/CTM.

  13. Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

    Science.gov (United States)

    Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

    2011-12-01

    Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

  14. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  15. Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay

    Science.gov (United States)

    Stanton, Jeffrey J.; Zong, Jian-Chao; Latimer, Erin; Tan, Jie; Herron, Alan; Hayward, Gary S.; Ling, Paul D.

    2013-01-01

    Objective To investigate the pathogenesis and transmission of elephant endotheliotropic herpesvirus (EEHV1) by analyzing various elephant fluid samples with a novel EEHV1-specific real-time PCR assay. Animals 5 apparently healthy captive Asian elephants (Elephas maximus) from the same herd. Procedures A real-time PCR assay was developed that specifically detects EEHV1. The assay was used to evaluate paired whole blood and trunk-wash samples obtained from the 5 elephants during a 15-week period. Deoxyribonucleic acid sequencing and viral gene subtyping analysis were performed on trunk-wash DNA preparations that had positive results for EEHV1. Viral gene subtypes were compared with those associated with past fatal cases of herpesvirus-associated disease within the herd. Results The PCR assay detected viral DNA to a level of 1,200 copies/mL of whole blood. It was used to detect EEHV1 in trunk secretions of 3 of the 5 elephants surveyed during the 15-week period. Viral gene subtyping analysis identified 2 distinct elephant herpesviruses, 1 of which was identical to the virus associated with a previous fatal case of herpesvirus-associated disease within the herd. Conclusions and Clinical Relevance EEHV1 was shed in the trunk secretions of healthy Asian elephants. Trunk secretions may provide a mode of transmission for this virus. Results of this study may be useful for the diagnosis, treatment, and management of EEHV1-associated disease and the overall management of captive elephant populations. PMID:20673092

  16. Fundamentals of passive nondestructive assay of fissionable material: laboratory workbook

    International Nuclear Information System (INIS)

    Reilly, T.D.; Augustson, R.H.; Parker, J.L.; Walton, R.B.; Atwell, T.L.; Umbarger, C.J.; Burns, C.E.

    1975-02-01

    This workbook is a supplement to LA-5651-M, ''Fundamentals of Passive Nondestructive Assay of Fissionable Material'' which is the text used during the Nondestructive Assay Training Session given by Group A-1 of the Los Alamos Scientific Laboratory. It contains the writeups used during the six laboratory sessions covering basic gamma-ray principles, quantitative gamma-ray measurements, uranium enrichment measurements, equipment holdup measurements, basic neutron principles, and quantitative neutron assay

  17. Fundamentals of passive nondestructive assay of fissionable material: laboratory workbook

    Energy Technology Data Exchange (ETDEWEB)

    Reilly, T.D.; Augustson, R.H.; Parker, J.L. Walton, R.B.; Atwell, T.L.; Umbarger, C.J.; Burns, C.E.

    1975-02-01

    This workbook is a supplement to LA-5651-M, ''Fundamentals of Passive Nondestructive Assay of Fissionable Material'' which is the text used during the Nondestructive Assay Training Session given by Group A-1 of the Los Alamos Scientific Laboratory. It contains the writeups used during the six laboratory sessions covering basic gamma-ray principles, quantitative gamma-ray measurements, uranium enrichment measurements, equipment holdup measurements, basic neutron principles, and quantitative neutron assay.

  18. Optical assay for biotechnology and clinical diagnosis.

    Science.gov (United States)

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  19. Quantitation of (beta)N-Alkanoyl-5-hydroxytryptamides in coffee by means of LC-MS/MS-SIDA and assessment of their gastric acid secretion potential using the HGT-1 cell assay.

    Science.gov (United States)

    Lang, Roman; Bardelmeier, Ina; Weiss, Carola; Rubach, Malte; Somoza, Veronika; Hofmann, Thomas

    2010-02-10

    A straightforward stable isotope dilution analysis (SIDA) for the reliable quantitative determination of (beta)N-C(18:0)- to (beta)N-C(24:0)-alkanoyl-5-hydroxytryptamides (C5HTs) in coffee powder and beverages by means of LC-MS/MS was developed. The developed SIDA showing accuracy values of 92.6-107% and precision between 0.5 and 7% relative standard deviation for the individual derivatives allowed the sensitive and selective quantification of the target compounds in coffee beverages. Depending on the type of coffee, quantitation revealed C5HT levels between 65 and 144 microg/L in filtered coffee and up to 3500 microg/L in a French press beverage, thus indicating that about 0.3 or 7.2% of the C5HTs were extracted from the coffee powder into the beverage when using the cellulose filter method or the French press, respectively. To estimate the potential contribution of the C5HTs to the phenomenon of stomach irritation after ingestion of coffee brew, in vitro cell studies were performed with pure individual 5-hydroxytryptamides and a mixture of the predominating derivatives in ratios matching those found in coffee. All substances tested induced a decrease in the intracellular proton index (IPX) coined as an indicator of stomach acid secretion. While the biomimetic C5HT mixture was highest in its inducing effect, the individual stearic acid, oleic acid, and linoleic acid 5-hydroxytryptamide did not differ significantly from each other, but showed a less pronounced effect compared to arachinic acid 5-hydroxytryptamide. In conclusion, not the grade of saturation seems to determine the C5HT's mode of action in driving the stomach acid secretion, rather than the fatty acid chain length.

  20. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil; Al Kilani, Alia; Hamdan, Samir; Sakashita, Kosuke; Gadhoum, Samah Z.; Merzaban, Jasmeen

    2015-01-01

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

    Science.gov (United States)

    Loh, W K W; Bond, P; Ashton, K J; Roberts, D T; Tibbetts, I R

    2014-08-01

    The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples. © 2014 The Fisheries Society of the British Isles.

  2. A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

    Science.gov (United States)

    Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

    2015-10-01

    Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. Copyright © 2015. Published by Elsevier Ltd.

  3. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2015-06-29

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a [3H]estradiol binding assay based on isoelectric focusing in polyacrylamide gel

    International Nuclear Information System (INIS)

    Pousette, A.; Gustafsson, S.A.; Thoernblad, A.M.N.; Nordgren, A.; Saellstroem, J.Li.; Lindgren, A.; Sundelin, P.; Gustafsson, J.A.

    1986-01-01

    Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of [ 3 H]estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure

  5. Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

    Science.gov (United States)

    Tissier, Adeline; Denis, Martine; Hartemann, Philippe

    2012-01-01

    Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter. PMID:22138985

  6. Computer-assisted assessment of the Human Epidermal Growth Factor Receptor 2 immunohistochemical assay in imaged histologic sections using a membrane isolation algorithm and quantitative analysis of positive controls

    International Nuclear Information System (INIS)

    Hall, Bonnie H; Ianosi-Irimie, Monica; Javidian, Parisa; Chen, Wenjin; Ganesan, Shridar; Foran, David J

    2008-01-01

    Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2) are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra- and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens. Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+) cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC) in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than manual scoring, and significantly less false positive rate when used to evaluate immunohistochemically equivocal cases. It has been shown that by incorporating both a membrane isolation algorithm and analysis of known positive controls a computer-assisted diagnostic algorithm was

  7. Computer-assisted assessment of the Human Epidermal Growth Factor Receptor 2 immunohistochemical assay in imaged histologic sections using a membrane isolation algorithm and quantitative analysis of positive controls

    Directory of Open Access Journals (Sweden)

    Ianosi-Irimie Monica

    2008-06-01

    Full Text Available Abstract Background Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2 are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra- and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens. Methods Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+ cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC analysis. Results A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than manual scoring, and significantly less false positive rate when used to evaluate immunohistochemically equivocal cases. Conclusion It has been shown that by incorporating both a membrane isolation algorithm and analysis of known

  8. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  9. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  10. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  11. Quantitative research.

    Science.gov (United States)

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  12. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  13. Quantitative habitability.

    Science.gov (United States)

    Shock, Everett L; Holland, Melanie E

    2007-12-01

    A framework is proposed for a quantitative approach to studying habitability. Considerations of environmental supply and organismal demand of energy lead to the conclusions that power units are most appropriate and that the units for habitability become watts per organism. Extreme and plush environments are revealed to be on a habitability continuum, and extreme environments can be quantified as those where power supply only barely exceeds demand. Strategies for laboratory and field experiments are outlined that would quantify power supplies, power demands, and habitability. An example involving a comparison of various metabolisms pursued by halophiles is shown to be well on the way to a quantitative habitability analysis.

  14. Quantitating cellular immune responses to cancer vaccines.

    Science.gov (United States)

    Lyerly, H Kim

    2003-06-01

    While the future of immunotherapy in the treatment of cancer is promising, it is difficult to compare the various approaches because monitoring assays have not been standardized in approach or technique. Common assays for measuring the immune response need to be established so that these assays can one day serve as surrogate markers for clinical response. Assays that accurately detect and quantitate T-cell-mediated, antigen-specific immune responses are particularly desired. However, to date, increases in the number of cytotoxic T cells through immunization have not been correlated with clinical tumor regression. Ideally, then, a T-cell assay not only needs to be sensitive, specific, reliable, reproducible, simple, and quick to perform, it must also demonstrate close correlation with clinical outcome. Assays currently used to measure T-cell response are delayed-type hypersensitivity testing, flow cytometry using peptide major histocompatibility complex tetramers, lymphoproliferation assay, enzyme-linked immunosorbant assay, enzyme-linked immunospot assay, cytokine flow cytometry, direct cytotoxicity assay, measurement of cytokine mRNA by quantitative reverse transcriptase polymerase chain reaction, and limiting dilution analysis. The purpose of this review is to describe the attributes of each test and compare their advantages and disadvantages.

  15. Comparison of Different Promoter Methylation Assays in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Karijn P. M. Suijkerbuijk

    2010-01-01

    Full Text Available Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance.

  16. Restriction Inhibition Assay: A Qualitative and Quantitative Method to ...

    African Journals Online (AJOL)

    Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index ... inclination towards plant derived drug discovery ..... small biological samples [13].

  17. Quantitative Assay for Starch by Colorimetry Using a Desktop Scanner

    Science.gov (United States)

    Matthews, Kurt R.; Landmark, James D.; Stickle, Douglas F.

    2004-01-01

    The procedure to produce standard curve for starch concentration measurement by image analysis using a color scanner and computer for data acquisition and color analysis is described. Color analysis is performed by a Visual Basic program that measures red, green, and blue (RGB) color intensities for pixels within the scanner image.

  18. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    National Research Council Canada - National Science Library

    Merrill, Gerald A; Rivera, Victor R; Neal, Dwayne D; Young, Charles; Poli, Mark A

    2006-01-01

    .... Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru...

  19. Restriction Inhibition Assay: A Qualitative and Quantitative Method to ...

    African Journals Online (AJOL)

    rich fractions (PRFs) with high affinity for EcoRI and HindIII restriction sequences and correlate their interaction to an anticancer activity. Methods: pBR322 linear plasmid DNA was used as a template to screen the sequence-selective inhibition of ...

  20. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    Science.gov (United States)

    2006-08-10

    Doyle, L.R. Beuchat, T.J. Montville (Eds.), Food Microbiology : Fundamentals and Fron- tiers, Second ed., ASM Press, Washington, D.C., 2001, pp. 351...D.E. Lorant, A.E. Bryant, G.A. Zimmerman, T.M. McIn- tyre, D.L. Stevens, S.M. Prescott , Alpha toxin from Clostridium per- fringens induces

  1. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  2. A highly sensitive and specific assay for vertebrate collagenase

    International Nuclear Information System (INIS)

    Sodek, J.; Hurum, S.; Feng, J.

    1981-01-01

    A highly sensitive and specific assay for vertebrate collagenase has been developed using a [ 14 C]-labeled collagen substrate and a combination of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and fluorography to identify and quantitate the digestion products. The assay was sufficiently sensitive to permit the detection and quantitation of collagenase activity in 0.1 μl of gingival sulcal fluid, and in samples of cell culture medium without prior concentration. The assay has also been used to detect the presence of inhibitors of collagenolytic enzymes in various cell culture fluids. (author)

  3. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  4. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  5. Factor II assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  6. Factor VII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  7. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  8. Quantitative Finance

    Science.gov (United States)

    James, Jessica

    2017-01-01

    Quantitative finance is a field that has risen to prominence over the last few decades. It encompasses the complex models and calculations that value financial contracts, particularly those which reference events in the future, and apply probabilities to these events. While adding greatly to the flexibility of the market available to corporations and investors, it has also been blamed for worsening the impact of financial crises. But what exactly does quantitative finance encompass, and where did these ideas and models originate? We show that the mathematics behind finance and behind games of chance have tracked each other closely over the centuries and that many well-known physicists and mathematicians have contributed to the field.

  9. Quantitation of 4-Methyl-4-sulfanylpentan-2-one (4MSP) in Hops by a Stable Isotope Dilution Assay in Combination with GC×GC-TOFMS: Method Development and Application To Study the Influence of Variety, Provenance, Harvest Year, and Processing on 4MSP Concentrations.

    Science.gov (United States)

    Reglitz, Klaas; Steinhaus, Martin

    2017-03-22

    A stable isotope dilution assay was developed for quantitation of 4-methyl-4-sulfanylpentan-2-one (4MSP) in hops. The approach included the use of 4-( 13 C)methyl-4-sulfanyl(1,3,5- 13 C 3 )pentan-2-one as internal standard, selective isolation of hop thiols by mercurated agarose, and GC×GC-TOFMS analysis. Application of the method to 53 different hop samples revealed 4MSP concentrations between <1 and 114 μg/kg. Notably high concentrations were associated with United States varieties such as Citra, Eureka, Simcoe, and Apollo, whereas 4MSP was absent from traditional German and English varieties. Further experiments showed that besides the variety, also harvest year and storage vitally influenced 4MSP concentrations, whereas the impact of provenance was less pronounced. Hop processing such as drying and pelletizing had only a minor impact on 4MSP concentrations. Like the majority of other hop volatiles, 4MSP is predominantly located in the lupulin glands.

  10. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  11. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  12. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  13. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  14. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  15. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  16. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  17. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  18. Quantitative radiography

    International Nuclear Information System (INIS)

    Brase, J.M.; Martz, H.E.; Waltjen, K.E.; Hurd, R.L.; Wieting, M.G.

    1986-01-01

    Radiographic techniques have been used in nondestructive evaluation primarily to develop qualitative information (i.e., defect detection). This project applies and extends the techniques developed in medical x-ray imaging, particularly computed tomography (CT), to develop quantitative information (both spatial dimensions and material quantities) on the three-dimensional (3D) structure of solids. Accomplishments in FY 86 include (1) improvements in experimental equipment - an improved microfocus system that will give 20-μm resolution and has potential for increased imaging speed, and (2) development of a simple new technique for displaying 3D images so as to clearly show the structure of the object. Image reconstruction and data analysis for a series of synchrotron CT experiments conducted by LLNL's Chemistry Department has begun

  19. Quantitative lymphography

    International Nuclear Information System (INIS)

    Mostbeck, A.; Lofferer, O.; Kahn, P.; Partsch, H.; Koehn, H.; Bialonczyk, Ch.; Koenig, B.

    1984-01-01

    Labelled colloids and macromolecules are removed lymphatically. The uptake of tracer in the regional lymphnodes is a parameter of lymphatic flow. Due to great variations in patient shape - obesity, cachexia - and accompanying variations in counting efficiencies quantitative measurements with reasonable accuracy have not been reported to date. A new approach to regional absorption correction is based on the combination of transmission and emission scans for each patient. The transmission scan is used for calculation of an absorption correction matrix. Accurate superposition of the correction matrix and the emission scan is achieved by computing the centers of gravity of point sources and - in the case of aligning opposite views - by cross correlation of binary images. In phantom studies the recovery was high (98.3%) and the coefficient of variation of repeated measurement below 1%. In patient studies a standardized stress is a prerequisite for reliable and comparable results. Discrimination between normals (14.3 +- 4.2D%) and patients with lymphedema (2.05 +- 2.5D%) was highly significant using praefascial lymphography and sc injection. Clearence curve analysis of the activities at the injection site, however, gave no reliable data for this purpose. In normals, the uptake in lymphnodes after im injection is by one order of magnitude lower then the uptake after sc injection. The discrimination between normals and patients with postthromboic syndrome was significant. Lymphography after ic injection was in the normal range in 2/3 of the patients with lymphedema and is therefore of no diagnostic value. The difference in uptake after ic and sc injection demonstrated for the first time by our quantitative method provides new insights into the pathophysiology of lymphedema and needs further investigation. (Author)

  20. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  1. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  2. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  3. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  4. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  5. Quantitative Thermochronology

    Science.gov (United States)

    Braun, Jean; van der Beek, Peter; Batt, Geoffrey

    2006-05-01

    Thermochronology, the study of the thermal history of rocks, enables us to quantify the nature and timing of tectonic processes. Quantitative Thermochronology is a robust review of isotopic ages, and presents a range of numerical modeling techniques to allow the physical implications of isotopic age data to be explored. The authors provide analytical, semi-analytical, and numerical solutions to the heat transfer equation in a range of tectonic settings and under varying boundary conditions. They then illustrate their modeling approach built around a large number of case studies. The benefits of different thermochronological techniques are also described. Computer programs on an accompanying website at www.cambridge.org/9780521830577 are introduced through the text and provide a means of solving the heat transport equation in the deforming Earth to predict the ages of rocks and compare them directly to geological and geochronological data. Several short tutorials, with hints and solutions, are also included. Numerous case studies help geologists to interpret age data and relate it to Earth processes Essential background material to aid understanding and using thermochronological data Provides a thorough treatise on numerical modeling of heat transport in the Earth's crust Supported by a website hosting relevant computer programs and colour slides of figures from the book for use in teaching

  6. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  7. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  8. The quantitative Morse theorem

    OpenAIRE

    Loi, Ta Le; Phien, Phan

    2013-01-01

    In this paper, we give a proof of the quantitative Morse theorem stated by {Y. Yomdin} in \\cite{Y1}. The proof is based on the quantitative Sard theorem, the quantitative inverse function theorem and the quantitative Morse lemma.

  9. An improved method for staining cell colonies in clonogenic assays

    OpenAIRE

    Guda, Kishore; Natale, Leanna; Markowitz, Sanford D.

    2007-01-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we ...

  10. PAME: plasmonic assay modeling environment

    Directory of Open Access Journals (Sweden)

    Adam Hughes

    2015-08-01

    Full Text Available Plasmonic assays are an important class of optical sensors that measure biomolecular interactions in real-time without the need for labeling agents, making them especially well-suited for clinical applications. Through the incorporation of nanoparticles and fiberoptics, these sensing systems have been successfully miniaturized and show great promise for in-situ probing and implantable devices, yet it remains challenging to derive meaningful, quantitative information from plasmonic responses. This is in part due to a lack of dedicated modeling tools, and therefore we introduce PAME, an open-source Python application for modeling plasmonic systems of bulk and nanoparticle-embedded metallic films. PAME combines aspects of thin-film solvers, nanomaterials and fiber-optics into an intuitive graphical interface. Some of PAME’s features include a simulation mode, a database of hundreds of materials, and an object-oriented framework for designing complex nanomaterials, such as a gold nanoparticles encased in a protein shell. An overview of PAME’s theory and design is presented, followed by example simulations of a fiberoptic refractometer, as well as protein binding to a multiplexed sensor composed of a mixed layer of gold and silver colloids. These results provide new insights into observed responses in reflectance biosensors.

  11. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    Science.gov (United States)

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

  12. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    Levin, G.V.; Straat, P.A.

    1981-01-01

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  13. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  14. A novel clot lysis assay for recombinant plasminogen activator.

    Science.gov (United States)

    Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

    2015-03-01

    Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

  15. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  16. Quantitative imaging as cancer biomarker

    Science.gov (United States)

    Mankoff, David A.

    2015-03-01

    The ability to assay tumor biologic features and the impact of drugs on tumor biology is fundamental to drug development. Advances in our ability to measure genomics, gene expression, protein expression, and cellular biology have led to a host of new targets for anticancer drug therapy. In translating new drugs into clinical trials and clinical practice, these same assays serve to identify patients most likely to benefit from specific anticancer treatments. As cancer therapy becomes more individualized and targeted, there is an increasing need to characterize tumors and identify therapeutic targets to select therapy most likely to be successful in treating the individual patient's cancer. Thus far assays to identify cancer therapeutic targets or anticancer drug pharmacodynamics have been based upon in vitro assay of tissue or blood samples. Advances in molecular imaging, particularly PET, have led to the ability to perform quantitative non-invasive molecular assays. Imaging has traditionally relied on structural and anatomic features to detect cancer and determine its extent. More recently, imaging has expanded to include the ability to image regional biochemistry and molecular biology, often termed molecular imaging. Molecular imaging can be considered an in vivo assay technique, capable of measuring regional tumor biology without perturbing it. This makes molecular imaging a unique tool for cancer drug development, complementary to traditional assay methods, and a potentially powerful method for guiding targeted therapy in clinical trials and clinical practice. The ability to quantify, in absolute measures, regional in vivo biologic parameters strongly supports the use of molecular imaging as a tool to guide therapy. This review summarizes current and future applications of quantitative molecular imaging as a biomarker for cancer therapy, including the use of imaging to (1) identify patients whose tumors express a specific therapeutic target; (2) determine

  17. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  18. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  19. Quantitative multiplex detection of pathogen biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

    2016-02-09

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  20. Quantitative multiplex detection of pathogen biomarkers

    Science.gov (United States)

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

    2014-10-14

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  1. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

    Full Text Available An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease. Keywords: Prions, Thickness shear mode quartz sensor

  2. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  3. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    Science.gov (United States)

    An ELISA assay for heme oxygenase (HO-l ) Abstract A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  4. Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

    DEFF Research Database (Denmark)

    Engle, Ronald E; Russell, Rodney S; Purcell, Robert H

    2008-01-01

    A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first...

  5. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  6. The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

    Science.gov (United States)

    Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

    2016-02-01

    Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, PLumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

    International Nuclear Information System (INIS)

    Mehdi, S.; Wiseman, J.S.

    1989-01-01

    An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection

  8. Evaluating In Vitro DNA Damage Using Comet Assay.

    Science.gov (United States)

    Lu, Yanxin; Liu, Yang; Yang, Chunzhang

    2017-10-11

    DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

  9. Battery operated preconcentration-assisted lateral flow assay.

    Science.gov (United States)

    Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

    2017-07-11

    Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

  10. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. An environmental DNA assay for detecting Arctic grayling in the upper Missouri River basin, North America

    Science.gov (United States)

    K. J. Carim; J. C. S. Dysthe; Michael Young; Kevin McKelvey; Michael Schwartz

    2016-01-01

    The upper Missouri River basin in the northwestern US contains disjunct Arctic grayling (Thymallus arcticus) populations of conservation concern. To assist efforts aimed at understanding Artic grayling distribution, we developed a quantitative PCR assay to detect the presence of Arctic grayling DNA in environmental samples. The assay amplified low...

  12. GC-MS DETERMINATION OF RATIOS OF STABLE-ISOTOPE LABELED TO NATURAL UREA USING [(CN2)-C-13-N-15]UREA FOR STUDYING UREA KINETICS IN SERUM AND AS A MEANS TO VALIDATE ROUTINE METHODS FOR THE QUANTITATIVE ASSAY OF UREA IN DIALYSATE

    NARCIS (Netherlands)

    WOLTHERS, BG; TEPPER, T; WITHAG, A; NAGEL, GT; DEHAAN, THY; VANLEEUWEN, JJ; STEGEMAN, CA; HUISMAN, RM

    A GC-MS determination of urea in serum or spent dialysate is described, using (CN2)-C-13-N-15-labelled urea and assaying the area ratio of labelled to natural urea by mass fragmentographic monitoring of fragments m/e 153 and 156, after its eventual conversion into the trimethylsilylether-derivative

  13. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  14. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  15. A rapid colorimetric assay for mold spore germination using XTT tetrazolium salt

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2011-01-01

    Current laboratory test methods to measure efficacy of new mold inhibitors are time consuming, some require specialized test equipment and ratings are subjective. Rapid, simple quantitative assays to measure the efficacy of mold inhibitors are needed. A quantitative, colorimetric microassay was developed using XTT tetrazolium salt to metabolically assess mold spore...

  16. Quantitative investment analysis

    CERN Document Server

    DeFusco, Richard

    2007-01-01

    In the "Second Edition" of "Quantitative Investment Analysis," financial experts Richard DeFusco, Dennis McLeavey, Jerald Pinto, and David Runkle outline the tools and techniques needed to understand and apply quantitative methods to today's investment process.

  17. Rigour in quantitative research.

    Science.gov (United States)

    Claydon, Leica Sarah

    2015-07-22

    This article which forms part of the research series addresses scientific rigour in quantitative research. It explores the basis and use of quantitative research and the nature of scientific rigour. It examines how the reader may determine whether quantitative research results are accurate, the questions that should be asked to determine accuracy and the checklists that may be used in this process. Quantitative research has advantages in nursing, since it can provide numerical data to help answer questions encountered in everyday practice.

  18. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  19. Solution assay instrument operations manual

    International Nuclear Information System (INIS)

    Li, T.K.; Marks, T.; Parker, J.L.

    1983-09-01

    An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

  20. Quantitation: clinical applications

    International Nuclear Information System (INIS)

    Britton, K.E.

    1982-01-01

    Single photon emission tomography may be used quantitatively if its limitations are recognized and quantitation is made in relation to some reference area on the image. Relative quantitation is discussed in outline in relation to the liver, brain and pituitary, thyroid, adrenals, and heart. (U.K.)

  1. Radioligand assay in reproductive biology

    International Nuclear Information System (INIS)

    Korenman, S.G.; Sherman, B.M.

    1975-01-01

    Radioligand assays have been developed for the principal reproductive steroids and peptide hormones. Specific binding reagents have included antibodies, plasma binders, and intracellular receptors. In each assay, problems of specificity, sensitivity, and nonspecific inhibitors were encountered. Many features of the endocrine physiology in childhood, during puberty, and in adulthood have been characterized. Hormonal evaluations of endocrine disorders of reproduction are characterized on the basis of their characteristic pathophysiologic alterations. (U.S.)

  2. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  3. Trofile HIV co-receptor usage assay.

    Science.gov (United States)

    Low, Andrew J; McGovern, Rachel A; Harrigan, P Richard

    2009-03-01

    The introduction of CCR5 antagonists increases the options available for constructing therapeutic drug regimens for HIV-positive patients. However, as these drugs do not inhibit HIV variants that use the CXCR4 co-receptor, a pretreatment test is required to determine accurately HIV co-receptor usage (tropism) before initiating CCR5 antagonist-based therapy. To discuss the Monogram Trofile assay as a diagnostic tool for determining HIV tropism by critically reviewing reported literature and available data. Monogram Trofile has become, largely by default, the de facto standard for HIV tropism assay. However, there is significant room for improvement in the speed, cost and availability of the test. Furthermore, the test is not quantitative, requires high-input HIV RNA viral loads, and produces results that are less biologically stable than expected. These technical considerations may limit the use of CCR5 antagonists in therapy. Nevertheless, this test is likely to remain the most widely used tropism diagnostic for the short term. We expect that a more practical and possibly more accurate method for measuring HIV tropism can be developed.

  4. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  5. Detection and quantification of serum or plasma HCV RNA: mini review of commercially available assays.

    Science.gov (United States)

    Le Guillou-Guillemette, Helene; Lunel-Fabiani, Francoise

    2009-01-01

    The treatment schedule (combination of compounds, doses, and duration) and the virological follow-up for management of antiviral treatment in patients chronically infected by HCV is now well standardized, but to ensure good monitoring of the treated patients, physicians need rapid, reproducible, and sensitive molecular virological tools with a wide range of detection and quantification of HCV RNA in blood samples. Several assays for detection and/or quantification of HCV RNA are currently commercially available. Here, all these assays are detailed, and a brief description of each step of the assay is provided. They are divided into two categories by method: those based on signal amplification and those based on target amplification. These two categories are then divided into qualitative, quantitative, and quantitative detection assays. The real-time reverse-transcription polymerase chain reaction (RT-PCR)-based assays are the most promising strategy in the HCV virological area.

  6. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  7. Bioavailability of two oral formulations of triazolam using radioreceptor assay

    Energy Technology Data Exchange (ETDEWEB)

    Lapka, R; Cepelakova, H; Rejholec, V; Franc, Z

    1986-04-01

    The radioreceptor assay (RRA) was used to quantitate plasma triazolam concentration in eight female volunteers following single 0.5 mg doses of two tablet formulations in a cross-over study. Bioavailability in terms of area under the plasma concentration versus time curve, maximum plasma concentration, time to maximum, and mean residence time was not statistically significantly different from one formulation to the other.

  8. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  9. Development of robotic plasma radiochemical assays for positron emission tomography

    International Nuclear Information System (INIS)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J.

    1995-01-01

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [ 11 C]Benztropine, [ 11 C]cocaine, [ 11 C]clorgyline, [ 11 C]deprenyl, [ 11 C]methadone, [ 11 C]methylphenidate, [ 11 C]raclorpride, and [ 11 C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers

  10. Application of radioreceptor assay of benzodiazepines for toxicology

    International Nuclear Information System (INIS)

    Aaltonen, L.; Scheinin, M.

    1982-01-01

    A radioreceptor assay (RRA) for determining benzodiazepines (BZ) has been developed and applied to toxicological analysis of serum samples from 21 patients with acute BZ overdosage. The method was sensitive (e.g., lorazepam 17 nM, diazepam 41 nM), and specific for pharmacologically active BZ derivatives. The reproducibility of the results was good (intra-assay variation < 8%, inter-assay variation < 10%). Concentrations measured by the RRA showed a good correlation with those obtained by gas-liquid chromatographic analysis of the same samples. The quantitative results represent the sum of one or several parent substances and all biologically active metabolites, in proportion to their receptor binding affinities. (author)

  11. Application of statistical process control to qualitative molecular diagnostic assays.

    Science.gov (United States)

    O'Brien, Cathal P; Finn, Stephen P

    2014-01-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  12. A sensitive competitive binding assay for exogenous and endogenous heparins

    International Nuclear Information System (INIS)

    Dawes, J.; Pepper, D.S.

    1982-01-01

    A new type of assay for heparins has been devised, in which the test material competes with 125 I-labelled heparin for binding to protamine-Sepharose. The assay is very sensitive and will measure heparin concentrations down to 10 ng ml-1. It responds to both the degree of sulphation and the molecular weight of acidic polysaccharides, but is independent of their biological activities. It can be used to quantitate heparins in biological fluids after pretreatment of the samples with protease. In this way endogenous heparins were measured in normal human serum, plasma and urine. The assay is extremely versatile and has great potential for the investigation of endogenous and exogenous heparins

  13. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Eustaquio, Trisha; Leary, James F

    2012-01-01

    Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

  14. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    Science.gov (United States)

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

  15. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  16. SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

    Science.gov (United States)

    Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.

    2016-01-01

    The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

  17. Quantitative analysis chemistry

    International Nuclear Information System (INIS)

    Ko, Wansuk; Lee, Choongyoung; Jun, Kwangsik; Hwang, Taeksung

    1995-02-01

    This book is about quantitative analysis chemistry. It is divided into ten chapters, which deal with the basic conception of material with the meaning of analysis chemistry and SI units, chemical equilibrium, basic preparation for quantitative analysis, introduction of volumetric analysis, acid-base titration of outline and experiment examples, chelate titration, oxidation-reduction titration with introduction, titration curve, and diazotization titration, precipitation titration, electrometric titration and quantitative analysis.

  18. Quantitative Algebraic Reasoning

    DEFF Research Database (Denmark)

    Mardare, Radu Iulian; Panangaden, Prakash; Plotkin, Gordon

    2016-01-01

    We develop a quantitative analogue of equational reasoning which we call quantitative algebra. We define an equality relation indexed by rationals: a =ε b which we think of as saying that “a is approximately equal to b up to an error of ε”. We have 4 interesting examples where we have a quantitative...... equational theory whose free algebras correspond to well known structures. In each case we have finitary and continuous versions. The four cases are: Hausdorff metrics from quantitive semilattices; pWasserstein metrics (hence also the Kantorovich metric) from barycentric algebras and also from pointed...

  19. Quantitative autoradiography of neurochemicals

    International Nuclear Information System (INIS)

    Rainbow, T.C.; Biegon, A.; Bleisch, W.V.

    1982-01-01

    Several new methods have been developed that apply quantitative autoradiography to neurochemistry. These methods are derived from the 2-deoxyglucose (2DG) technique of Sokoloff (1), which uses quantitative autoradiography to measure the rate of glucose utilization in brain structures. The new methods allow the measurement of the rate of cerbral protein synthesis and the levels of particular neurotransmitter receptors by quantitative autoradiography. As with the 2DG method, the new techniques can measure molecular levels in micron-sized brain structures; and can be used in conjunction with computerized systems of image processing. It is possible that many neurochemical measurements could be made by computerized analysis of quantitative autoradiograms

  20. Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia trachomatis by Quantitative High-Resolution Melt Analysis (HRMA)

    Science.gov (United States)

    Stevens, Matthew P.; Garland, Suzanne M.; Zaia, Angelo M.; Tabrizi, Sepehr N.

    2012-01-01

    A quantitative high-resolution melt analysis assay was developed to differentiate lymphogranuloma venereum-causing serovars of Chlamydia trachomatis (L1 to L3) from other C. trachomatis serovars (D to K). The detection limit of this assay is approximately 10 copies per reaction, comparable to the limits of other quantitative-PCR-based methods. PMID:22933594

  1. Operating characteristics of a qualitative troponin assay for the diagnosis of acute coronary syndrome.

    Science.gov (United States)

    Farsi, Davood; Pishbin, Elham; Abbasi, Saeed; Hafezimoghadam, Peyman; Fathi, Marzieh; Zare, Mohammad Amin

    2013-04-01

    The troponin I serum level is widely used in acute coronary syndrome patients for their classification. The qualitative assay is faster and more available than the quantitative assay. The objective was to determine the operating characteristics of a qualitative troponin I assay compared with a quantitative method. This is a prospective observational study and patients suspected to have acute coronary syndrome were enrolled. A rapid troponin I test and a quantitative assay were carried out for each patient on arrival and 6 h after admission. A total of 262 patients were enrolled. The degree of agreement between the second rapid qualitative and quantitative troponin I was excellent (κ=0.946; 95% confidence interval, 0.903-0.989). The sensitivity, specificity, negative predictive value, and positive predictive value of the rapid qualitative troponin I test were 92.6, 100, 96.8, and 100%, respectively. In conclusion, this study reveals an excellent agreement between quantitative and qualitative bedside assays 6 h after admission in a sample of Iranian patients in the emergency department.

  2. Drosophila comet assay: insights, uses, and future perspectives

    Science.gov (United States)

    Gaivão, Isabel; Sierra, L. María

    2014-01-01

    The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

  3. Parallel force assay for protein-protein interactions.

    Science.gov (United States)

    Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  4. Parallel force assay for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Daniela Aschenbrenner

    Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  5. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  6. Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay.

    Science.gov (United States)

    Randhawa, A S; Stanton, G J; Green, J A; Baron, S

    1977-05-01

    A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.

  7. Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

    International Nuclear Information System (INIS)

    Bullock, S.; Hayward, C.; Manson, J.; Brock, D.J.H.; Raeburn, J.A.

    1982-01-01

    Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis. (author)

  8. Quantification of methanogenic biomass by enzyme-linked immunosorbent assay and by analysis of specific methanogenic cofactors

    Energy Technology Data Exchange (ETDEWEB)

    Gorris, L G.M.; Kemp, H A; Archer, D B

    1987-01-01

    The reliability and accuracy with which enzyme-linked immunosorbent assay (ELISA) and an assay of methanogenic cofactors detect and quantify methanogenic species were investigated. Both assays required standardization with laboratory cultures of methanogenic bacteria and were applied to mixtures of pure cultures and samples from anaerobic digesters. ELISA was shown to be a simple method for detecting and quantifying individual methanogenic species. The range of species which can be assayed is limited by the range of antisera available but, potentially, ELISA can be applied to all methanogens. Although the cofactor assay is not species-specific it can distinguish hydrogenotrophic and acetotrophic methanogens and is quantitative.

  9. Automation of the dicentric chromosome assay and related assays

    International Nuclear Information System (INIS)

    Balajee, Adayabalam S.; Dainiak, Nicholas

    2016-01-01

    Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

  10. Assay strategies and methods for phospholipases

    International Nuclear Information System (INIS)

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

  11. A molecular assay for sensitive detection of pathogen-specific T-cells.

    Directory of Open Access Journals (Sweden)

    Victoria O Kasprowicz

    Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

  12. Nondestructive assay of sale materials

    International Nuclear Information System (INIS)

    Rodenburg, W.W.; Fleissner, J.G.

    1981-01-01

    This paper covers three primary areas: (1) reasons for performing nondestructive assay on SALE materials; (2) techniques used; and (3) discussion of investigators' revised results. The study shows that nondestructive calorimetric assay of plutonium offers a viable alternative to traditional wet chemical techniques. For these samples, the precision ranged from 0.4 to 0.6% with biases less than 0.2%. Thus, for those materials where sampling errors are the predominant source of uncertainty, this technique can provide improved accuracy and precision while saving time and money as well as reducing the amount of liquid wastes to be handled. In addition, high resolution gamma-ray spectroscopy measurements of solids can provide isotopic analysis data in a cost effective and timely manner. The timeliness of the method can be especially useful to the plant operator for production control and quality control measurements

  13. Comet Assay in Cancer Chemoprevention.

    Science.gov (United States)

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  14. Radioreceptor assay for somatomedin A

    Energy Technology Data Exchange (ETDEWEB)

    Takano, K [Tokyo Women' s Medical Coll. (Japan)

    1975-04-01

    Measurement method of somatomedian A by radioreceptor assay using the human placenta membrane was described and discussed. Binding rate of /sup 125/I-somatomedin A to its receptors was studied under various conditions of time and temperature of the incubation, and pH of the system. The influence of somatomedin A, porcine insulin, and porcine calcitonin, on /sup 125/I-somatomedin A bound receptors was studied, and these hormones showed the competitive binding to somatomedin A receptors in some level. The specificity, recovery rate, and clinical applications of somatomedin A were also discussed. Radioreceptor assay for somatomedine A provided easier, faster, and more accurate measurements than conventional bioassay. This technique would be very useful to study somatomedin A receptor and functions of insulin.

  15. An improved 96-well turbidity assay for T4 lysozyme activity.

    Science.gov (United States)

    Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

  16. Assay of vitamin B12

    International Nuclear Information System (INIS)

    Tovey, K.C.; Carrick, D.T.

    1982-01-01

    A radioassay is described for vitamin B12 which involves denaturing serum protein binding proteins with alkali. In the denaturation step a dithiopolyol and cyanide are used and in the intrinsic factor assay step a vitamin B12 analogue such as cobinamide is used to bind with any remaining serum proteins. The invention also includes a kit in which the dithiopolyol is provided in admixture with the alkali. The dithiopolyol may be dithiothreitol or dithioerythritol. (author)

  17. Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

    OpenAIRE

    Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

    2005-01-01

    A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) ...

  18. Assay of ribulose bisphosphate carboxylase

    International Nuclear Information System (INIS)

    Pike, C.; Berry, J.

    1987-01-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, [ 14 C]-NaHCO 3 , and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30 0 . The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number

  19. Quantitative immunochemistry using IgG and IgY against plant ...

    African Journals Online (AJOL)

    Quantitative immunochemistry using IgG and IgY against plant hormones. ... both of animal origin, using chemical couplings to their respective functional groups. ... enzyme-linked immunosorbent assay; ELISA) to detect and quantify these ...

  20. Optimization of a colorimetric assay for glycosylated human serum albumin

    International Nuclear Information System (INIS)

    Bohney, J.P.; Feldhoff, R.C.

    1986-01-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

  1. Quantitative phenotyping via deep barcode sequencing.

    Science.gov (United States)

    Smith, Andrew M; Heisler, Lawrence E; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J; Chee, Mark; Roth, Frederick P; Giaever, Guri; Nislow, Corey

    2009-10-01

    Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

  2. Transuranic waste assay instrumentation: new developments and directions at the Los Alamos Scientific Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Close, D.A.; Umbarger, C.J.; West, L.; Smith, W.J.; Cates, M.R.; Noel, B.W.; Honey, F.J.; Franks, L.A.; Pigg, J.L.; Trundle, A.S.

    1978-01-01

    The Los Alamos Scientific Laboratory is developing assay instrumentation for the quantitative analysis of transuranic materials found in bulk solid wastes generated by Department of Energy facilities and by the commercial nuclear power industry. This also includes wastes generated in the decontamination and decommissioning of facilities and wastes generated during burial ground exhumation. The assay instrumentation will have a detection capability for the transuranics of less than 10 nCi of activity per gram of waste whenever practicable.

  3. Transuranic waste assay instrumentation: new developments and directions at the Los Alamos Scientific Laboratory

    International Nuclear Information System (INIS)

    Close, D.A.; Umbarger, C.J.; West, L.; Smith, W.J.; Cates, M.R.; Noel, B.W.; Honey, F.J.; Franks, L.A.; Pigg, J.L.; Trundle, A.S.

    1978-01-01

    The Los Alamos Scientific Laboratory is developing assay instrumentation for the quantitative analysis of transuranic materials found in bulk solid wastes generated by Department of Energy facilities and by the commercial nuclear power industry. This also includes wastes generated in the decontamination and decommissioning of facilities and wastes generated during burial ground exhumation. The assay instrumentation will have a detection capability for the transuranics of less than 10 nCi of activity per gram of waste whenever practicable

  4. Quantitative film radiography

    International Nuclear Information System (INIS)

    Devine, G.; Dobie, D.; Fugina, J.; Hernandez, J.; Logan, C.; Mohr, P.; Moss, R.; Schumacher, B.; Updike, E.; Weirup, D.

    1991-01-01

    We have developed a system of quantitative radiography in order to produce quantitative images displaying homogeneity of parts. The materials that we characterize are synthetic composites and may contain important subtle density variations not discernible by examining a raw film x-radiograph. In order to quantitatively interpret film radiographs, it is necessary to digitize, interpret, and display the images. Our integrated system of quantitative radiography displays accurate, high-resolution pseudo-color images in units of density. We characterize approximately 10,000 parts per year in hundreds of different configurations and compositions with this system. This report discusses: the method; film processor monitoring and control; verifying film and processor performance; and correction of scatter effects

  5. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    Science.gov (United States)

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  6. Dyes assay for measuring physicochemical parameters.

    Science.gov (United States)

    Moczko, Ewa; Meglinski, Igor V; Bessant, Conrad; Piletsky, Sergey A

    2009-03-15

    A combination of selective fluorescent dyes has been developed for simultaneous quantitative measurements of several physicochemical parameters. The operating principle of the assay is similar to electronic nose and tongue systems, which combine nonspecific or semispecific elements for the determination of diverse analytes and chemometric techniques for multivariate data analysis. The analytical capability of the proposed mixture is engendered by changes in fluorescence signal in response to changes in environment such as pH, temperature, ionic strength, and presence of oxygen. The signal is detected by a three-dimensional spectrofluorimeter, and the acquired data are processed using an artificial neural network (ANN) for multivariate calibration. The fluorescence spectrum of a solution of selected dyes allows discreet reading of emission maxima of all dyes composing the mixture. The variations in peaks intensities caused by environmental changes provide distinctive fluorescence patterns which can be handled in the same way as the signals collected from nose/tongue electrochemical or piezoelectric devices. This optical system opens possibilities for rapid, inexpensive, real-time detection of a multitude of physicochemical parameters and analytes of complex samples.

  7. Prandiology of Drosophila and the CAFE assay

    Science.gov (United States)

    Ja, William W.; Carvalho, Gil B.; Mak, Elizabeth M.; de la Rosa, Noelle N.; Fang, Annie Y.; Liong, Jonathan C.; Brummel, Ted; Benzer, Seymour

    2007-01-01

    Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7× their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant. PMID:17494737

  8. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Directory of Open Access Journals (Sweden)

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  9. Development and optimization of a direct plaque assay for human and avian metapneumoviruses

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Li, Junan; Li, Jianrong

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses. PMID:22684013

  10. Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

    DEFF Research Database (Denmark)

    Riber, Ulla; Boesen, Henriette Toft; Jakobsen, Jeanne Toft

    2011-01-01

    The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evalu......The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult...

  11. Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

    Science.gov (United States)

    Kim, Dong-Hyeon; Chon, Jung-Whan; Lim, Jong-Soo; Kim, Hong-Seok; Kang, Il-Byeong; Jeong, Dana; Song, Kwang-Young; Kim, Hyunsook; Kim, Kwang-Yup; Seo, Kun-Ho

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.

  12. Radioligand binding assay of cortisol using horse transcortin

    International Nuclear Information System (INIS)

    Dash, R.J.; Sharma, B.R.; Lata, V.

    1979-01-01

    A modified radioligand binding assay was developed to measure cortisol in a single methylene chloride extract of human plasma/serum. The assay utilises 5 percent horse serum for cortisol binding and 3 H-cortisol as tracer. Except for a cross reaction of 13.3 percent for cortisone and 7 percent for prednisolone that for other steroids tested was negligible. The assay sensitivity at the lower 95 percent inhibition of buffer controls was 20 pg/tube. The log dose logit response standard curve was linear between 80 pg and 5 ng/tube. Recovery(Y) of cortisol added (x) to male and pregnant female plasma was quantitated (y = 0.983 X-0.47, r = 0.98). Regression analysis of cortisol estimates obtained in 51 plasma/serum samples with this assay system and a specific radioimmunoassay (using cortisol-3-BSA antiserum) for plasma cortisol gave a coefficient of correlation (r) of 0.95 and a regression coefficient (b) of 0.97. The method was found to be simple and highly reproducible. Availability of all reagents in the aqueous phase permitted handling of a large number of samples by a single technician. (auth.)

  13. Some quantitative studies on the transplantation of human tissues into nude mice

    International Nuclear Information System (INIS)

    Zietman, A.; Suit, H.D.; Sedlacek, R.

    1987-01-01

    Quantitative cell transplantation assays (TD/sub 50/) were performed for human tumors xenografted into athymic NCr(nμ/nμ) nude mice. Transplantation assays for FaDu when transplanted into brain and when transplanted into subcutaneous tissues are compared. Effects of immunization are discussed and results are given

  14. Establishment of a quantitative ELISA capable of determining peptide - MHC class I interaction

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Kristensen, N; Blicher, T

    2002-01-01

    dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards...

  15. Radiosotopic assay and binder therefor

    International Nuclear Information System (INIS)

    Caston, J.D.; Kamen, B.A.

    1976-01-01

    A rapid and less costly radioisotopic assay for measuring the concentration of folate in blood serum is described. This procedure utilizes 3 H-pteroylmonoglutamate, unlabeled 5-methyltetrahydrofolic acid, and a partially purified folate binder, such as for example a folate binder extracted from hog kidney. The procedure involves radioisotopically relating the bound amounts of a labeled folate and a known folate at various concentrations of the known folate in a system containing a predetermined amount of the labeled folate, a predetermined amount of the binder factor for the folates, and a predetermined amount of defolated test serum. 16 claims, 8 drawing figures

  16. The local lymph node assay and skin sensitization testing.

    Science.gov (United States)

    Kimber, Ian; Dearman, Rebecca J

    2010-01-01

    The mouse local lymph node assay (LLNA) is a method for the identification and characterization of skin sensitization hazards. In this context the method can be used both to identify contact allergens, and also determine the relative skin sensitizing potency as a basis for derivation of effective risk assessments.The assay is based on measurement of proliferative responses by draining lymph node cells induced following topical exposure of mice to test chemicals. Such responses are known to be causally and quantitatively associated with the acquisition of skin sensitization and therefore provide a relevant marker for characterization of contact allergic potential.The LLNA has been the subject of exhaustive evaluation and validation exercises and has been assigned Organization for Economic Cooperation and Development (OECD) test guideline 429. Herein we describe the conduct and interpretation of the LLNA.

  17. A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum

    DEFF Research Database (Denmark)

    Lloyd, Yukie M.; Ngati, Elise P.; Salanti, Ali

    2017-01-01

    Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation....... A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads...

  18. Assessment of the Microscreen phage-induction assay for screening hazardous wastes

    Energy Technology Data Exchange (ETDEWEB)

    Houk, V.S.; DeMarini, D.M.

    1987-09-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

  19. Quantitative fluorescence microscopy and image deconvolution.

    Science.gov (United States)

    Swedlow, Jason R

    2013-01-01

    Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

  20. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    International Nuclear Information System (INIS)

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

  1. Comparative evaluation of two radioenzymatic procedures designed to determine noradrenaline in the plasma (COMT assay and PNMT assay)

    International Nuclear Information System (INIS)

    Barth, A.

    1984-01-01

    A comparative evaluation of two radioenzymatic procedures to determine the concentration of noradrenaline in the plasma - with linearity, sensitivity, specifity and accuracy serving as test criteria - led to the following results: In view of a probability of error in the order of 2% both methods were judged to show a satisfactory sensitivity. The specific of the COMT assay, by contrast with that of the PNMT assay, was found to be wanting, as the noradrenaline measurements in the presence of other biogenic amines were biassed in such a way that the values determined were higher than the actual concentrations. During antihypertensive treatment even minimal changes in the noradrenaline concentration can be ascertained on a quantitative basis. If suitable hardware is available, the COMT assay permits up to 25 single determinations to be carried out per day, while the number of double determinations is restricted to 7 per day. One advantage, however, lies in the fact that several catecholamines in the plasma can be detected simultaneously, if required. In cases where the noradrenaline concentration alone is to be determined for clinical purposes, preference should be given to the PNMT assay, as both tests showed equal linearity and sensitivity. (TRV) [de

  2. Antioxidants and the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

    It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

  3. Rotor assembly and assay method

    Science.gov (United States)

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  4. Direct Assay of Filter Media following DEOX Testing

    International Nuclear Information System (INIS)

    R.P. Lind; J.J. Giglio; D.G. Cummings; M.W. Huntley; C.D. Morgan; K.J. Bateman; D.L. Wahlquist; D.A. Sell

    2007-01-01

    The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs-137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media

  5. Direct assay of filter media following DEOX testing

    International Nuclear Information System (INIS)

    Westphal, B.R.; Lind, R.P.; Giglio, J.J.; Cummings, D.G.; Huntley, M.W.; Morgan, C.D.; Bateman, K.J.; Wahlquist, D.L.; Sell, D.A.

    2007-01-01

    The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs- 137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media. (authors)

  6. Data transformation methods for multiplexed assays

    Science.gov (United States)

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  7. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert

    2016-01-01

    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  8. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  9. Quantitative secondary electron detection

    Science.gov (United States)

    Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

    2018-05-08

    Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

  10. [Methods of quantitative proteomics].

    Science.gov (United States)

    Kopylov, A T; Zgoda, V G

    2007-01-01

    In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

  11. Quantitative Decision Support Requires Quantitative User Guidance

    Science.gov (United States)

    Smith, L. A.

    2009-12-01

    Is it conceivable that models run on 2007 computer hardware could provide robust and credible probabilistic information for decision support and user guidance at the ZIP code level for sub-daily meteorological events in 2060? In 2090? Retrospectively, how informative would output from today’s models have proven in 2003? or the 1930’s? Consultancies in the United Kingdom, including the Met Office, are offering services to “future-proof” their customers from climate change. How is a US or European based user or policy maker to determine the extent to which exciting new Bayesian methods are relevant here? or when a commercial supplier is vastly overselling the insights of today’s climate science? How are policy makers and academic economists to make the closely related decisions facing them? How can we communicate deep uncertainty in the future at small length-scales without undermining the firm foundation established by climate science regarding global trends? Three distinct aspects of the communication of the uses of climate model output targeting users and policy makers, as well as other specialist adaptation scientists, are discussed. First, a brief scientific evaluation of the length and time scales at which climate model output is likely to become uninformative is provided, including a note on the applicability the latest Bayesian methodology to current state-of-the-art general circulation models output. Second, a critical evaluation of the language often employed in communication of climate model output, a language which accurately states that models are “better”, have “improved” and now “include” and “simulate” relevant meteorological processed, without clearly identifying where the current information is thought to be uninformative and misleads, both for the current climate and as a function of the state of the (each) climate simulation. And thirdly, a general approach for evaluating the relevance of quantitative climate model output

  12. Making transuranic assay measurements using modern controllers

    International Nuclear Information System (INIS)

    Kuckertz, T.H.; Caldwell, J.T.; Medvick, P.A.; Kunz, W.E.; Hastings, R.D.

    1987-01-01

    This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

  13. Non destructive assay (NDA) techniques

    International Nuclear Information System (INIS)

    Mafra Guidicini, Olga; Llacer, Carlos D.; Rojo, Marcelo

    2001-01-01

    In the IAEA Safeguards System the basic verification method used is nuclear material accountancy, with containment and surveillance as important complementary measures. If nuclear material accountancy is to be effective, IAEA inspectors have to make independent measurements to verify declared material quantities. Most of the equipment available to the inspectors is designed to measure gamma rays and/or neutrons emitted by various nuclear materials. Equipment is also available to measure the gross weight of an item containing nuclear material. These types of measurement techniques are generally grouped under the title of nondestructive assay (NDA). The paper describes the NDA techniques and instruments used to verify the total amount of nuclear material held at a nuclear facility. (author)

  14. Assay of cysteine dioxygenase activity

    International Nuclear Information System (INIS)

    Bagley, P.J.; Stipanuk, M.H.

    1990-01-01

    It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD + and has a pH optimum of 6.8 and one which is not stimulated by NAD + and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac trademark PA1 (4x250 mm)) was used to separate the [ 35 S]cysteinesulfinic acid produced from [ 35 S]cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine

  15. Extending Quantitative Easing

    DEFF Research Database (Denmark)

    Hallett, Andrew Hughes; Fiedler, Salomon; Kooths, Stefan

    The notes in this compilation address the pros and cons associated with the extension of ECB quantitative easing programme of asset purchases. The notes have been requested by the Committee on Economic and Monetary Affairs as an input for the February 2017 session of the Monetary Dialogue....

  16. Quantitative Moessbauer analysis

    International Nuclear Information System (INIS)

    Collins, R.L.

    1978-01-01

    The quantitative analysis of Moessbauer data, as in the measurement of Fe 3+ /Fe 2+ concentration, has not been possible because of the different mean square velocities (x 2 ) of Moessbauer nuclei at chemically different sites. A method is now described which, based on Moessbauer data at several temperatures, permits the comparison of absorption areas at (x 2 )=0. (Auth.)

  17. Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

    Science.gov (United States)

    Blanchard, Philippe; Regnault, Julie; Schurr, Frank; Dubois, Eric; Ribière, Magali

    2012-03-01

    Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Critical Quantitative Inquiry in Context

    Science.gov (United States)

    Stage, Frances K.; Wells, Ryan S.

    2014-01-01

    This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

  19. Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron

    NARCIS (Netherlands)

    Suaréz-Pantaleón, C.; Wichers, J.H.; Abad-Somovilla, A.; Amerongen, van A.

    2013-01-01

    Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator

  20. A powerful selection assay for mixture libraries of DNA alkylating agents.

    Science.gov (United States)

    Ham, Young-Wan; Boger, Dale L

    2004-08-04

    A simple and powerful selection assay that permits the separation (rpHPLC), quantitation (ELSD), and identification (ESI-MS) of thermally released adenine adducts derived from duocarmycin analogues is detailed that can establish the most effective DNA alkylating agents in synthetic combinatorial mixtures.

  1. In vivo assay to identify bacteria with β-glucosidase activity

    Directory of Open Access Journals (Sweden)

    Erwin Strahsburger

    2017-11-01

    Conclusion: This in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity.

  2. The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence

    DEFF Research Database (Denmark)

    Ignatiadis, Michail; Azim, Hatem A; Desmedt, Christine

    2016-01-01

    Importance: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay...

  3. High-pressure liquid chromatographic assay of Bay n 7133 in human serum.

    OpenAIRE

    Fasching, C E; Hughes, C E; Hector, R F; Peterson, L R

    1984-01-01

    A high-pressure liquid chromatographic method that includes a Sep-Pak (Waters Associates, Inc., Milford , Mass.) preparation of human serum was employed for the quantitative assay of Bay n 7133. Drug levels of 0.1 to 20 micrograms/ml could be detected. No interference from amphotericin B was found in the chromatographic analysis of Bay n 7133.

  4. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

    NARCIS (Netherlands)

    Kusters, J. G.; Reuland, E. A.; Bouter, S.; Koenig, P.; Dorigo-Zetsma, J. W.

    2015-01-01

    A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus

  5. Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

    Directory of Open Access Journals (Sweden)

    Mary T. Bausch-Jurken

    2017-06-01

    Full Text Available Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay. The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay.

  6. Development of a heavy metals enzymatic-based assay using papain

    International Nuclear Information System (INIS)

    Shukor, Yunus; Baharom, Nor Azlan; Rahman, Fadhil Abd.; Abdullah, Mohd. Puad; Shamaan, Nor Aripin; Syed, Mohd. Arif

    2006-01-01

    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC 50 (concentration of toxicant giving 50% inhibition) of Hg 2+ , Ag 2+ , Pb 2 , Zn 2+ is 0.39, 0.40, 2.16, 2.11 mg l -1 , respectively. For Cu 2+ and Cd 2+ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l -1 , respectively. The IC 50 and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox TM , 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis

  7. Investigation into the dissolution and direct assay of high-fired plutonium dioxide

    International Nuclear Information System (INIS)

    Patterson, J.K.

    1976-01-01

    A fusion-melt and dissolution assay method has been developed and tested for the quantitative analysis of high-fired plutonium dioxide. The method employs fusion of the plutonium dioxide at temperatures greater than the melting point of an eutectic mixture of potassium pyrosulfate plus sodium peroxide. The resultant melt is then titrated directly by either controlled potential coulometry or a gravimetric titration, using standardized ceric sulfate as the titrant. It has been concluded from these investigations that by using the techniques described, high-fired plutonium dioxide (stochiometric) can be quantitatively dissolved and assayed to a degree heretofore beyond the state-of-the-art, while showing direct traceability to the Federal standards. After fusion, the dissolution and direct assay is applicable to existing routine analytical procedures. The method was designed so as to minimize physical handling, simplify the chemical operations, and maximize the personal safety of the analyst at an appreciable cost savings per analysis

  8. Precise quantitation of PAIgG: A new radiometric microtechnique

    International Nuclear Information System (INIS)

    Schwartz, K.A.; Gauger, J.A.; Davis, J.M.

    1990-01-01

    We report the development of a radiometric assay for platelet-bound IgG that is both sensitive and quantitative. The assay utilized 96-well millititer plates incorporating a 0.2 microns filter membrane in the bottom. A 125I-labeled monoclonal antihuman IgG, as a secondary antibody, detected the platelet-bound human IgG. Since 5 x 10(6) platelets were used for each assay, tests for platelet-bound IgG can be performed on persons with severe thrombocytopenia. For the detection of circulating antiplatelet alloantibodies, as little as 10 microliters of platelet-free plasma per assay is required. Antiplatelet IgG was quantitated by using anti-PIA1 antibody that was purified with affinity and elution and DEAE chromatography. This purified antiplatelet antibody was labeled with 125I and was used to determine the binding ratio of secondary antibody to primary antibody. Under our standard conditions, this ratio was found to be stable at approximately 0.35 over the sensitivity range of the assay. The assay can detect approximately 200 molecules of human IgG per platelet (0.1 ng of secondary antibody bound per 5 x 10(6) platelets). It has a linear range from 0 to 7,000 molecules per platelet. Quantitation of anti-PIA1 binding for platelets stored for up to 6 months under refrigeration showed no change in number of PIA1 binding sites. Clinical studies showed that 18 of 19 ITP patients had an increased number of IgG molecules per platelet as did patients with malignancy and drug-induced immune thrombocytopenia. Patients who had received multiple platelet transfusions had antiplatelet antibody in their plasma. Normal amounts of PAIgG were observed in platelets and plasma of patients with nonimmune thrombocytopenia

  9. Expert system for transuranic waste assay

    Energy Technology Data Exchange (ETDEWEB)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  10. Expert system for transuranic waste assay

    International Nuclear Information System (INIS)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

  11. Radiometric assays for glycerol, glucose, and glycogen

    International Nuclear Information System (INIS)

    Bradley, D.C.; Kaslow, H.R.

    1989-01-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

  12. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    Prasanna, Pataje G.

    2014-01-01

    The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

  13. Applied quantitative finance

    CERN Document Server

    Chen, Cathy; Overbeck, Ludger

    2017-01-01

    This volume provides practical solutions and introduces recent theoretical developments in risk management, pricing of credit derivatives, quantification of volatility and copula modeling. This third edition is devoted to modern risk analysis based on quantitative methods and textual analytics to meet the current challenges in banking and finance. It includes 14 new contributions and presents a comprehensive, state-of-the-art treatment of cutting-edge methods and topics, such as collateralized debt obligations, the high-frequency analysis of market liquidity, and realized volatility. The book is divided into three parts: Part 1 revisits important market risk issues, while Part 2 introduces novel concepts in credit risk and its management along with updated quantitative methods. The third part discusses the dynamics of risk management and includes risk analysis of energy markets and for cryptocurrencies. Digital assets, such as blockchain-based currencies, have become popular b ut are theoretically challenging...

  14. Quantitative skeletal scintiscanning

    International Nuclear Information System (INIS)

    Haushofer, R.

    1982-01-01

    330 patients were examined by skeletal scintiscanning with sup(99m)Tc pyrophosphate and sup(99m)methylene diphosphonate in the years between 1977 and 1979. Course control examinations were carried out in 12 patients. The collective of patients presented with primary skeletal tumours, metastases, inflammatory and degenerative skeletal diseases. Bone scintiscanning combined with the ''region of interest'' technique was found to be an objective and reproducible technique for quantitative measurement of skeletal radioactivity concentrations. The validity of nuclear skeletal examinations can thus be enhanced as far as diagnosis, course control, and differential diagnosis are concerned. Quantitative skeletal scintiscanning by means of the ''region of interest'' technique has opened up a new era in skeletal diagnosis by nuclear methods. (orig./MG) [de

  15. Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

    Science.gov (United States)

    Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

    2015-07-01

    Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

  16. Quantitative FDG in depression

    Energy Technology Data Exchange (ETDEWEB)

    Chua, P.; O`Keefe, G.J.; Egan, G.F.; Berlangieri, S.U.; Tochon-Danguy, H.J.; Mckay, W.J.; Morris, P.L.P.; Burrows, G.D. [Austin Hospital, Melbourne, VIC (Australia). Dept of Psychiatry and Centre for PET

    1998-03-01

    Full text: Studies of regional cerebral glucose metabolism (rCMRGlu) using positron emission tomography (PET) in patients with affective disorders have consistently demonstrated reduced metabolism in the frontal regions. Different quantitative and semi-quantitative rCMRGlu regions of interest (ROI) comparisons, e.g. absolute metabolic rates, ratios of dorsolateral prefrontal cortex (DLPFC) to ipsilateral hemisphere cortex, have been reported. These studies suffered from the use of a standard brain atlas to define ROls, whereas in this case study, the individual``s magnetic resonance imaging (MRI) scan was registered with the PET scan to enable accurate neuroanatomical ROI definition for the subject. The patient is a 36-year-old female with a six-week history of major depression (HAM-D = 34, MMSE = 28). A quantitative FDG PET study and an MRI scan were performed. Six MRI-guided ROls (DLPFC, PFC, whole hemisphere) were defined. The average rCMRGlu in the DLPFC (left = 28.8 + 5.8 mol/100g/min; right = 25.6 7.0 mol/100g/min) were slightly reduced compared to the ipsilateral hemispherical rate (left = 30.4 6.8 mol/100g/min; right = 29.5 7.2 mol/100g/min). The ratios of DLPFC to ipsilateral hemispheric rate were close to unity (left = 0.95 0.29; right 0.87 0.32). The right to left DLPFC ratio did not show any significant asymmetry (0.91 0.30). These results do not correlate with earlier published results reporting decreased left DLPFC rates compared to right DLPFC, although our results will need to be replicated with a group of depressed patients. Registration of PET and MRI studies is necessary in ROI-based quantitative FDG PET studies to allow for the normal anatomical variation among individuals, and thus is essential for accurate comparison of rCMRGlu between individuals.

  17. Quantitative FDG in depression

    International Nuclear Information System (INIS)

    Chua, P.; O'Keefe, G.J.; Egan, G.F.; Berlangieri, S.U.; Tochon-Danguy, H.J.; Mckay, W.J.; Morris, P.L.P.; Burrows, G.D.

    1998-01-01

    Full text: Studies of regional cerebral glucose metabolism (rCMRGlu) using positron emission tomography (PET) in patients with affective disorders have consistently demonstrated reduced metabolism in the frontal regions. Different quantitative and semi-quantitative rCMRGlu regions of interest (ROI) comparisons, e.g. absolute metabolic rates, ratios of dorsolateral prefrontal cortex (DLPFC) to ipsilateral hemisphere cortex, have been reported. These studies suffered from the use of a standard brain atlas to define ROls, whereas in this case study, the individual''s magnetic resonance imaging (MRI) scan was registered with the PET scan to enable accurate neuroanatomical ROI definition for the subject. The patient is a 36-year-old female with a six-week history of major depression (HAM-D = 34, MMSE = 28). A quantitative FDG PET study and an MRI scan were performed. Six MRI-guided ROls (DLPFC, PFC, whole hemisphere) were defined. The average rCMRGlu in the DLPFC (left = 28.8 + 5.8 mol/100g/min; right = 25.6 7.0 mol/100g/min) were slightly reduced compared to the ipsilateral hemispherical rate (left = 30.4 6.8 mol/100g/min; right = 29.5 7.2 mol/100g/min). The ratios of DLPFC to ipsilateral hemispheric rate were close to unity (left = 0.95 0.29; right 0.87 0.32). The right to left DLPFC ratio did not show any significant asymmetry (0.91 0.30). These results do not correlate with earlier published results reporting decreased left DLPFC rates compared to right DLPFC, although our results will need to be replicated with a group of depressed patients. Registration of PET and MRI studies is necessary in ROI-based quantitative FDG PET studies to allow for the normal anatomical variation among individuals, and thus is essential for accurate comparison of rCMRGlu between individuals

  18. Automated amperometric plutonium assay system

    International Nuclear Information System (INIS)

    Burt, M.C.

    1985-01-01

    The amperometric titration for plutonium assay has been used in the nuclear industry for over twenty years and has been in routine use at the Hanford Engineering Development Laboratory since 1976 for the analysis of plutonium oxide and mixed oxide fuel material for the Fast Flux Test Facility. It has proven itself to be an accurate and reliable method. The method may be used as a direct end point titration or an excess of titrant may be added and a back titration performed to aid in determination of the end point. Due to the slowness of the PuVI-FeII reaction it is difficult to recognize when the end point is being approached and is very time consuming if the current is allowed to decay to the residual value after each titrant addition. For this reason the back titration in which the rapid FeII-CrVI reaction occurs is used by most laboratories. The back titration is performed by the addition of excess ferrous solution followed by two measured aliquots of standard dichromate with measurement of cell current after each addition

  19. TRU assay system and measurements

    International Nuclear Information System (INIS)

    Brodzinski, R.L.

    1984-02-01

    The measurement of the transuranic content of nuclear products or process residues has become increasingly important for the recovery of fissionable material from spent fuel elements, the identification of commercial fuel elements which have not yet reached full burnup, the measurement and recovery of transuranics from discarded or stored waste materials, the determination of the transuranic content in high gamma activity waste material scheduled for disposal, compliance with 10CFR61 by land burial operators/shippers, and the satisfaction of accountability requirements. Active neutron interrogation techniques measure either the prompt neutrons or the beta delayed neutrons from fission products following induced fission. These techniques normally only measure fissile transuranics ( 235 U, 239 Pu, and 241 Pu) and are commonly applied only to contact handleable waste. Passive neutron interrogation techniques, on the other hand, are capable of measuring all transuranics except 235 U with adequate sensitivity and will work on both contact handleable and high gamma activity wastes. Since the passive techniques are senstitive to a wider spectrum of transuranic isotopes than the active techniques, substantially less complex and less expensive than the active systems, and they have proven techniques for measuring small quantities of TRU in high gamma activity packages, the passive neutron TRU assay technology was chosen for development into the instruments discussed in this paper

  20. Micronucleus assay for radiation workers

    International Nuclear Information System (INIS)

    Balasem, A.N.; Ali, A.S.K.

    1997-01-01

    Micronucleus assay was performed on 49 radiation workers and 22 healthy volunteers. Radiation workers were subdivided into two groups according to their employments durations in the radiation field. Group a consisted of 18 radiation workers who have been in this work between 5 and 22 years. Group b included 31 employees who have been classified as radiation workers for 1 to 4.5 years. Statistical analysis showed significant variations between the yields of micronuclei in groups A and B as well as between group A and a group of healthy controls. Meanwhile no significant difference was noticed between the yields of micronuclei in group B and the corresponding values in the healthy controls. The possible effect of age in the induction of micronuclei was discussed and a comparison with the yield of chromosomal aberrations was described. It seems that cytokinesis- blocking method may be used to detect the radiation-induced micronuclei in workers exposed occupationally to ionizing radiation in levels below the maximum permissible limit of 0.05 Sv per year

  1. Quantitative traits and diversification.

    Science.gov (United States)

    FitzJohn, Richard G

    2010-12-01

    Quantitative traits have long been hypothesized to affect speciation and extinction rates. For example, smaller body size or increased specialization may be associated with increased rates of diversification. Here, I present a phylogenetic likelihood-based method (quantitative state speciation and extinction [QuaSSE]) that can be used to test such hypotheses using extant character distributions. This approach assumes that diversification follows a birth-death process where speciation and extinction rates may vary with one or more traits that evolve under a diffusion model. Speciation and extinction rates may be arbitrary functions of the character state, allowing much flexibility in testing models of trait-dependent diversification. I test the approach using simulated phylogenies and show that a known relationship between speciation and a quantitative character could be recovered in up to 80% of the cases on large trees (500 species). Consistent with other approaches, detecting shifts in diversification due to differences in extinction rates was harder than when due to differences in speciation rates. Finally, I demonstrate the application of QuaSSE to investigate the correlation between body size and diversification in primates, concluding that clade-specific differences in diversification may be more important than size-dependent diversification in shaping the patterns of diversity within this group.

  2. Tumor markers kits development for use in radioimmunometric assays

    International Nuclear Information System (INIS)

    Ahmed, B.

    1997-01-01

    The immunoassays such as RIA and IRMA are now widely used through the world for the quantitation of a variety of substances in the biological fluid for their high sensibility and specificity which required simple equipments. These techniques are also very used in Algeria for an effective amelioration of public heath The assays kits of RIA/IRMA of thyroid hormones are the most used, followed by peptidic hormones, steroids hormones and IRMA Tumor Markers (T.M) kits. In spite of the important demand, of tumor markers kits for the diagnosis and follow up of cancers their use are always insufficient due to the high cost. The research contract programme proposed by IAEA on the theme 'The Developments of IRMA Tumor Markers Kits' of prostate specific Antigen (PSA) and Tissue Polypeptide Specific Antigen (TPS) will allowed us to produce locally with best quality-price, the main reagents for PSA and TPS IRMA assays kits for diagnosis and follow up the prostate and breast cancers which are very spready in the country. This report include the following points: Generalities on the use of tumor markers in Algeria, programme for the Development of the PSA IRMA assay (schedule of protocols applied for each reagents; annual planning for assessing the programme activities) and conclusion

  3. Application of statistical process control to qualitative molecular diagnostic assays.

    Directory of Open Access Journals (Sweden)

    Cathal P O'brien

    2014-11-01

    Full Text Available Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control. Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply statistical process control to assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater samples with a resultant protracted time to detection. Modelled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of statistical process control to qualitative laboratory data.

  4. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Lago-Cachón, D., E-mail: dlagocachon@gmail.com [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Rivas, M., E-mail: rivas@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Martínez-García, J.C., E-mail: jcmg@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Oliveira-Rodríguez, M., E-mail: oliveiramyriam@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); Blanco-López, M.C., E-mail: cblanco@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); García, J.A., E-mail: joseagd@uniovi.es [Dpto. de Física, Universidad de Oviedo, Escuela de Marina, Campus de Viesques, 33204 Gijón (Spain)

    2017-02-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

  5. A comparison of assays measuring the viability of Legionella ...

    Science.gov (United States)

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

  6. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    International Nuclear Information System (INIS)

    Lago-Cachón, D.; Rivas, M.; Martínez-García, J.C.; Oliveira-Rodríguez, M.; Blanco-López, M.C.; García, J.A.

    2017-01-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

  7. Normalization methods in time series of platelet function assays

    Science.gov (United States)

    Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

    2016-01-01

    Abstract Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM). The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques. In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series. Normalization was calculated per assay (test) for all time points and per time point for all tests. Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

  8. Optimization of a colorimetric assay for glycosylated human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Bohney, J.P.; Feldhoff, R.C.

    1986-05-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

  9. Effective implementation of novel MET pharmacodynamic assays in translational studies.

    Science.gov (United States)

    Srivastava, Apurva K; Navas, Tony; Herrick, William G; Hollingshead, Melinda G; Bottaro, Donald P; Doroshow, James H; Parchment, Ralph E

    2017-01-01

    MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control pre-analytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.

  10. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    Shackleton, D.R.; Hulmes, D.J.

    1990-01-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

  11. Assay development status report for total cyanide

    International Nuclear Information System (INIS)

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

  12. Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

    Science.gov (United States)

    Netzel, Brian C; Grebe, Stefan K G; Carranza Leon, B Gisella; Castro, M Regina; Clark, Penelope M; Hoofnagle, Andrew N; Spencer, Carole A; Turcu, Adina F; Algeciras-Schimnich, Alicia

    2015-08-01

    Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

  13. Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

    Energy Technology Data Exchange (ETDEWEB)

    Houk, V.S.; DeMarini, D.M.

    1988-01-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

  14. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  15. New automated pellet/powder assay system

    International Nuclear Information System (INIS)

    Olsen, R.N.

    1975-01-01

    This paper discusses an automated, high precision, pellet/ powder assay system. The system is an active assay system using a small isotopic neutron source and a coincidence detection system. The handling of the pellet powder samples has been automated and a programmable calculator has been integrated into the system to provide control and data analysis. The versatile system can assay uranium or plutonium in either active or passive modes

  16. Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

    International Nuclear Information System (INIS)

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

  17. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    Science.gov (United States)

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

  18. Quantitative luminescence imaging system

    Science.gov (United States)

    Erwin, David N.; Kiel, Johnathan L.; Batishko, Charles R.; Stahl, Kurt A.

    1990-01-01

    The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopie imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber.

  19. 233U Assay A Neutron NDA System

    Energy Technology Data Exchange (ETDEWEB)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  20. 233U Assay A Neutron NDA System

    International Nuclear Information System (INIS)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-01-01

    The assay of highly enriched 233 U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched 235 U do not convert easily over to the assay of 233 U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with γ ray isotopics information should give a good overall determination of 233 U material now stored in bldg. 3019 at the Oak Ridge National Laboratory

  1. Safeguards and Non-destructive Assay

    International Nuclear Information System (INIS)

    Carchon, R.; Bruggeman, M.

    2001-01-01

    SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

  2. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

    Science.gov (United States)

    Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

    2005-02-01

    The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

  3. Electrokinetically-controlled RNA-DNA hybridization assay for foodborne pathogens

    International Nuclear Information System (INIS)

    Weng, X.; Jiang, H.; Li, D.

    2012-01-01

    We have developed a microfluidic chip for use in an RNA-DNA hybridization assay for foodborne pathogens. Automatic sequential reagent dispensing and washing was realized with a programmable DC voltage sequencer. Signal detection was achieved with a miniaturized optical detection module. Salmonella and Listeria monocytogenes bacteria in different concentrations were quantitatively determined by this RNA-DNA hybridization assay in the microfluidic chip. The detection limit for the Salmonella and Listeria monocytogenes bacteria is 10 3 to 10 4 CFU mL -1 . The method excels by a significant reduction in the consumption of sample and reagent, and a short assay time. This automatic-operating microfluidic RNA-DNA hybridization assay is promising for on-site pathogen detection. (author)

  4. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  5. Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

    2017-02-21

    Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

  6. Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

    International Nuclear Information System (INIS)

    Stack, G.; Fricker, L.D.; Snyder, S.H.

    1984-01-01

    A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of 3 H-labeled substrate and product in chloroform. The substrates 3 H-benzoyl-Phe-Ala-Arg or 3 H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, 3 H-benzoyl-Phe-Ala or 3 H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate

  7. Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Stack, G.; Fricker, L.D.; Snyder, S.H.

    1984-01-09

    A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of /sup 3/H-labeled substrate and product in chloroform. The substrates /sup 3/H-benzoyl-Phe-Ala-Arg or /sup 3/H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, /sup 3/H-benzoyl-Phe-Ala or /sup 3/H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.

  8. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

    Science.gov (United States)

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

    Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  9. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

    Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  10. Quantitative ion implantation

    International Nuclear Information System (INIS)

    Gries, W.H.

    1976-06-01

    This is a report of the study of the implantation of heavy ions at medium keV-energies into electrically conducting mono-elemental solids, at ion doses too small to cause significant loss of the implanted ions by resputtering. The study has been undertaken to investigate the possibility of accurate portioning of matter in submicrogram quantities, with some specific applications in mind. The problem is extensively investigated both on a theoretical level and in practice. A mathematical model is developed for calculating the loss of implanted ions by resputtering as a function of the implanted ion dose and the sputtering yield. Numerical data are produced therefrom which permit a good order-of-magnitude estimate of the loss for any ion/solid combination in which the ions are heavier than the solid atoms, and for any ion energy from 10 to 300 keV. The implanted ion dose is measured by integration of the ion beam current, and equipment and techniques are described which make possible the accurate integration of an ion current in an electromagnetic isotope separator. The methods are applied to two sample cases, one being a stable isotope, the other a radioisotope. In both cases independent methods are used to show that the implantation is indeed quantitative, as predicted. At the same time the sample cases are used to demonstrate two possible applications for quantitative ion implantation, viz. firstly for the manufacture of calibration standards for instrumental micromethods of elemental trace analysis in metals, and secondly for the determination of the half-lives of long-lived radioisotopes by a specific activity method. It is concluded that the present study has advanced quantitative ion implantation to the state where it can be successfully applied to the solution of problems in other fields

  11. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    Hood, R.L.

    1975-01-01

    A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

  12. Q2 - a very low level quantitative and qualitative waste assay and release certification system

    International Nuclear Information System (INIS)

    Bronson, F.L.

    1990-01-01

    Low level radioactive waste disposal is very expensive, especially when all of the handling transportation and documentation costs are included. However for most generators, a large fraction of this low level waste is not contaminated at all, or only slightly so. The paper describes the development and performance of a low level counter that is convenient to use, and that can accurately identify and quantify the radioactivity of any gamma emitter thing that can be placed in a 55 gallon (200 liter) container. These measurement results can be used to verify the absence of radioactivity at a very low levels (10 nCi (370 Bq)/sample), and to identify the nuclides and quantities present, while differentiating against natural radioactivity (Radium, Thorium, Potassium). These results can be used as part of a 10CFR20.302 waste stream exemption program, and thus allow significant savings and a less than 1 year payback at a typical nuclear power plant. The Q1 system is fully shielded to allow it's use in the low level radwaste storage area. The detectors are either 3 Intrinsic Germanium detectors or 2 large NaI detectors. The software is fully automated for simple operation. Correlation factors can be entered to estimate non-gamma emitters from pre-established correlations to other nuclides. Typical Ge detector sensitivities are 8 nCi (300 Bq) LLD for Cs-137 at 0.8 g/cc for a 10 minute count time. NaI detector systems can achieve the same LLD in a 1 minute count. 5 figs., 1 tab

  13. Development and application of a real-time quantitative PCR assay ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... and a gel band purification kit (GE Health Care, UK), the PCR products obtained were ... amino acid sequences from various species, sequence alignment ..... sequence weighting, positions-specific gap penalties and weight.

  14. Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement

    Science.gov (United States)

    Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, cr...

  15. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Ogrinc, Nina [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); LOTRIČ Metrology, Selca 163, SI-4227 Selca (Slovenia); Pelicon, Primož, E-mail: primoz.pelicon@ijs.si [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Vavpetič, Primož; Kelemen, Mitja; Grlj, Nataša; Jeromel, Luka [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Tomić, Sergej [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Čolić, Miodrag [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Medical Faculty, University of Niš, Boulevard of Dr. Zoran Djindjić 81, 18000 Niš (Serbia); Beran, Alfred [Dipartimento di Oceanografia Biologica, Istituto Nazionale di Oceanografia e Geofisica Sperimentale, Via Auguste Piccard 54, 34151 Trieste (Italy)

    2013-07-01

    Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10{sup −12} g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

  16. Rapid, quantitative and sensitive immunochromatographic assay based on stripping voltammetric detection of a metal ion label

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Fang; Wang, Kaihua; Lin, Yuehe

    2005-10-10

    A novel, sensitive immunochromatographic electrochemical biosensor (IEB) which combines an immunochromatographic strip technique with an electrochemical detection technique is demonstrated. The IEB takes advantages of the speed and low-cost of the conventional immunochromatographic test kits and high-sensitivity of stripping voltammetry. Bismuth ions (Bi3+) have been coupled with the antibody through the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA). After immunoreactions, Bi3+ was released and quantified by anodic stripping voltammetry at a built-in single-use screen-printed electrode. As an example for the applications of such novel device, the detection of human chorionic gonadotronphin (HCG) in a specimen was performed. This biosensor provides a more user-friendly, rapid, clinically accurate, and less expensive immunoassay for such analysis in specimens than currently available test kits.

  17. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    Niesters, H. G.; van Esser, J.; Fries, E.; Wolthers, K. C.; Cornelissen, J.; Osterhaus, A. D.

    2000-01-01

    With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using

  18. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    H.G.M. Niesters (Bert); E. Fries; K.C. Wolthers (Katja); A.D.M.E. Osterhaus (Albert); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost)

    2000-01-01

    textabstractWith the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and

  19. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911, Saudi Arabia,. 2Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, ..... Sambrook J, Russell DW. Molecular cloning: a laboratory manual: New York: Cold Spring. Harbor Laboratory Press ...

  20. Early detection of airborne inoculum of Magnaporthe oryzae in turfgrass fields using a quantitative LAMP assay

    Science.gov (United States)

    Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid diagnostic protoc...