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Sample records for biological assay

  1. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  2. Gas Plasma Surface Chemistry for Biological Assays.

    Science.gov (United States)

    Sahagian, Khoren; Larner, Mikki

    2015-01-01

    Biological systems respond to and interact with surfaces. Gas plasma provides a scalable surface treatment method for designing interactive surfaces. There are many commercial examples of plasma-modified products. These include well plates, filtration membranes, dispensing tools, and medical devices. This chapter presents an overview of gas plasma technology and provides a guide to using gas plasma for modifying surfaces for research or product development. PMID:26160577

  3. Chemiluminescence assay for the detection of biological warfare agents

    Energy Technology Data Exchange (ETDEWEB)

    Langry, K; Horn, J

    1999-11-05

    A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. This chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.

  4. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    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)

  5. An improved in vitro micronucleus assay to biological dosimetry

    International Nuclear Information System (INIS)

    The biological dosimetry is widely used to estimate the absorbed dose in people occupationally or accidentally exposed to the radiation for a better medical treatment, minimizing the harmful effects. Many techniques and methods have been proposed to detect and quantify the radioinduced lesions in genetic material, among them, the micronucleus (MN) assay. In the present study, we proposed an improved in vitro micronucleus technique that is rapid, sensitive and with minor cell manipulations. Assays were carried out with human tumor cells (MCF-7) seeded (3x104 cells) in slides placed into Petri dishes. Adherent cells were maintained with RPMI medium, supplemented with fetal calf serum, 1 % antibiotics, cytochalasin B (2 μg/mL), and incubated at 37 deg C in the presence of 5% CO2 for 72h. Cells were pre-treated for 24h with aminoguanidine, a nitric oxide synthase inhibitor. Nitric oxide is an intracellular free-radical, involved in DNA double-strand break repair mechanisms. After incubation, adherent cells on slides were briefly fixed with paraformaldehyde and stained with acridine orange (100 μg/mL) for analysis through fluorescence microscopy. Dye fluorescence permitted accurate discrimination between nuclei and micronuclei (bright green) and cytoplasm (red), and made possible a faster counting of binucleated cells. Aminoguanidine (2 mM) induced significant increase (p< 0.05) in frequencies of binucleated cells with micronuclei and in the number of micronuclei per binucleated cell. Data showed that proposed modifications permit to understand an early aspect of NO inhibition and suggested an improved protocol to MN assays. (author)

  6. A rapid assay for the biological evaluation of helicase activity.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dimitrios Vlachakis, Andrea Brancale, Colin Berry & Sophia Kossida ### Abstract A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. T...

  7. The influence factors in micronucleus assay for biological dosimetry

    International Nuclear Information System (INIS)

    The factors influencing the yield of micronuclei in the cycokinesis blocked micronucleus assay with human lymphocytes have been determined. It was found that 6 μg/mL of cycochalasin B in culture was a proper concentration and 72 hours was a proper culture time, though a shorter time of 66 hours is permitted. Significant interindividual variations were detected for both spontaneous and induced micronucleus frequency, but no correlation was found between them. An age effect of 1.9% increase per year was found for spontaneous micronucleus

  8. Specific methods for theophylline assay in biological samples

    International Nuclear Information System (INIS)

    The objective of monitoring theophylline plasma levels in in therapeutic work is well established, and the consequences of using unspecific analytical methods are obvious. For the determination of pharmacokinetic parameters, concentrations far below the therapeutic range must often be measured. Interferences acceptable in therapeutic monitoring of theophylline could cause severe inaccuracy at these drug levels. The term specificity and its meaning are discussed in general and in relation to the different steps of the analytical procedure (e.g., sampling and sample work-up). Different analytical methods for theophylline are discussed in terms of specificity. Plasma concentrations of paraxanthine (1,7-dimethylxanthine) - a metabolite of caffeine - as high as 3 μg/ml have been observed, and the compound can interfere in various theophylline assay methods. An example is given of the pharmacokinetic consequences. (author)

  9. Insects as biological models to assay spider and scorpion venom toxicity

    OpenAIRE

    M. F. Manzoli-Palma; N. GOBBI; Palma, M. S.

    2003-01-01

    This study was undertaken to develop an experimental protocol using insects as biological models to assay venom toxicity of the following spiders Loxosceles gaucho, Phoneutria nigriventer, Nephilengys cruentata and Tityus serrulatus scorpion. Three different insect species were bioassayed: Apis mellifera (Hymenoptera), Grillus assimilis (Orthoptera), and Diatraea saccharalis (Lepidoptera). Venoms were injected into the hemocele of insects with a microsyringe at concentrations that caused dose...

  10. Measurement of biologically active interleukin-1 by a soluble receptor binding assay

    International Nuclear Information System (INIS)

    A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants

  11. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    OpenAIRE

    Federica Martini; José V. Tarazona; M. Victoria Pablos

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetra...

  12. KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay.

    Science.gov (United States)

    Toro, Tasha B; Watt, Terry J

    2015-12-01

    Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions. PMID:26402585

  13. Similarity criterion analysis of dose-response curves in biological assay and radioimmunoassay of hormones

    International Nuclear Information System (INIS)

    The difficulties involved in the control of biological and radioimmunological assay systems, and in the maintenance of standard, as well as, the usual heterogeneity of assayed samples require some evidence of similarity between the dose-response curves obtained with the standard and the sample. Nowadays the parallelism test is used to provide such evidence. However, some indications of non-normal errors distribution, such as the presence of out layers, render the parallelism test both conceptually implausible and statistically inefficient. In such a manner we suggest the non-parametric 'frequencial' test as a more sounding option. (author)

  14. A Biology Laboratory Exercise Using Macromolecule Assays to Distinguish Four Types of Milk

    Directory of Open Access Journals (Sweden)

    Charlotte W. Pratt

    2011-03-01

    Full Text Available One of the drawbacks of cookbook-style laboratory exercises for General Biology courses is that students are not challenged to develop skills in scientific reasoning, such as formulating hypotheses and designing and carrying out experiments. Several traditional laboratory curricula include exercises involving semi-quantitative colorimetric assays to detect proteins (biuret test, reducing sugars (Benedict’s test, starch (Lugol’s test, and lipids (Sudan red test in a variety of easily prepared solutions (glucose, albumin, glycine, etc. and familiar food items (lemon juice, cornstarch, egg white, etc.. An extension of this lab exercise was developed to allow students to use their knowledge of the macromolecule assays to design an experiment to distinguish four types of “milk”: whole milk, skim milk, cream, and soy milk (rice milk or almond milk could also be included.

  15. Insects as biological models to assay spider and scorpion venom toxicity

    Directory of Open Access Journals (Sweden)

    M. F. Manzoli-Palma

    2003-01-01

    Full Text Available This study was undertaken to develop an experimental protocol using insects as biological models to assay venom toxicity of the following spiders Loxosceles gaucho, Phoneutria nigriventer, Nephilengys cruentata and Tityus serrulatus scorpion. Three different insect species were bioassayed: Apis mellifera (Hymenoptera, Grillus assimilis (Orthoptera, and Diatraea saccharalis (Lepidoptera. Venoms were injected into the hemocele of insects with a microsyringe at concentrations that caused dose/weight-dependent effects; doses causing either paralysis (ED50 or death (LD50 were recorded for each venom and insect test-species. T. serrulatus and L. gaucho venoms were lethal to all tested species, while P. nigriventer venom caused paralysis and death, and N. cruentata venom caused only paralysis at the doses assayed. A comparison between the insect test species described above revealed that A. mellifera was highly sensitive to all venoms tested; even a tiny amount of N. cruentata non-lethal venom caused a change in the walking pattern leading to transient paralysis. D. saccharalis larvae were very resistant to all four venoms.

  16. A novel bench-scale column assay to investigate site-specific nitrification biokinetics in biological rapid sand filters

    DEFF Research Database (Denmark)

    Tatari, Karolina; Smets, Barth F.; Albrechtsen, Hans-Jørgen

    2013-01-01

    A bench-scale assay was developed to obtain site-specific nitrification biokinetic information from biological rapid sand filters employed in groundwater treatment. The experimental set-up uses granular material subsampled from a full-scale filter, packed in a column, and operated with controlled....../m3 packed sand/h could easily be determined at 7.5 g NH4+–N/m3 packed sand/h. This assay, with conditions reflecting full-scale observations, and where the biological activity is subject to minimal physical disturbance, provides a simple and fast, yet powerful tool to gain insight in nitrification...

  17. Enhanced Photoelectrochemical Proximity Assay for Highly Selective Protein Detection in Biological Matrixes.

    Science.gov (United States)

    Wen, Guangming; Ju, Huangxian

    2016-08-16

    This work proposes the first photoelectrochemical proximity assay (PECPA) method via the sensitization of CdTe quantum dots (QDs) on photoelectrochemical response of ITO/TiO2/CdS electrode for highly selective and sensitive detection of proteins. This detection was performed on a sensing interface formed via the hybridization of capture DNA immobilized on ITO/TiO2/CdS electrode with labeled antibody-DNA (DNA-Ab1). Upon the recognition of Ab1 to target protein, the immunocomplex of DNA-Ab1, target, and the detection antibody-DNA (DNA-Ab2) was formed, which led to the proximity hybridization of the DNA in DNA-Ab2, capture DNA, and signal DNA-CdTe QDs, and brought CdTe QDs to the ITO/TiO2/CdS electrode to produce a sensitized photocurrent. The photocurrent intensity increased with the increasing concentration of the specific target protein. Using insulin as a target, this sensitized method showed a detectable range of 10 fM to 10 nM and a detection limit of 3.0 fM without the need of a washing step. It possessed high selectivity and good accuracy for detection of proteins in biological matrixes. This method is extremely flexible and can be extended to varieties of protein targets. PMID:27464227

  18. Translating tumor biology into personalized treatment planning: analytical performance characteristics of the Oncotype DX® Colon Cancer Assay

    International Nuclear Information System (INIS)

    The Oncotype DX® Colon Cancer Assay is a new diagnostic test for determining the likelihood of recurrence in stage II colon cancer patients after surgical resection using fixed paraffin embedded (FPE) primary colon tumor tissue. Like the Oncotype DX Breast Cancer Assay, this is a high complexity, multi-analyte, reverse transcription (RT) polymerase chain reaction (PCR) assay that measures the expression levels of specific cancer-related genes. By capturing the biology underlying each patient's tumor, the Oncotype DX Colon Cancer Assay provides a Recurrence Score (RS) that reflects an individualized risk of disease recurrence. Here we describe its analytical performance using pre-determined performance criteria, which is a critical component of molecular diagnostic test validation. All analytical measurements met pre-specified performance criteria. PCR amplification efficiency for all 12 assays was high, ranging from 96% to 107%, while linearity was demonstrated over an 11 log2 concentration range for all assays. Based on estimated components of variance for FPE RNA pools, analytical reproducibility and precision demonstrated low SDs for individual genes (0.16 to 0.32 CTs), gene groups (≤0.05 normalized/aggregate CTs) and RS (≤1.38 RS units). Analytical performance characteristics shown here for both individual genes and gene groups in the Oncotype DX Colon Cancer Assay demonstrate consistent translation of specific biology of individual tumors into clinically useful diagnostic information. The results of these studies illustrate how the analytical capability of the Oncotype DX Colon Cancer Assay has enabled clinical validation of a test to determine individualized recurrence risk after colon cancer surgery

  19. Biological dosimetry by the triage dicentric chromosome assay - Further validation of international networking

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Ruth C., E-mail: Ruth.Wilkins@hc-sc.gc.ca [Health Canada, Ottawa, ON K1A 0K9 (Canada); Romm, Horst; Oestreicher, Ursula [Bundesamt fur Strahlenschutz, 38226 Salzgitter (Germany); Marro, Leonora [Health Canada, Ottawa, ON K1A 0K9 (Canada); Yoshida, Mitsuaki A. [Biological Dosimetry Section, Dept. of Dose Assessment, Research Center for Radiation Emergency Medicine, NIRS, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Department Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564 (Japan); Suto, Y. [Biological Dosimetry Section, Dept. of Dose Assessment, Research Center for Radiation Emergency Medicine, NIRS, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Prasanna, Pataje G.S. [National Cancer Institute, Division of Cancer Treatment and Diagnosis, Radiation Research Program, 6130 Executive Blvd., MSC 7440, Bethesda, MD 20892-7440 (United States)

    2011-09-15

    Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific dicentric chromosomes in exposed individuals' peripheral blood lymphocytes. However, this method is labor-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the dicentric chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labor-demanding and time-consuming metaphase spread analysis to 20 - 50 metaphase spreads instead of routine 500 - 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.

  20. Cytokines-block micronucleus assay and its use for biological dosimetry

    International Nuclear Information System (INIS)

    The micronucleus (MN) assay in lymphocytes has been a convenient tool for the assessment of genetic damage induced by irradiation or chemical agents. This method is proved to be simpler, cheaper and rapid than the classical chromosome aberration assay. In the present paper, the influence factors in MN assay and established MN dose-response curve for X rays and γ rays has been studied

  1. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    Directory of Open Access Journals (Sweden)

    Federica Martini

    2012-01-01

    Full Text Available Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim and essential metals (zinc, copper, manganese, and selenium that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50 and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians.

  2. Are fish and standardized FETAX assays protective enough for amphibians? A case study on Xenopus laevis larvae assay with biologically active substances present in livestock wastes.

    Science.gov (United States)

    Martini, Federica; Tarazona, José V; Pablos, M Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC(50)) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  3. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    Science.gov (United States)

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  4. Radioreceptor assay for analysis of fentanyl and its analogs in biological samples

    International Nuclear Information System (INIS)

    The assay is based on the competition of these drugs with [3H] fentanyl for opioid receptors in membrane preparations of rat forebrain in vitro. The binding in stereospecific, reversible and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Morphine and hydromorphone complete with [3H]fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of [3H]fentanyl. Many other commonly abused drugs do not compete with [3H]fentanyl for the opioid receptors. Urine samples from animals injected with fentanyl, (±)-cis-3-methylfentanyl, alpha-methylfentanyl, butyrylfentanyl and benzylfentanyl were analyzed by radioreceptor assay, radioimmunoassay, and gas chromatography/mass spectrometry. Urinary analysis of fentanyl showed a good correlation with these three methods; however, discrepancies were observed in the analysis of fentanyl analogs. This radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine with good correlation with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs. The implications of these discrepancies are discussed

  5. Radioreceptor assay for analysis of fentanyl and its analogs in biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Alburges, M.E.

    1988-01-01

    The assay is based on the competition of these drugs with ({sup 3}H) fentanyl for opioid receptors in membrane preparations of rat forebrain in vitro. The binding in stereospecific, reversible and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Morphine and hydromorphone complete with ({sup 3}H)fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of ({sup 3}H)fentanyl. Many other commonly abused drugs do not compete with ({sup 3}H)fentanyl for the opioid receptors. Urine samples from animals injected with fentanyl, ({plus minus})-cis-3-methylfentanyl, alpha-methylfentanyl, butyrylfentanyl and benzylfentanyl were analyzed by radioreceptor assay, radioimmunoassay, and gas chromatography/mass spectrometry. Urinary analysis of fentanyl showed a good correlation with these three methods; however, discrepancies were observed in the analysis of fentanyl analogs. This radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine with good correlation with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs. The implications of these discrepancies are discussed.

  6. High-throughput assay for optimising microbial biological control agent production and delivery

    Science.gov (United States)

    Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialization. A myriad of variables associated with BCA cultivation, formulation, drying, storage, and reconstitution processes complicates agent quality maximization. An efficie...

  7. Biological Variations of Lupus Anticoagulant, Antithrombin, Protein C, Protein S, and von Willebrand Factor Assays.

    Science.gov (United States)

    Shou, Weiling; Chen, Qian; Wu, Wei; Cui, Wei

    2016-02-01

    The results of lupus anticoagulant (LA), antithrombin (AT), protein C (PC), and protein S (PS) testing, and the values of von Willebrand factor antigen (VWF:Ag) are important in diagnosis and therapeutic monitoring of thrombosis and hemostasis diseases. Till now, no published study has focused on the biological variations in LA testing, and only a few studies have examined the biological variations of AT, PC, PS, and VWF:Ag. With the latest fully automated instruments and improved reagents, the analytical, within-subject, and between-subject biological variations were estimated for these five coagulant parameters in a cohort of 25 apparently healthy subjects. Blood specimens were collected at 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5. The analytical biological variation (CV(A)) values of all the parameters were less than 3%. The within-subject biological variation (CV(W)) and between-subject biological variation (CV(G)) values of the LA normalized ratio were 4.64 and 6.83%, respectively. No significant differences were observed in the intraday and interday biological variations of LA tests, or in AT, PC, PS, and VWF:Ag values. Additionally, the utility of the conventional population-based reference intervals of the five coagulation parameters was evaluated by the index of individuality, and data on CV(W) and CV(A) were used to calculate the reference change value to identify the significance of changes in serial results from the same individual. PMID:26516946

  8. Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

    OpenAIRE

    Broccolo, Francesco; Locatelli, Giuseppe; Sarmati, Loredana; Piergiovanni, Sara; Veglia, Fabrizio; Andreoni, Massimo; Buttò, Stefano; Ensoli, Barbara; Lusso, Paolo; Malnati, Mauro S.

    2002-01-01

    Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the pres...

  9. High content screening as high quality assay for biological evaluation of photosensitizers in vitro.

    Directory of Open Access Journals (Sweden)

    Gisela M F Vaz

    Full Text Available A novel single step assay approach to screen a library of photdynamic therapy (PDT compounds was developed. Utilizing high content analysis (HCA technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4. The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT. The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells. A high correlation was found between the high content screening (HCS and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR study for photosensitizer libraries.

  10. An Approach to the Use of Modern Biological Assay Methods as a Fast Criterion for the Surface Cleaning/Assay Decision (LPS and ATP)

    Science.gov (United States)

    Kern, R.; Wainwright, N.; Kazarians, G.; Kuhlman, G.; Kempf, M.; Chen, F.; Venkateswaran, K.

    NASA has an ongoing research effort to introduce new methodologies to evaluate trace levels of biological contamination on spacecraft outbound from Earth to the surface of Mars. Present NASA regulations call for the evaluation of bioburden on spacecraft surfaces by the determination of aerobic spore-forming bacteria as a proxy for the total bioburden actually present. We are currently investigating molecular based methodologies that assess bioburden in a more rapid manner than the NASA standard technique. These are expected to find initial use, not for regulatory purposes, but for assisting the engineering team during the assembly of spacecraft by providing a rapid indicator of bioburden. We have evaluated two complementary methodologies and found they correlate to a significant degree with the presence of bacterial spores. A bioluminescence based detection method is capable of rapidly monitoring ATP levels from 10-13 M to 10-10 M. Similarly a Limulus amoebocyte lysate assay is capable of rapidly monitoring levels of bacterial lipopolysaccharide (LPS) from 0.005 to 50 endotoxin units (approx. 5x10-13 to 5x10-10 g E. coli LPS). Both methods have been tested during the course of the Mars Exploration Rover's assembly with a total of over 500 samples taken. Not only do these methods give one an independent measure of bioburden, they can also reasonably predict the presence of spores. There appears to be a significant correlation between samples containing ATP, LPS and spores.

  11. Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation.

    Science.gov (United States)

    Shinde, Vaibhav; Klima, Stefanie; Sureshkumar, Perumal Srinivasan; Meganathan, Kesavan; Jagtap, Smita; Rempel, Eugen; Rahnenführer, Jörg; Hengstler, Jan Georg; Waldmann, Tanja; Hescheler, Jürgen; Leist, Marcel; Sachinidis, Agapios

    2015-01-01

    Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically. PMID:26132533

  12. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including early detection of cancers

    Science.gov (United States)

    Martinez, Jennifer S.; Swanson, Basil I.; Shively, John E.; Li, Lin

    2009-06-02

    An assay element is described including recognition ligands adapted for binding to carcinoembryonic antigen (CEA) bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of CEA is described including injecting a possible CEA-containing sample into a sensor cell including the assay element, maintaining the sample within the sensor cell for time sufficient for binding to occur between CEA present within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  13. Hazard evaluation of soil contaminants from an abandoned oil refinery site with chemical and biological assays

    International Nuclear Information System (INIS)

    The phytotoxic characteristics of soil and leachates of soil from an abandoned oil refinery site were evaluated with rice (Oryza sativa L.) seed germinations and root elongation assays. Toxicity of soil leachates to aquatic animals was determined with acute and martial chronic toxicity tests with Ceriodaphnia dubia, fathead minnows, and Microtox reg-sign. Soil samples from uncontaminated (control) and selected contaminated areas within the old refinery were extracted with Toxic Characteristics Leachate Procedure (TCLP), an aqueous procedure and a supercritical carbon dioxide method. Aqueous extracts of soil from the oil leaded gasoline storage area exhibited greatest effects in all tests. Aqueous extracts from this site also caused a significant reduction in rice root development. Supercritical carbon dioxide extraction proved to be a quick and non-toxic procedure for isolating non-polar organics for assay with aquatic toxicity tests. Subsequent supercritical extracts collected in solvent can help characterize the class of toxicants through HPLC and Gas Chromatography. The toxic constituents were characterized with a Toxicity Identification/Toxicity Reduction Evaluation protocol to fractionate the contaminants into conventional non-polar organics, weak acids, base-neutrals, or heavy metals for subsequent analysis

  14. Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli

    Science.gov (United States)

    Heistermann, Michael; Santema, Peter; Dantzer, Ben; Mausbach, Jelena; Ganswindt, Andre; Manser, Marta B.

    2016-01-01

    In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach. PMID:27077741

  15. Analytical detection and biological assay of antileukemic drug using gold nanoparticles

    OpenAIRE

    V. Selvaraj; Alagar, M.; Hamerton, I

    2006-01-01

    Gold nanoparticles are reported and evaluated as probes for the detection of anticancer drug 5-fluorouracil (5FU). The nature of binding between 5FU and gold nanoparticles via complexation is investigated using ultraviolet visible spectrophotometry, cyclic voltammetry, transmission electron microscopy, fluorescence and Fourier transform infrared (FTIR) spectroscopy. The bound antileukemic drug is fluorescent and the quenching property of gold nanoparticles could be exploited for biological in...

  16. Analytical approaches for assaying metallodrugs in biological samples: recent methodological developments and future trends.

    Science.gov (United States)

    Timerbaev, Andrei; Sturup, Stefan

    2012-03-01

    Contemporary medicine increasingly relies on metal-based drugs and correspondingly growing in importance is the monitoring of the drugs and their metabolites in biological samples. Over the last decade, a range of analytical techniques have been developed in order to improve administration strategies for clinically approved compounds and understand pharmacokinetics, pharmacodynamics, and metabolism of new drugs so as ultimately to make their clinical development more effective. This paper gives an overview of various separation and detection methods, as well as common sample preparation strategies, currently in use to achieve the intended goals. The critical discussion of existing analytical technologies encompasses notably their detection capability, ability to handle biological matrices with minimum pretreatment, sample throughput, and cost efficiency. The main attention is devoted to those applications that are progressed to real-world biosamples and selected examples are given to illustrate the overall performance and applicability of advanced analytical systems. Also emphasized is the emerging role of inductively coupled plasma mass spectrometry (ICP-MS), both as a standalone instrument (for determination of metals originating from drug compounds) and as an element-specific detector in combinations with liquid chromatography or capillary electrophoresis (for drug metabolism studies). An increasing number of academic laboratories are using ICP-MS technology today, and this review will focus on the analytical possibilities of ICP-MS which would before long provide the method with the greatest impact on the clinical laboratory. PMID:21838702

  17. Adsorptive stripping voltammetric assay of phenazopyridine hydrochloride in biological fluids and pharmaceutical preparations.

    Science.gov (United States)

    Sabry, S M

    1999-08-23

    A sensitive method for the measurement of phenazopyridine hydrochloride (PAP) by differential pulse polarography (DPP) based on adsorptive stripping technique, using a hanging mercury drop electrode (HMDE) is described. The voltammetric peak is obtained at -0.760 V, which corresponds to the reduction of the azo group in Britton-Robinson buffer. The redox behaviour is reversible. Optimum conditions were found to be: accumulation potential -50 mV (vs. Ag/AgCl), accumulation time 60 s, scan rate 5 mV s(-1), pulse amplitude -100 mV and supporting electrolyte Britton-Robinson buffer (0.04 M, pH=11). The relative standard deviation (at 20 ng ml(-1) level) was +/-0.6% for six measurements. The calculated detection limit was 0.0299 ng ml(-1) with a 60-s accumulation time. The applicability of such a method was evaluated through the assay of PAP in human plasma and urine samples after a simple extraction procedure and in pharmaceutical preparation. The mean recovery was 97+/-2 (100 ng ml(-1) plasma). PMID:18967703

  18. 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.

  19. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    International Nuclear Information System (INIS)

    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 10−12 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

  20. Biological dosimetry of X-rays using in-Vitro micronucleus assay in human lymphocytes

    International Nuclear Information System (INIS)

    Micronuclei (Mn) are products of fragmented chromosomes which are recently being used as an alternative approach to the chromosome aberrations analysis for the estimation of biological absorbed dose in the case of occupationally exposed radiation workers including those working in industrial radiography, diagnostic radiology, nuclear medicine departments, as well as in nuclear installations. In the present work human blood samples were taken and after in- vitro irradiation with various doses of X-rays, in the dose range of 5-50 and 50-300 cgs were studied. Micronucleus test was performed using cytokinesis blocked cells. The best fit was obtained by linear quadratic model, Y=C +αD +βD2. Moreover we have observed X-ray induced micronucleus in lymphocytes from 7 exposed hospital X-ray workers. The micronucleus counts in these persons were 3-7 fold higher than those in control

  1. Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays

    DEFF Research Database (Denmark)

    Molina, Lazaro; Rodriguez, Cayo Juan Ramos; Ronchel, Maria C.;

    1998-01-01

    Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn5 transposon bearing a fusion of the P(lac) promoter to the gef killing gene and a...... planted and unplanted soils with and without 3-methylbenzoate. In unplanted soils the numbers of both the contained strain and the uncontained strain per gram of soil tended to decrease, but the numbers of the contained strain decreased faster in soils without 3-methylbenzoate. The decrease in the number......(6) CFU/g of soil regardless of the presence of 3-methylbenzoate. In the bulk soil the numbers of bacterial cells were 2 to 3 orders of magnitude lower. In planted soils the contained strain tended to disappear, but this tendency was more pronounced in the absence of 3-methylbenzoate and occurred faster...

  2. Analytical detection and biological assay of antileukemic drug using gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Selvaraj, V. [Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600025 (India)]. E-mail: rajselva_77@yahoo.co.in; Alagar, M. [Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600025 (India)]. E-mail: mkalagar@yahoo.com; Hamerton, I. [Chemistry Division, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH (United Kingdom)

    2006-11-12

    Gold nanoparticles are reported and evaluated as probes for the detection of anticancer drug 6-mercaptopurine (6-MP). The nature of binding between 6-MP and the gold nanoparticles via complexation is investigated using ultraviolet-visible spectrum, cyclic voltammetry, transmission electron microscopy, fluorescence and Fourier transform infrared (FT-IR) spectroscopy. The bound antileukemic drug is fluorescent and the quenching property of gold nanoparticles could be exploited for biological investigations. The 6-MP-colloidal gold complex is observed to have appreciable antibacterial and antifungal activity against Micrococcus luteus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Aspergillus fumigatus, and Aspergillus niger. The experimental studies suggest that gold nanoparticles have the potential to be used as effective carriers for anticancer drugs.

  3. Ultrasensitive detection in optically dense physiological media: applications to fast reliable biological assays

    Science.gov (United States)

    Matveeva, Evgenia G.; Gryczynski, Ignacy; Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt

    2006-02-01

    We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.

  4. Application of the alkaline comet assay to rat alveolar macrophages after homogeneous or heterogeneous irradiation: a biological dosimetry method

    International Nuclear Information System (INIS)

    The alkaline comet assay, also called alkaline single cell gel electrophoresis, is a simple technique to assess single strand breaks, double strand breaks and alkali sites. It is based on the ability of broken DNA to migrate more easily in an electric field than normal DNA. This method is well adapted for the assessment of the ionising radiations effects on single cells. The aim of this study is to develop a biological dosimetry method to estimate the dose delivered to the respiratory tract by an homogeneous (60Co) or an heterogeneous (radon) irradiation. The animal model chosen is the rat because it has been validated for the study of the carcinogenic role of radon in man. Alveolar macrophages have been selected for there homogeneous distribution in the deep lung. After an in vivo thoracic 60Co gamma irradiation or a radon inhalation, it can be considered that these cells received a dose which is representative from the whole dose received by the lung. The comet assay is performed on alveolar macrophages recovered by broncho-alveolar lavage, and comet moment is measured with an epi-fluorescence microscope coupled to an image acquisition and analysis computing system. The results show the residence of a dose - comet moment relationship after in vivo 60Co gamma and radon irradiations. The technique used enabled us to show differences between homogeneous and heterogeneous irradiations in term of comet moments distributions. Although these results are promising, this technique has to be improved for the detection of biological effects induced by low doses of irradiation in order to detect potential effects of indoor radon exposure. (authors)

  5. Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay

    Directory of Open Access Journals (Sweden)

    Zahra YADEGARI

    2015-10-01

    Full Text Available Background: Amelogenins are the major components of enamel matrix proteins. Enamel matrix derivatives (EMD can be used in periodontal diseases to regenerate periodontal tissues. The main aim of this study was to evaluate ex-pression of full-length functional recombinant human amelogenin (rhAm in Iranian lizard Leishmania (I.L.L. as an alternative eukaryotic expression system.Methods: Human cDNA encoding a 175-amino acid amelogenin expression cassette was sub cloned into a pLEXSY vector. The construct was transferred into Leishmania cells by electroporation. The protein production was surveyed in the transcription and the translation levels. The expressed protein was purified and some of its biological properties were investigated in comparison to EMD and negative control.Results: Expression of rhAm was confirmed by RT-PCR and western blot test in Leishmania cells. Purified rhAm sig-nificantly inhibited the formation of tartrate-resistant acid phosphatase positive (TRAP+ multinuclear cells in calcitriol stimulated mouse marrow cultures. Moreover, it significantly promoted proliferation and DNA synthesis in L929 mouse fibroblast cells.Conclusion: Functional rhAm was successfully expressed in I.L.L. Easy handling and post translation modification were the main advantages of this expression system. It is suggested to investigate molecular properties of this rhAm in the future.

  6. [Biological assay for galactose-1 phosphate measurement application in subjects with galactosemia].

    Science.gov (United States)

    Braham, Imene; Charfeddine, Bassem; Ben Othmene, Leila; Neffati, Souhir; Mtar, Aida; Ben Abdallah, Jihene; Ali Smach, Med; Dridi, Hedi; Limem, Khalifa

    2012-01-01

    Congenital galactosemia is a hereditary, autosomal recessive and metabolic disease. It is linked to an enzyme deficiency, more commonly known by the deficiency of galactose-1- phosphate uridyltransferase (GALT), which is responsible for an accumulation of galactose-1- phosphate in the blood. Clinical symptoms appear early in infancy from the second week of life. They generally manifested by some disorders within liver, kidney, eye, gastrointestinal, neurological and also with cataracts. Currently, the clinical diagnosis remains difficult hence the importance of further investigations based on effective biological assessments to highlight the disease. The diagnosis of galactosemia is made by the laboratory test. The latter includes the determination of Gal-1-P which is done by a fluorometric method spot test. This study was conducted in order to assess the repeatability, reproducibility, accuracy, and effectiveness of the techniques used. We have found the CV for a repeatability (CV = 5 %), reproducibility (CV = 4 %) which confirms the accuracy of the method proceeded in this study. This method allows us to have a degree of inaccuracy less than 1%. According to the study of the effectiveness of "spot test", we found that our technique is specific (Sp = 93 %) and sensitive (Se = 83 %). PMID:22294140

  7. SU-E-T-253: Open-Source Automatic Software for Quantifying Biological Assays of Radiation Effects

    International Nuclear Information System (INIS)

    Purpose: Clonogenic cell survival is a common assay for quantifying the effect of drugs and radiation. Manual counting of surviving colonies can take 30–90seconds per plate, a major limitation for large studies. Currently available automatic counting tools are not easily modified for radiation oncology research. Our goal is to provide an open-source toolkit for precise, accurate and fast analysis of biological assays in radiation oncology. Methods: As an example analysis, we used HeLa cells incubated with gadolinium nanoparticles prior to irradiation. After treatment, the cells are grown for 14days to allow for colony formation. To analyze the colony growth, we capture images of each dish for archiving and automatic computer-based analysis. A FujifilmX20 camera is placed at the top of a box setup, 20cm above the sample, which is backlit by a LED lamp placed at the bottom of the box. We use a Gaussian filter (width=1.3mm) and color threshold (19–255). The minimum size for a colony to be counted is 1mm. For this example, 20 dishes with a large range of colonies were analyzed. Each dish was counted 3 times manually by 3 different users and then compared to our counter. Results: Automatic counting of cell colonies takes an average of 7seconds, enabling the analysis process to be accelerated 4–12 times. The average precision of the automatic counter was 1.7%. The Student t-test demonstrated the non-significant differences between the two counting methods (p=0.64). The ICC demonstrated the reliability of each method with ICC>0.999 (automatic) and ICC=0.95 (manual). Conclusion: We developed an open-source automatic toolkit for the analysis of biological assays in radiation oncology and demonstrated the accuracy, precision and effort savings for clonogenic cell survival quantification. This toolkit is currently being used in two laboratories for routine experimental analysis and will be made freely available on our departmental website

  8. Magnetic core/shell nanoparticle thin films deposited by MAPLE: Investigation by chemical, morphological and in vitro biological assays

    Energy Technology Data Exchange (ETDEWEB)

    Cristescu, R., E-mail: rodica.cristescu@inflpr.ro [National Institute for Lasers, Plasma and Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest-Magurele (Romania); Popescu, C.; Socol, G.; Iordache, I.; Mihailescu, I.N. [National Institute for Lasers, Plasma and Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest-Magurele (Romania); Mihaiescu, D.E.; Grumezescu, A.M. [Faculty of Applied Chemistry and Materials Science, ' Politehnica' University of Bucharest, 1-7 Polizu Street, 011061 Bucharest (Romania); Balan, A.; Stamatin, I. [University of Bucharest, 3Nano-SAE Research Center, PO Box MG-38, Bucharest-Magurele (Romania); Chifiriuc, C. [Faculty of Biology, University of Bucharest, Microbiology Immunology Department, Aleea Portocalilor 1-3, Sector 5, 77206 Bucharest (Romania); Bleotu, C. [Stefan S. Nicolau Institute of Virology, 285 Mihai Bravu, 030304 Bucharest (Romania); Saviuc, C.; Popa, M. [Faculty of Biology, University of Bucharest, Microbiology Immunology Department, Aleea Portocalilor 1-3, Sector 5, 77206 Bucharest (Romania); Chrisey, D.B. [Rensselaer Polytechnic Institute, School of Engineering, Departments of Materials Science and Biomedical Engineering, Troy, 12180-3590, NY (United States)

    2012-09-15

    Highlights: Black-Right-Pointing-Pointer We deposit magnetic Fe{sub 3}O{sub 4}/oleic acid/cephalosporin nanoparticle thin films by MAPLE. Black-Right-Pointing-Pointer Thin films have a chemical structure similar to the starting material. Black-Right-Pointing-Pointer Cephalosporins have an additive effect on the grain size and induce changes in grain shape. Black-Right-Pointing-Pointer MAPLE can be used to develop novel strategies for fighting medical biofilms associated with chronic infections. - Abstract: We report on thin film deposition of nanostructured Fe{sub 3}O{sub 4}/oleic acid/ceftriaxone and Fe{sub 3}O{sub 4}/oleic acid/cefepime nanoparticles (core/shell/adsorption-shell) were fabricated by matrix assisted pulsed laser evaporation (MAPLE) onto inert substrates. The thin films were characterized by profilometry, Fourier transform infrared spectroscopy, atomic force microscopy, and investigated by in vitro biological assays. The biological properties tested included the investigation of the microbial viability and the microbial adherence to the glass coverslip nanoparticle film, using Gram-negative and Gram-positive bacterial strains with known antibiotic susceptibility behavior, the microbial adherence to the HeLa cells monolayer grown on the nanoparticle pellicle, and the cytotoxicity on eukaryotic cells. The proposed system, based on MAPLE, could be used for the development of novel anti-microbial materials or strategies for fighting pathogenic biofilms frequently implicated in the etiology of biofilm associated chronic infections.

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

    OpenAIRE

    Pascal E. Saikaly; 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...

  10. Elucidation of the binding mechanism of renin using a wide array of computational techniques and biological assays.

    Science.gov (United States)

    Tzoupis, Haralambos; Leonis, Georgios; Avramopoulos, Aggelos; Reis, Heribert; Czyżnikowska, Żaneta; Zerva, Sofia; Vergadou, Niki; Peristeras, Loukas D; Papavasileiou, Konstantinos D; Alexis, Michael N; Mavromoustakos, Thomas; Papadopoulos, Manthos G

    2015-11-01

    We investigate the binding mechanism in renin complexes, involving three drugs (remikiren, zankiren and enalkiren) and one lead compound, which was selected after screening the ZINC database. For this purpose, we used ab initio methods (the effective fragment potential, the variational perturbation theory, the energy decomposition analysis, the atoms-in-molecules), docking, molecular dynamics, and the MM-PBSA method. A biological assay for the lead compound has been performed to validate the theoretical findings. Importantly, binding free energy calculations for the three drug complexes are within 3 kcal/mol of the experimental values, thus further justifying our computational protocol, which has been validated through previous studies on 11 drug-protein systems. The main elements of the discovered mechanism are: (i) minor changes are induced to renin upon drug binding, (ii) the three drugs form an extensive network of hydrogen bonds with renin, whilst the lead compound presented diminished interactions, (iii) ligand binding in all complexes is driven by favorable van der Waals interactions and the nonpolar contribution to solvation, while the lead compound is associated with diminished van der Waals interactions compared to the drug-bound forms of renin, and (iv) the environment (H2O/Na(+)) has a small effect on the renin-remikiren interaction. PMID:26421414

  11. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including protein markers, pathogens and cellular debris

    Science.gov (United States)

    Martinez, Jennifer S.; Swanson, Basil I.; Grace, Karen M.; Grace, Wynne K.; Shreve, Andrew P.

    2009-06-02

    An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  12. ABSORPTION AND BIOAVAILABILITY OF THE MINERALS IN THE MULTI-MIXTURE FOOD SUPPLEMENT: BIOLOGICAL ASSAY IN RATS

    Directory of Open Access Journals (Sweden)

    E. HELBIG

    2008-12-01

    Full Text Available

    The absorption and bioavailability of the minerals Ca, Fe and Zn in balanced and mineral restricted diets with the addition of the food supplement multi-mixture (MM, was evaluated. A biological assay was carried out for 28 days with recently weaned Wistar rats, using 3 treatments and 8 animals in each group. The diets offered to each group were distributed as follows: CD – Control Diet (AIN-93G; CcD/MM – Control Diet + Supplement 5% MM; DRMIXc/MM – Control Diet with Mineral Restricted + Supplement 5% MM. The animals were monitored for weight; amount of diet consumed and amount of faeces excreted. They were sacrifi ced at the end of the experimental period and the pair of femurs, pancreas and liver removed for the determination of Ca, Fe and Zn. The CcD/MM diet presented absorption of Ca and Fe and bioavailability of Ca, Fe and Zn equal to that of CD, showing no statistical difference between the treatments. However the DRMIXcD/MM diet presented an increase in Ca absorption, equal absorption of Fe and an increase in the bioavailability of Ca, Fe and Zn when compared to CD. It was concluded that when fed on a balanced diet supplemented with 5% MM, there was no increase in the bioavailability of Ca, Fe or Zn, whereas when fed on a mineral defi cient diet, the group of animals that received supplementation with 5% MM, presented greater absorption and bioavailability of these minerals.

  13. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography.

    OpenAIRE

    Foulstone, M; Reading, C

    1982-01-01

    Augmentin is a new antibacterial formulation comprised of amoxicillin and the beta-lactamase inhibitor clavulanic acid. In the present paper, the use of high-performance liquid chromatography (HPLC) to provide a rapid assay of the components of Augmentin in body fluids is described. Clavulanic acid was assayed by reacting the sample with imidazole, which readily produces a derivative absorbing at 311 nm. This derivative chromatographs on reverse-phase HPLC columns clear of interfering compone...

  14. Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach

    Energy Technology Data Exchange (ETDEWEB)

    Carr, Steven A.; Abbateillo, Susan E.; Ackermann, Bradley L.; Borchers, Christoph H.; Domon, Bruno; Deutsch, Eric W.; Grant, Russel; Hoofnagle, Andrew N.; Huttenhain, Ruth; Koomen, John M.; Liebler, Daniel; Liu, Tao; MacLean, Brendan; Mani, DR; Mansfield, Elizabeth; Neubert, Hendrik; Paulovich, Amanda G.; Reiter, Lukas; Vitek, Olga; Aebersold, Ruedi; Anderson, Leigh N.; Bethem, Robert; Blonder, Josip; Boja, Emily; Botelho, Julianne; Boyne, Michael; Bradshaw, Ralph A.; Burlingame, Alma S.; Chan, Daniel W.; Keshishian, Hasmik; Kuhn, Eric; Kingsinger, Christopher R.; Lee, Jerry S.; Lee, Sang-Won; Moritz, Robert L.; Oses-Prieto, Juan; Rifai, Nader; Ritchie, James E.; Rodriguez, Henry; Srinivas, Pothur R.; Townsend, Reid; Van Eyk , Jennifer; Whiteley, Gordon; Wiita, Arun; Weintraub, Susan

    2014-01-14

    Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and

  15. Methods of acute biological assays in guinea-pigs for the study of toxicity and innocuity of drugs and chemicals

    OpenAIRE

    Gui Mi Ko; Adela Rosenkranz; Clélia Rejane Antonio Bertoncini; Neide Hyppolito Jurkiewicz; Mirian Ghiraldini Franco; Aron Jurkiewicz

    2010-01-01

    In this study, 602 samples were tested by the following assays performed at the animal facilities (Cedeme) of the Federal University of São Paulo (UNIFESP): 385 for dermal irritability, 90 for ocular irritability (discontinued in 1995), 31 for systemic toxicity by injection, 26 for oral acute toxicity, 15 for toxicity by intracutaneous injection, 15 for skin sensitization, 15 for toxicity of serum and vaccines for human use, 14 for toxicity by intramuscular implantation, 7 for pyrogens, 2 for...

  16. Application of an objective biological assay of human interferons to clinical specimens and a survey of a normal population.

    OpenAIRE

    Forti, R L; Moldovan, R A; Mitchell, W M; Callicoat, P; Schuffman, S S; Davies, H A; Smith, D. M.

    1985-01-01

    A rapid, quantitative, and nonsubjective method of interferon assay is described, which can be readily applied to clinical specimens. Automated data acquisition and data reduction allowed a significant increase in volume per unit of time over existing methodologies. Plasma always yielded higher (usually 2:1) interferon values than did serum obtained simultaneously. Ranges of interferon levels in plasma in normal control populations are reported as well as ranges for clinical virology laborato...

  17. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    OpenAIRE

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e....

  18. Electrochemical studies of nevirapine, an anti-HIV drug, and its assay in tablets and biological samples

    Directory of Open Access Journals (Sweden)

    JALDAPPAGARI SEETHARAMAPPA

    2012-06-01

    Full Text Available The electrochemical oxidation of nevirapine, an anti-HIV drug, at a glassy carbon electrode has been studied by voltammetric techniques. Nevirapine showed one well defined irreversible oxidation peak with a potential of 0.749 V in phosphate buffer at pH 10. The effects of different electrolytes, pH and scan rate on the electrochemical behaviour of nevira¬pine were examined to determine the optimum reaction conditions. The oxidation peak current was found to vary linearly with the concentration of nevirapine in the range of 5.0 – 350 µM. The limit of detection and limit of quantification values were calculated and found to be 1.026 µM and 3.420 µM, respectively. The low relative standard deviation values of inter-day and intra-day assays highlighted the good reproducibility of the proposed m¬ethod for assay of nevirapine. Further, a sensitive and accurate differential pulse voltammetric method was developed for the determination of nevirapine concentrations in pharma¬ceutical formulations.

  19. Methods for Precise Molecular Detection of Probiotic Microflora: Using Adjusted Molecular Biology Protocols, Primer Sets and PCR Assays

    Directory of Open Access Journals (Sweden)

    F. Abu Bakar

    2010-01-01

    Full Text Available Lactobacillus sp. is probiotic bacteria for which many detection methods were envisaged. However, culture-based methods failed to achieve specific detection of this bacterium due to its presence in mixed bacterial complex communities. The PCR assay was optimized to detect and quantify Lactobacillus sp. specifically in complex microbial community of mixed bacteria. Four DNA extraction methods, DNA integrity, primers specificity and optimized PCR procedure were all tested. It was shown that extracted genomic DNA using Wizard® Genomic DNA Purification Kit showed the highest yield, quality and performance in gel electrophoresis. Moreover, the specificity of the primer set, Lacto-16S-F /Lacto-16S-R, specific for Lactobacillus sp. was checked and found highly specific. In conclusion, the best DNA extraction protocol, working specific primer set and working PCR assay were achieved for achieving efficient, specific and reliable molecular-based, culture-independent, method of detection of lactobacillus sp. in PCR-suppressor highly protein-complex environment of mixed bacteria community.

  20. Validated capillary electrophoretic assays for disaccharide composition analysis of galactosaminoglycans in biologic samples and drugs/nutraceuticals.

    Science.gov (United States)

    Asimakopoulou, Athanasia P; Malavaki, Christina; Afratis, Nikolaos A; Theocharis, Achilleas D; Lamari, Fotini N; Karamanos, Nikos K

    2015-01-01

    Capillary electrophoresis is a separation technique with high resolving power and sensitivity with applications in glycosaminoglycan analysis. In this chapter, we present validated protocols for determining the variously sulfated chondroitin or dermatan sulfate-derived disaccharides. These approaches involve degradation of the polysaccharides with specific chondro/dermato-lyases and electrophoretic analysis with capillary zone electrophoresis in a low pH operating buffer and reversed polarity. This methodology has been applied to drug/nutraceutical formulations or to biologic samples (blood serum, lens capsule) and has been validated. Analysis of biologic tissue samples is often more demanding in terms of detection sensitivity, and thus concentration pretreatment steps and/or a derivatization step with 2-aminoacridone are often advisable. PMID:25325950

  1. First Evaluation of the Biologically Active Substances and Antioxidant Potential of Regrowth Velvet Antler by means of Multiple Biochemical Assays

    Directory of Open Access Journals (Sweden)

    Yujiao Tang

    2015-01-01

    Full Text Available We investigated the biologically active substances contained in RVA (regrowth velvet antler by comparing the composition of biologically active substances and antioxidant potential of different antler segments. RVA was subjected to extraction using DW (distilled water. RVA was divided into 3 segments: T-RVA (top RVA, M-RVA (middle RVA, and B-RVA (base RVA. The T-RVA section possessed the greatest amounts of uronic acid (36.251 mg/g, sulfated GAGs (sulfated glycosaminoglycans (555.76 mg/g, sialic acid (111.276 mg/g, uridine (0.957 mg/g, uracil (1.084 mg/g, and hypoxanthine (1.2631 mg/g. In addition, the T-RVA section possessed the strongest antioxidant capacity as determined by DPPH, H2O2 (hydrogen peroxide, hydroxyl, and ABTS (2,2′-azinobis-3-ethylbenzthiazoline-6-sulphonate radical scavenging activity as well as FRAP (ferric reducing antioxidant power and ORAC (oxygen radical absorbance capacity. The values of those were 53.44, 23.09, 34.12, 60.31, and 35.81 TE/μM at 1 mg/mL and 113.57 TE/μM at 20 μg/mL. These results indicate that the T-RVA section possesses the greatest amount of biologically active substances and highest antioxidant potential. This is the first report on the biologically active substances and antioxidant potential of RVA.

  2. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    Science.gov (United States)

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum. PMID:16825391

  3. Retention of biological activity following radioiodination of human interleukin 2: comparison with biosynthetically labeled growth factor in receptor binding assays

    International Nuclear Information System (INIS)

    Human interleukin 2 (IL-2) was radioiodinated by a modified form of the chloramine-T reaction. Comparison with biosynthetically radiolabeled IL-2 demonstrated that the iodinated molecule retained similar receptor binding characteristics and proliferation-inducing ability. The iodinated molecule also possessed the distinct advantages of a higher specific radioactivity and a reduced processing time for the assay samples. The majority of the iodine was incorporated at the tyrosine in position 45 of the polypeptide chain. Evidently, this residue is unimportant for the molecule's association with its receptor. The development of active radioiodinated IL-2 should facilitate routine measurement of the high-affinity IL-2 binding sites which mediate the physiological response to this lymphokine. (Auth.)

  4. High-performance liquid chromatographic assay for acetaminophen and phenacetin in the presence of their metabolites in biological fluids

    International Nuclear Information System (INIS)

    The authors propose a method in which tracer amounts of a radiolabeled compound are used as the internal standard for the same unlabeled compound in high-performance liquid chromatography. The approach is valuable when a response from the internal standard becomes undesirable due to the presence of interference by the metabolites. The authors tested their approach with phenacetin and its metabolites, 2-hydroxyphenacetin, N-hydroxyphenacetin, phenetidine, acetaminophen sulfate conjugate and acetaminophen glucuronide conjugate in biological fluids with the use of [14C] phenacetin and [3H] acetaminophen as the internal standards, and were able to quantitate both phenacetin and acetaminophen simultaneously. They also tested the alternative approach in which the unlabeled drug was used as internal standard for tracer amounts of the same radiolabeled compound, with phenacetin and acetaminophen as the internal standards for tracer amounts of [14C] phenacetin and [3H] acetaminophen. Again, they were able to quantiate the two tracer radiolabeled compounds simultaneously. (Auth.)

  5. Biological efficiency of the Brookhaven Medical Research Reactor mixed neutron beam estimated from gene mutations in Tradescantia stamen hair cells assay

    International Nuclear Information System (INIS)

    The relative biological effectiveness (RBE) of low energy neutrons for the induction of various abnormalities in Tradescantia stamen hair mutation (Trad-SH) assay was studied using two clones (T-4430 and T-02), heterozygous for flower color. Dose response relationship for gene mutations induced in somatic cells of Trad-SH were investigated after irradiation with a mixed neutron beam of the Brookhaven Medical Research Reactor (BMRR), currently used in a clinical trial of boron neutron capture therapy (BNCT) for glioblastoma. To establish the RBE of the BMRR beam in the induction of various biological end-points in Tradescantia, irradiation with various doses of γ-rays was also performed. After irradiation all plants were cultivated several days at Brookhaven National Laboratory (BNL), then transported to Poland for screening the biological end-points. Due to the post-exposure treatment, all plants showed high levels of lethal events and alteration of the cell cycle. Plants of clone 4430 were more reactive to post-treatment conditions, resulting in decreased blooming efficiency that affected the statistics. Slope coefficients estimated from the dose response curves for gene mutation frequencies allowed the evaluation of ranges for the maximal RBE values of the applied beam vs. γ rays as 6.0 and 5.4 for the cells of T-02 and T-4430, respectively. Estimated fraction of doses from neutrons and corresponding biological effects for the clones T-02 and T-4430 allowed to evaluate the RBE values for neutrons part in the beam as 32.3 and 45.4, respectively. (author)

  6. Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

    Science.gov (United States)

    Martins Alho, Miriam A; Marrero-Ponce, Yovani; Barigye, Stephen J; Meneses-Marcel, Alfredo; Machado Tugores, Yanetsy; Montero-Torres, Alina; Gómez-Barrio, Alicia; Nogal, Juan J; García-Sánchez, Rory N; Vega, María Celeste; Rolón, Miriam; Martínez-Fernández, Antonio R; Escario, José A; Pérez-Giménez, Facundo; Garcia-Domenech, Ramón; Rivera, Norma; Mondragón, Ricardo; Mondragón, Mónica; Ibarra-Velarde, Froylán; Lopez-Arencibia, Atteneri; Martín-Navarro, Carmen; Lorenzo-Morales, Jacob; Cabrera-Serra, Maria Gabriela; Piñero, Jose; Tytgat, Jan; Chicharro, Roberto; Arán, Vicente J

    2014-03-01

    which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses. PMID:24513185

  7. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  8. Biologic

    CERN Document Server

    Kauffman, L H

    2002-01-01

    In this paper we explore the boundary between biology and the study of formal systems (logic). In the end, we arrive at a summary formalism, a chapter in "boundary mathematics" where there are not only containers but also extainers ><, entities open to interaction and distinguishing the space that they are not. The boundary algebra of containers and extainers is to biologic what boolean algebra is to classical logic. We show how this formalism encompasses significant parts of the logic of DNA replication, the Dirac formalism for quantum mechanics, formalisms for protein folding and the basic structure of the Temperley Lieb algebra at the foundations of topological invariants of knots and links.

  9. Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 2. The biological variability of the murine Local Lymph Node Assay (LLNA) for skin sensitisation.

    Science.gov (United States)

    Roberts, David W; Api, Anne Marie; Aptula, Aynur O

    2016-10-01

    The Local Lymph Node Assay (LLNA) is the most common in vivo regulatory toxicology test for skin sensitisation, quantifying potency as the EC3, the concentration of chemical giving a threefold increase in thymidine uptake in the local lymph node. Existing LLNA data can, along with clinical data, provide useful comparator information on the potency of sensitisers. Understanding of the biological variability of data from LLNA studies is important for those developing non-animal based risk assessment approaches for skin allergy. Here an existing set of 94 EC3 values for 12 chemicals, all tested at least three times in the same vehicle have been analysed by calculating standard deviations (SD) for logEC3 values. The SDs range from 0.08 to 0.22. The overall SD for the 94 logEC3 values is 0.147. Thus the 95% confidence limits (2xSD) for LLNA EC3 values are within a factor of 2, comparable to those for physico-chemical measurements such as partition coefficients and solubility. The residual SDs of Quantitative Mechanistic Models (QMMs) based on physical organic chemistry parameters are similar to the overall SD of the LLNA, indicating that QMMs of this type are unlikely to be bettered for predictive accuracy. PMID:27470439

  10. Methods of acute biological assays in guinea-pigs for the study of toxicity and innocuity of drugs and chemicals

    Directory of Open Access Journals (Sweden)

    Gui Mi Ko

    2010-06-01

    Full Text Available In this study, 602 samples were tested by the following assays performed at the animal facilities (Cedeme of the Federal University of São Paulo (UNIFESP: 385 for dermal irritability, 90 for ocular irritability (discontinued in 1995, 31 for systemic toxicity by injection, 26 for oral acute toxicity, 15 for toxicity by intracutaneous injection, 15 for skin sensitization, 15 for toxicity of serum and vaccines for human use, 14 for toxicity by intramuscular implantation, 7 for pyrogens, 2 for acute dermal toxicity, and 2 for irritation of mucous membrane. The following agents were tested: cosmetics and related substances (42.0%, chemicals used in industry (32.9%, plastics, rubber, and other polymers (15.9%, agrotoxics (4.0%, medicines (2.7%, and vaccines (2.5%. In the present description, emphasis was given to tests of dermal irritability and sensitization. This work was conducted entirely in animal facilities, according to our general belief that animal facilities at universities, while considering ethic principles and sanitary, genetic, nutritional, and pathophysiological controls, also require laboratories specialized in areas such as transgenics, cryopreservation, ambiental physiology, functional genomics, alternative models, and mainly activities and research on methods in toxicology, as focused in this study.Descrevemos os testes usados em ensaios biológicos de curta duração para estudo de toxicidade e inocuidade de cosméticos, fármacos e outras substâncias químicas, feitos no Biotério Central/Cedeme da Unifesp, de 1986 a 2000. Testamos 602 amostras nos seguintes ensaios: 385 de irritação cutânea, 90 de irritação ocular (até 1995, 31 de toxicidade sistêmica por injeção, 26 de toxicidade oral aguda, 15 de toxicidade por aplicação intracutânea, 15 de sensibilização da pele, 15 de toxicidade de soros e vacinas de uso humano, 14 de toxicidade por implantação intramuscular, 7 de pirogênio, 2 de toxicidade dérmica aguda e

  11. Biological Efficiency of Californium-252 Source Evaluated by Comet Assay, Classical Cytogenetics and FISH in Human Lymphocytes Irradiated without and with BSH Pretreatment

    International Nuclear Information System (INIS)

    Biological effectiveness of californium-252 source was evaluated after irradiations in vitro of normal or pre-treated with compound enriched in B-10 ion cells. Peripheral blood lymphocytes were used as a model for human cells. DNA and chromosomal damage were studied to compare biological effectiveness of irradiation. Human blood samples or isolated lymphocytes were irradiated with the isotopic source of 252Cf, at the Faculty of Physics and Nuclear Techniques at the University of Mining and Metallurgy (both neutron source and samples were placed in ''infinite'' polyethylene block). Chemical pretreatment with Na210B12H11SH (BSH) was performed to introduce boron-10 ion into cells in order to check any enhancement effect due to the process of boron neutron capture. Single cell gel electrophoresis also known as the Comet assay was done to investigate the DNA damage. Classical cytogenetic analysis was applied to assess the frequencies of unstable aberrations (dicentrics, rings and a centric fragments). To evaluate the frequencies of stable aberrations the fluorescence in situ hybridisation (FISH) with probes for chromosomes 1, 4 (14.3% of the whole genome) was performed. Linear (or close to linear) increase with radiation dose were observed for the DNA damage and aberration frequencies in lymphocytes both untreated or pre-treated with BSH. Levels of translocations evaluated for the whole genome were comparable with the frequencies of dicentrics and rings. No significant differences were detected due to radiation dose in the frequencies of sister chromatid exchanges (SCE) detected in the second mitosis. No statistically significant differences were observed in various biological end-points between normal or boron pre-treated cells. (author)

  12. 肝素生物测定法(血浆法)方法学改进研究%Methodology Improvement for Biological Assay of Heparin

    Institute of Scientific and Technical Information of China (English)

    吴超权; 周智; 覃君良; 方珍文

    2015-01-01

    Objective:To improve the plasma method for biological assay of heparin based on China Pharmacopeia currently in effect.Methods:National standard compound of heparin and a batch of sodium heparin injection were chosen, and the coagulation time of the standard heparin and heparin injection was tested by platelet aggregation and coagulation analyzer using 1∶1 diluted rabbit plasma with sodium chloride injection, according to the method collected in appendix XII B of China Pharmacopeia Volume II (version 2010) .Results: Compared with the standard target potency, the recovery rate of the standard was in the range of 98.88%~101.86%, while the recovery rate of the sodium heparin injection was in the range of 102.3%~103.6%.Conclusion:This new method for heparin determination is easy to operate, with objective end-point high accuracy and repetitiveness, and the conifdence limits meet the requirement, we therefore suggest this new method as the improved method for biological assay of heparin.%目的:针对现行《中国药典》附录中使用的肝素生物测定法的血浆法方法学进行改进。方法:选用肝素钠国家标准品和肝素钠注射液1批,采用1∶1氯化钠注射液稀释的兔血浆,用血小板聚集凝血因子分析仪,按《中国药典》2010年版二部附录XⅡ B方法对肝素钠标准品和肝素钠注射液的凝结时间进行测定。结果:已知靶值的标准品效价的回收率为98.88%~101.86%,肝素钠注射液加样回收率为102.30%~103.60%。结论:该肝素测定方法操作简单,终点客观,重现性和准确性好,可信限满足要求,推荐使用该方法作为肝素生物效价测定方法。

  13. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    Science.gov (United States)

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-07-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure.

  14. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    Science.gov (United States)

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-01-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure. PMID:27445126

  15. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats.

    Science.gov (United States)

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-01-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1-3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure. PMID:27445126

  16. Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

    Science.gov (United States)

    Weissbrodt, David G; Maillard, Julien; Brovelli, Alessandro; Chabrelie, Alexandre; May, Jonathan; Holliger, Christof

    2014-12-01

    Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC. PMID:24975745

  17. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    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.)

  18. The neutral comet assay

    International Nuclear Information System (INIS)

    The single-cell gel electrophoresis (or comet) assay is a rapid and sensitive fluorescence microscopic method that is used for the detection of DNA damage at the individual cell level. Comet assay under neutral conditions allows the detection of DNA double-strand breaks, considered to the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method and its use for studying DNA damage and its repair in irradiated human lymphocytes. (author)

  19. Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

    Science.gov (United States)

    We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

  20. Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC–MS assay for delayed analyses of pharmacokinetic samples in rats

    OpenAIRE

    Jones, Jessica M.; Raleigh, Michael D.; Pentel, Paul R.; Harmon, Theresa M.; Keyler, Daniel E.; Remmel, Rory P.; Birnbaum, Angela K

    2012-01-01

    Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain ...

  1. A Selective Assay to Detect Chitin and Biologically Active Nano-Machineries for Chitin-Biosynthesis with Their Intrinsic Chitin-Synthase Molecules

    Directory of Open Access Journals (Sweden)

    Hildgund Schrempf

    2010-09-01

    Full Text Available A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein, has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.

  2. Antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay

    International Nuclear Information System (INIS)

    A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2/sup b/ x H-2/sup d/) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2/sup d/) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2/sup b/ serum and complement, in order to selectively destroy H-2/sup bd/ host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2/sup d/ P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant

  3. Controlling variation in the comet assay

    OpenAIRE

    Collins, Andrew R; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of ...

  4. Hormone assay

    International Nuclear Information System (INIS)

    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. 125I-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)

  5. Differential risk assessments from five hypoxia specific assays: The basis for biologically adapted individualized radiotherapy in advanced head and neck cancer patients

    DEFF Research Database (Denmark)

    Nordsmark, Marianne; Eriksen, Jesper Grau; Gebski, Val; Alsner, Jan; Horsman, Michael Robert; Overgaard, Jens

    2007-01-01

    PURPOSE: Hypoxia adversely relates with prognosis in human tumours. Five hypoxia specific predictive marker assays were compared and correlated with definitive radiotherapy. PATIENTS AND METHODS: Sixty-seven patients with advanced head and neck carcinomas were studied for pre-treatment plasma...... osteopontin measured by ELISA, tumour oxygenation status using pO(2) needle electrodes and tumour osteopontin, hypoxia inducible factor 1alpha (HIF-1alpha) and carboxyanhydrase 9 (CA9) by immunohistochemistry. The primary treatment was radiotherapy and the hypoxic radiosensitizer nimorazole. Loco......-regional tumour control was evaluated at 5 years. RESULTS: All five markers showed inter-tumour variability. Inter-marker correlations were inconsistent. Only plasma osteopontin inversely correlated with median tumour pO(2), (p=0.02, r=0.28) and CA9 correlated with HIF-1alpha (p<0.01, r=0.45). In Kaplan...

  6. Assay method and compositions

    International Nuclear Information System (INIS)

    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(3H)-methyl. The O-methylated (3H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin-3H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin-3H and raising the pH of the aqueous periodate phase from which O-methylated (3H) 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.)

  7. Chemical composition of the essential oil of Phlomis linearis Boiss. & Bal., and biological effects on the CAM-assay: a safety evaluation.

    Science.gov (United States)

    Demirci, Betül; Dadandi, Mehmet Y; Paper, Dietrich H; Franz, Gerhard; Başer, Kemal Hüsnü Can

    2003-01-01

    Phlomis linearis Boiss. & Bal. of the Lamiaceae family growing in central, east and southeast Anatolia is an endemic species for Turkey. The essential oil obtained from the aerial parts by hydro distillation was subsequently analyzed by GC/MS. The main components of the oil were found as beta-caryophyllene (24.2%), germacrene D (22.3%) and caryophyllene oxide (9.2%), among 49 identified compounds, representing 94.5% of the total essential oil. The overall biological activity of the essential oil (100 microg/pellet) was tested on the chorioallantoic membrane (CAM) of the fertilized hen's egg in order to examine the anti-angiogenic and anti-inflammatory activity. None of the tests showed pronounced activity, toxicity or irritation at the tested concentration. PMID:14713159

  8. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    Science.gov (United States)

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  9. Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats.

    Science.gov (United States)

    Jones, Jessica M; Raleigh, Michael D; Pentel, Paul R; Harmon, Theresa M; Keyler, Daniel E; Remmel, Rory P; Birnbaum, Angela K

    2013-02-23

    Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105% of the target value. Variability was 4.0-8.9% for all analytes within the range of 5-200 ng/mL for heroin, 5-1000 ng/mL for 6-MAM, and 10-200 ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4 mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80°C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3% over one week in rat plasma under these conditions, and by <1-4.7% in bovine serum or human plasma. PMID:23245263

  10. Radioreceptor assays

    International Nuclear Information System (INIS)

    Radioreceptor assay (RRA) is an analytical method using the specific interaction of some pharmaceuticals and endogenic substances (ligands) with specific receptors present in certin tissues of living organisms. RRA uses the principle of isotope dilution. The method is described in detail of the preparation of receptors, samples and radioligands, conditions of incubation, the separation of free and bound radioligand, and the mathematical evaluation of RRA. The sensitivity of RRA is measured in units to tens of pg. The specificity of RRA relates to a group of substances with similar pharmacological effect. RRA may be used for identifying neuroleptics, antidepressants, anxiolytics, ergot alkaloids, beta blockers, anticholinergic drugs, certain hormones and neuropeptides. (M.D.)

  11. Radiopharmaceutical assays

    International Nuclear Information System (INIS)

    Under the laws in force, radiopharmaceuticals for human use must be among other features, non-pyrogenous and non-toxic. For this reason pyrogenity and toxicity assays are carried out. Pharmacokinetic studies may also be necessary in some cases. Products currently made at the Radiopharmaceutical Center, new products designed for certification and raw materials used to manufacture the above, were tested. A total 342 pyrogenity and toxicity tests, and four pharmacokinetic studies were conducted in 1996. To determine pyrogenity, the temperature animals were measured following intravenous administration of radiopharmaceuticals concerned: sodium pertechnetate, colloidal gold and sodium orthoradiohippurate from current production; pharmaceutic components of several new products, i.e. technetium generator, fibrinogen and microspheres. A total 327 products were tested, 96 percent of which met the requirements. To determine toxicity, the probit method was used, consisting of the administration of radiopharmaceutical doses for seven straight days, and checking for lethal effects. An overall 15 tests were carried out and 80 percent of products tested were found certifiable. Pharmacokinetic tests consisted of tropism on target organs and biodistribution in several organs using the tomographic method. (author)

  12. Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

    Science.gov (United States)

    Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

    2016-04-01

    A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. PMID:26746109

  13. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Science.gov (United States)

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  14. The chemistry behind antioxidant capacity assays.

    Science.gov (United States)

    Huang, Dejian; Ou, Boxin; Prior, Ronald L

    2005-03-23

    This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays. PMID:15769103

  15. Evaluation and validation of a single-dilution potency assay based upon serology of vaccines containing diphtheria toxoid: analysis for consistency in production and testing at the laboratory of the Control of Biological Products of the RIVM

    NARCIS (Netherlands)

    Akkermans AM; Hendriksen CFM; Marsman FR; de Jong WH; van de Donk HJM

    1993-01-01

    A single-dilution assay can be a valid procedure to demonstrate that a product exceeds the minimal requirement given for potency provided that consistency in production and testing has been proven. Information is presented justifying the use of a single dilution assay based upon quantitative respon

  16. Synthesizing Novel Anthraquinone Natural Product-Like Compounds to Investigate Protein-Ligand Interactions in Both an in Vitro and in Vivo Assay: An Integrated Research-Based Third-Year Chemical Biology Laboratory Course

    Science.gov (United States)

    McKenzie, Nancy; McNulty, James; McLeod, David; McFadden, Meghan; Balachandran, Naresh

    2012-01-01

    A new undergraduate program in chemical biology was launched in 2008 to provide a unique learning experience for those students interested in this interdisciplinary science. An innovative undergraduate chemical biology laboratory course at the third-year level was developed as a key component of the curriculum. The laboratory course introduces…

  17. 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.

  18. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.

    2001-08-01

    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  19. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    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.

  20. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2012-05-15

    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.

  1. Tricyclic antidepressant radioreceptor assay

    International Nuclear Information System (INIS)

    A receptor assay for tricyclic antidepressants described here is based on the ability of these drugs to compete with [3H]-3-guinuclidnyl benzilate (3H-QNB) for binding to muscarinic cholinergic receptors in rat brain membranes. The assay is sensitive, in that it can detect, for example, 2ng/ml nortriptyline in plasma. Seven plasma samples from depressed patients treated with nortriptyline were assayed with the radioreceptor and gas liquid chromatographic methods, and the results from these two methods were almost identical. This assay should be used cautiously, if at all, in patients treated with other drugs that have potent anticholinergic effects. (Auth.)

  2. Plants Bioassays: Comet Assay on Higher Plants

    Czech Academy of Sciences Publication Activity Database

    Mukherjee, A.; Gichner, Tomáš

    Houston: Studium Press, 2009 - (Sampietro, D.; Narwal, S.), s. 97-108 ISBN 1-933699-42-6 R&D Projects: GA ČR GA521/05/0500 Institutional research plan: CEZ:AV0Z50380511 Keywords : Comet assay * DNA damage * Plants Subject RIV: EB - Genetics ; Molecular Biology

  3. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    OpenAIRE

    KHUSNUL YAQIN

    2006-01-01

    Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The appli...

  4. CPTAC Assay Portal: a repository of targeted proteomic assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  5. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  6. Biological Threats

    Science.gov (United States)

    ... Workplace Plans School Emergency Plans Main Content Biological Threats Biological agents are organisms or toxins that can ... for Disease Control and Prevention . Before a Biological Threat Unlike an explosion, a biological attack may or ...

  7. A study of the process control and hydrolytic characteristics in a thermophilic hydrogen fermentor fed with starch-rich kitchen waste by using molecular-biological methods and amylase assay

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yu-Hsuan; Li, Shiue-Lin; Chen, I.-Chieh; Tseng, I.-Cheng; Cheng, Sheng-Shung [Department of Environmental Engineering, National Cheng Kung University, No. 1, University Road, Tainan 701 (China)

    2010-12-15

    Starch-rich kitchen waste was chosen as the feedstock in this study, and a 3-L intermittent-continuous stirred tank reactor (I-CSTR) was established. Within 240 days, the maximum average hydrogen production rate of 2.2 L-H{sup 2} L{sup -1} day{sup -1} and the highest average hydrogen yield of 2.1 mmol-H{sub 2} g-COD{sup -1} were both observed in run 3-2, which was operated at an eight-day hydraulic retention time (HRT) and 39 g-COD L{sup -1} day{sup -1} of organic loading rate. According to the analyses of amylase and reducing sugar, the maximum average amylase activity was about 11 U mL{sup -1} in run 1, but the maximum solid carbohydrate hydrolysis rate was about 45% in run 3. Some Michaealis-Menton kinetic parameters, such as K{sub M} (17 g L{sup -1}) and the maximum activity (1.5 U mL{sup -1}) of the amylase were obtained. The best amylase reacting temperature was 55 C, and the best reacting pH was 4.4 tested with acetate buffer. Twenty-seven operational taxonomic units (OTUs) were selected from this reactor by using a cloning method. According to the data of terminal restricted fragment length polymorphism (T-RFLP) and amylase assay, the OTUs that were related to Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. were in direct proportion to the amylase activity. (author)

  8. Automated Protein Assay Using Flow Injection Analysis

    Science.gov (United States)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  9. 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.

  10. Rover waste assay system

    International Nuclear Information System (INIS)

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235U 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 137Cs 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

  11. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  12. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  13. Automated phantom assay system

    International Nuclear Information System (INIS)

    This paper describes an automated phantom assay system developed for assaying phantoms spiked with minute quantities of radionuclides. The system includes a computer-controlled linear-translation table that positions the phantom at exact distances from a spectrometer. A multichannel analyzer (MCA) interfaces with a computer to collect gamma spectral data. Signals transmitted between the controller and MCA synchronize data collection and phantom positioning. Measured data are then stored on disk for subsequent analysis. The automated system allows continuous unattended operation and ensures reproducible results

  14. Recent Advances in In Vivo Genotoxicity Testing: Prediction of Carcinogenic Potential Using Comet and Micronucleus Assay in Animal Models

    OpenAIRE

    Kang, Seung Hun; Kwon, Jee Young; Lee, Jong Kwon; Seo, Young Rok

    2013-01-01

    Genotoxic events have been known as crucial step in the initiation of cancer. To assess the risk of cancer, genotoxicity assays, including comet, micronucleus (MN), chromosomal aberration, bacterial reverse, and sister chromatid exchange assay, can be performed. Compared with in vitro genotoxicity assay, in vivo genotoxicity assay has been used to verify in vitro assay result and definitely provide biological significance for certain organs or cell types. The comet assay can detect DNA strand...

  15. 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...

  16. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  17. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    Directory of Open Access Journals (Sweden)

    KHUSNUL YAQIN

    2006-09-01

    Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

  18. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins

    2014-10-01

    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  19. Abstracts of the 29. annual meeting of the Brazilian Society on Biochemistry and Molecular Biology

    International Nuclear Information System (INIS)

    Several aspects concerning biochemistry and molecular biology of either animals (including man), plants and microorganisms are studied. Topics such as cell membrane structures (including receptors), enzymatic assays, biological pathways, structural chemical analysis, metabolism, biological functions are focused. The use of radiolabelled compounds (radioassay, radioenzymatic assay, radioreceptor assay and nuclear magnetic resonance are the most applied techniques

  20. Introduction to radiation biology

    International Nuclear Information System (INIS)

    This book is arranged in a logical sequence, starting from radiation physics and radiation chemistry, followed by molecular, subcellular and cellular effects and going on to the level of organism. Topics covered include applied radiobiology like modifiers of radiosensitivity, predictive assay, health physics, human genetics and radiopharmaceuticals. The topics covered are : 1. Radiation Physics, 2. Detection and Measurement of Radiation, 3. Radiation Chemistry, 4. DNA Damage and Repair, 5. Chromosomal Aberrations and Gene Mutations, 6. Cellular Radiobiology 7. Acute Radiation Effects, 8. Delayed Effects of Radiation, 9. Biological Basis of Radiotherapy, 10. Chemical Modifiers of Radiosensitivity, 11. Hyperthermia, 12. High LET Radiations in Cancer, Therapy, 13. Predictive Assays, 14. Radiation Effects on Embryos, 15. Human Radiation Genetics, 16. Radiolabelled Compounds in Biology and Medicine and 17. Radiological Health

  1. Dual isotope assays

    International Nuclear Information System (INIS)

    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)

  2. Automated uranium assays

    International Nuclear Information System (INIS)

    Precise, timely inventories of enriched uranium stocks are vital to help prevent the loss, theft, or diversion of this material for illicit use. A wet-chemistry analyzer has been developed at LLL to assist in these inventories by performing automated analyses of uranium samples from different stages in the nuclear fuel cycle. These assays offer improved accuracy, reduced costs, significant savings in manpower, and lower radiation exposure for personnel compared with present techniques

  3. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller;

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......-65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings...

  4. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    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.)

  5. Radon assay for SNO+

    International Nuclear Information System (INIS)

    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+

  6. 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+.

  7. Radioreceptor assay for epidermal growth factor

    International Nuclear Information System (INIS)

    An established cell line of human lung fibroblasts with a high number of surface receptors for mouse epidermal growth factor (mEGF) was used to deveop a simple and highly sensitive radioreceptor assay for EGF. 125I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF

  8. Canberra waste assay systems

    International Nuclear Information System (INIS)

    Canberra Industries, Inc. (USA) offers complete solutions and turnkey systems for qualifying and quantifying drums of radioactive waste. Different Waste Assay Systems are available for low-, medium-, and high activity barrels. Such systems scan the segments of the drums. The gamma-emitting nuclides present in the radioactive waste are identified from the spectra of the gamma-rays emitted and their activities are quantified. The spectrum of each segment of the waste container is analysed separately and the results are summed for total drum inventory reporting. The Waste Assay specific Canberra software is an easy-to-use menu driven program. It offers automatic procedures for routine work but interactive mode is also available to investigate particular cases. The way of analysis and the format and contents of the report can be customized. Additional Quality Control program module monitors system parameters. The measured and calculated values of each waste container are saved into a relational database. Canberra offers mathematical efficiency calibration services for different drum types, with different waste densities and different activities. This new theoretical approach of efficiency calibration eliminates the need of calibration drums, thus saving costs. For field applications Canberra developed a portable In-Situ Object Counting System. This system determines efficiency calibration for any kind of objects after the user enters its size and density, furthermore the type of shielding and the distance between the detector and the object. (author)

  9. Assays measuring tumoral radio-sensibility in 1998

    International Nuclear Information System (INIS)

    Various assays measuring tumor radiosensitivity were developed in the early eighties. These assays were based on cell cultures derived from tumor biopsies. In most studies there was no correlation between measured radiosensitivity and treatment outcome. New assays using new techniques (such as measurement of radiation-induced apoptosis, or fluorescence in situ hybridization) have been developed recently. Preliminary results are encouraging, but correlation with treatment outcome is expected to be difficult to demonstrate as multi-various clinical and biological parameters are involved in loco-regional control. (author)

  10. 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...... adapted to receive one or more replaceable solid support(s) (40) onto which chemical entities (41) are attached, said device comprising a base (1, 60, 80, 300, 400, 10, 70, 140, 20, 90, 120, 150, 30, 100), one or more inlet(s) (5), one or more outlet(s) (6). The base and the solid support (40) defines......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  11. Assay of oestrogen

    International Nuclear Information System (INIS)

    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 125I-labelled oestriol during the assay procedure and the steroid-protein bound 125I-labelled oestriol is precipitated along with the antibody-bound 125I-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.)

  12. PathogenMip Assay: A Multiplex Pathogen Detection Assay

    OpenAIRE

    Akhras, Michael S.; Sreedevi Thiyagarajan; Villablanca, Andrea C.; Davis, Ronald W; Pål Nyrén; Nader Pourmand

    2007-01-01

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The ...

  13. DNA-PK assay

    Science.gov (United States)

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  14. Development of radiation biological dosimetry

    International Nuclear Information System (INIS)

    Up until now, only a few methods have been developed for radiation biological dosimetry such as conventional chromosome aberration and micronucleus in peripheral blood cell. However, because these methods not only can be estimated by the expert, but also have a little limitation due to need high technique and many times in the case of radiation accident, it is very difficult to evaluate the absorbed dose of victims. Therefore, we should develop effective, easy, simple and rapid biodosimetry and its guideline (triage) to be able to be treated the victims as fast as possible. We established the premature chromosome condensation assay and apoptotic fragment assay which was the significant relationship between dose and cell damages to evaluate the irradiation dose as correct and rapid as possible using lymphocytes and crypt cells, and compared with conventional chromosome aberration assay and micronuclei assay

  15. Development of radiation biological dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Chul Koo; Kim, Tae Hwan; Lee, Yun Sil; Son, Young Sook; Kim, Soo Kwan; Jang, Won Suk; Le, Sun Joo; Jee, Young Heun; Jung, Woo Jung

    1999-04-01

    Up until now, only a few methods have been developed for radiation biological dosimetry such as conventional chromosome aberration and micronucleus in peripheral blood cell. However, because these methods not only can be estimated by the expert, but also have a little limitation due to need high technique and many times in the case of radiation accident, it is very difficult to evaluate the absorbed dose of victims. Therefore, we should develop effective, easy, simple and rapid biodosimetry and its guideline (triage) to be able to be treated the victims as fast as possible. We established the premature chromosome condensation assay and apoptotic fragment assay which was the significant relationship between dose and cell damages to evaluate the irradiation dose as correct and rapid as possible using lymphocytes and crypt cells, and compared with conventional chromosome aberration assay and micronuclei assay.

  16. DNA repair in plants studied by comet assay

    Czech Academy of Sciences Publication Activity Database

    Angelis, Karel; Kozák, Jaroslav; Vágnerová, Radka; Holá, Marcela

    Lausanne: ICAW, 2015 - (Allison, D.) ISSN 1664-8021. [International Comet Assay Workshop /11./. Antwerpen (BE), 01.09.2015-04.09.2015] R&D Projects: GA ČR GA13-06595S Institutional support: RVO:61389030 ; RVO:61388963 Keywords : physcomitrella patens * Arabidopsis * alternative DSB repair Subject RIV: EB - Genetics ; Molecular Biology

  17. Quality control of antibodies for assay development.

    Science.gov (United States)

    Schumacher, Sarah; Seitz, Harald

    2016-09-25

    Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test. PMID:26873787

  18. A sensitive competitive binding assay for exogenous and endogenous heparins

    International Nuclear Information System (INIS)

    A new type of assay for heparins has been devised, in which the test material competes with 125I-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

  19. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Science.gov (United States)

    Elango, Chinnasamy

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  20. Biology Notes.

    Science.gov (United States)

    School Science Review, 1982

    1982-01-01

    Presents procedures, exercises, demonstrations, and information on a variety of biology topics including labeling systems, biological indicators of stream pollution, growth of lichens, reproductive capacity of bulbous buttercups, a straw balance to measure transpiration, interaction of fungi, osmosis, and nitrogen fixation and crop production. (DC)

  1. Use of a Brine Shrimp Assay to Study Herbal Teas in the Classroom.

    Science.gov (United States)

    Opler, Annette; Mizell, Rebecca; Robert, Alexander; Cervantes-Cervantes, Miguel; Kincaid, Dwight; Kennelly, Edward J.

    2002-01-01

    Introduces a brine shrimp assay to demonstrate the effects of the biological activity of herbal remedies. Describes two protocols, one using aqueous extracts and the other using methanol extracts. (Contains 21 references.) (YDS)

  2. From Antenna to Assay

    Science.gov (United States)

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  3. New Application of the Comet Assay: Chromosome–Comet Assay

    OpenAIRE

    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 use...

  4. Evidence of ATP assay as an appropriate alternative of MTT assay for cytotoxicity of secondary effluents from WWTPs.

    Science.gov (United States)

    Yang, Yang; Lu, Yun; Wu, Qian-Yuan; Hu, Hong-Ying; Chen, Ying-Hua; Liu, Wan-Li

    2015-12-01

    Biological tests are effective and comprehensive methods to assess toxicity of environmental pollutants to ensure the safety of reclaimed water. In this study, the canonical MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cytotoxicity of dissolved organic matters (DOMs) of secondary effluents from wastewater treatment plants (WWTPs). It was surprising that most concentrated DOMs treated HepG2 cells yielded much higher signal compared with vehicle control regardless of difference of treatment technologies and seasons. However, there was actually no obvious enhancement of the cell proliferation by microscopy. In order to find out potential reason for the discrepancy, another three assays were performed. The results of ATP assay and flow cytometry showed expected toxicity, which was consistent with microscopy and previous studies, while DNA assay did not exhibit apparent change in treated cells. The possible mechanisms of abnormal MTT signal could be that some materials in secondary effluents isolated by solid extraction with HLB resin directly reacted with MTT and/or enhanced the activity of mitochondrial dehydrogenase. Therefore, the MTT assay is not suitable to assess cytotoxicity of complex mixtures such as secondary effluents, while ATP assay is an optional sensitive method. This study also suggests the importance of choosing both suitable extraction methods and detection assays for toxicity evaluation of component-unknown environmental samples. PMID:26410194

  5. [Biologics and mycobacterial diseases].

    Science.gov (United States)

    Tsuyuguchi, Kazunari; Matsumoto, Tomoshige

    2013-03-01

    Various biologics such as TNF-alpha inhibitor or IL-6 inhibitor are now widely used for treatment of rheumatoid arthritis. Many reports suggested that one of the major issues is high risk of developing tuberculosis (TB) associated with using these agents, which is especially important in Japan where tuberculosis still remains endemic. Another concern is the risk of development of nontuberculous mycobacterial (NTM) diseases and we have only scanty information about it. The purpose of this symposium is to elucidate the role of biologics in the development of mycobacterial diseases and to establish the strategy to control them. First, Dr. Tohma showed the epidemiologic data of TB risks associated with using biologics calculated from the clinical database on National Database of Rheumatic Diseases by iR-net in Japan. He estimated TB risks in rheumatoid arthritis (RA) patients to be about four times higher compared with general populations and to become even higher by using biologics. He also pointed out a low rate of implementation of QuantiFERON test (QFT) as screening test for TB infection. Next, Dr. Tokuda discussed the issue of NTM disease associated with using biologics. He suggested the airway disease in RA patients might play some role in the development of NTM disease, which may conversely lead to overdiagnosis of NTM disease in RA patients. He suggested that NTM disease should not be uniformly considered a contraindication to treatment with biologics, considering from the results of recent multicenter study showing relatively favorable outcome of NTM patients receiving biologics. Patients with latent tuberculosis infection (LTBI) should receive LTBI treatment before starting biologics. Dr. Kato, a chairperson of the Prevention Committee of the Japanese Society for Tuberculosis, proposed a new LTBI guideline including active implementation of LTBI treatment, introducing interferon gamma release assay, and appropriate selection of persons at high risk for

  6. Quantum Biology

    Directory of Open Access Journals (Sweden)

    Alessandro Sergi

    2009-06-01

    Full Text Available A critical assessment of the recent developmentsof molecular biology is presented.The thesis that they do not lead to a conceptualunderstanding of life and biological systems is defended.Maturana and Varela's concept of autopoiesis is briefly sketchedand its logical circularity avoided by postulatingthe existence of underlying living processes,entailing amplification from the microscopic to the macroscopic scale,with increasing complexity in the passage from one scale to the other.Following such a line of thought, the currently accepted model of condensed matter, which is based on electrostatics and short-ranged forces,is criticized. It is suggested that the correct interpretationof quantum dispersion forces (van der Waals, hydrogen bonding, and so onas quantum coherence effects hints at the necessity of includinglong-ranged forces (or mechanisms for them incondensed matter theories of biological processes.Some quantum effects in biology are reviewedand quantum mechanics is acknowledged as conceptually important to biology since withoutit most (if not all of the biological structuresand signalling processes would not even exist. Moreover, it is suggested that long-rangequantum coherent dynamics, including electron polarization,may be invoked to explain signal amplificationprocess in biological systems in general.

  7. Abstracts of the 30. Annual meeting of the Brazilian Society on Biochemistry and Molecular Biology

    International Nuclear Information System (INIS)

    Several aspects concerning biochemistry and molecular biology of either animals, plants and microorganisms are studied. Topics such as cell membrane structures (including receptors), enzymatic assays, biological pathways, structural chemical analysis, metabolism, biological functions are focused. The use of radiolabelled compounds (radioassay, radioreceptor assay) and nuclear magnetic resonance are the most applied techniques

  8. Calorimetric assay of minor actinides

    Energy Technology Data Exchange (ETDEWEB)

    Rudy, C.; Bracken, D.; Cremers, T.; Foster, L.A.; Ensslin, N.

    1996-12-31

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques.

  9. Calorimetric assay of minor actinides

    International Nuclear Information System (INIS)

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques

  10. Localized irradiations, evaluation through 'Comet Assay'

    International Nuclear Information System (INIS)

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources of Cobalt 60, Cesium 137 or Iridium 192 at work placed for industrial gammagraphy and other radiation sources. Severe skin reaction may developed at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models 'Contaminated Poisson' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. There are also different biophysical techniques that can give response in localized irradiations. Biological dosimetry is a necessary complement to physical and clinical dosimetries. Thus, there is increasing interest in the assessment of biological markers that permit the detection of radiation induced damage in the localized irradiations. The 'Comet Assay' (single cell gel electrophoresis) is a sensitive, rapid and relatively inexpensive method for measuring DNA damage in individual cells. Single cells are embedded in agarose on microscope slides, lysed to remove the majority of the proteins, electrophoresed, then stained with ethidium bromide in order to visualize the DNA. When visualized using a fluorescent microscope, DNA of undamaged cells appears as a spherical mass occupying the cavity formed by the lysed cell. Following radiation damage, the smaller the fragment size and the grater the number of fragments of DNA, the grater the percentage of DNA that it is able to migrate in an electric field, forming a comet image. The assay can be performed under alkaline conditions to examine DNA single strand breaks (SSBs), or in non denaturing (neutral) conditions to measure double strand breaks (DSBs) in individual

  11. Quantum Biology

    CERN Document Server

    Sergi, Alessandro

    2009-01-01

    A critical assessment of the recent developments of molecular biology is presented. The thesis that they do not lead to a conceptual understanding of life and biological systems is defended. Maturana and Varela's concept of autopoiesis is briefly sketched and its logical circularity avoided by postulating the existence of underlying {\\it living processes}, entailing amplification from the microscopic to the macroscopic scale, with increasing complexity in the passage from one scale to the other. Following such a line of thought, the currently accepted model of condensed matter, which is based on electrostatics and short-ranged forces, is criticized. It is suggested that the correct interpretation of quantum dispersion forces (van der Waals, hydrogen bonding, and so on) as quantum coherence effects hints at the necessity of including long-ranged forces (or mechanisms for them) in condensed matter theories of biological processes. Some quantum effects in biology are reviewed and quantum mechanics is acknowledge...

  12. Third workshop on heavy charged particles in biology and medicine

    International Nuclear Information System (INIS)

    The book of abstracts contains 67 papers presented at the workshop. Main topics are: Physics, chemistry, DNA, cell biology, cellular and molecular repair, space biology, tumor and tissue biology, predictive assays, cancer therapy, and new projects. Separate entries in the database are prepared for all of these papers. (MG)

  13. A Specific Saturation Assay Technique for Serum Triiodothyronine

    International Nuclear Information System (INIS)

    Triiodothyronine (T3) exists in plasma at a concentration of approximately 0.3 μg/100 ml (representing 3-5% of the total thyroid hormone present); nevertheless it accounts for more than 20% of the secretion of hormonal iodine. Moreover, because of the relative biological potencies of thyroxine (T4) and T3, the latter compound represents roughly half the biologically effective hormone secreted by the thyroid gland. Serum T3 may, in principle, be assayed by saturation assay techniques relying on serum thyroxine binding protein as the saturable binding reagent, and at least one such method has been published. However, in our own thyroxine assay procedure T4 is approximately eight times more reactive than T3; this implies that, to measure serum T3 to an accuracy of better than 10% it is essential that not more than 0.05% of serum T4 should remain as a contaminant of T3 extracts. Observations of this kind raise doubts regarding the accuracy of published 13 estimates relying on T. B. P. /saturation assay techniques, and underline the desirability of an assay method designed to discriminate more effectively against T4. We have succeeded in raising specific antibodies directed against T3 which are essentially non-reaptive with T4. Using these as the saturable reagent, we have devised an assay technique sensitive to approximately 50 pg T3/ml of incubation mixture. Such sensitivity enables normal serum T3 values to be readily measured using starting volumes of blood of about 1-2 ml, and the physiological role of this hormone in health and disease to be elucidated. The application of the method to the assay of the iodinated amino acids MIT and DIT and to other similar compounds of a normally non- antigenic nature is also discussed. (author)

  14. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed. PMID:16834538

  15. Advancement in biochemical assays in andrology

    Institute of Scientific and Technical Information of China (English)

    Wolf-BernhardSchill; RaftHenkel

    1999-01-01

    Determination of maikers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variafion characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability .Biochemical parameters may be used in clinical practice to evaluate the sperm fertitizing capacity (acrosin, aniline blue,ROS), to characterize male accessory sex gland secretinns (fructose, a-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS). (As/an J Androl 1999 Jun; 1: 45-51)

  16. 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.

  17. A radioreceptor assay for pharmaceutical preparations of insulin

    International Nuclear Information System (INIS)

    A radioreceptor assay has been developed that is suitable for the measurement of the potency of crystalline insulin and pharmaceutical insulin formulations. It utilizes the well characterized and widely available IM-9 human lymphocyte cell line as the source of receptor. Bovine, porcine and human crystalline and formulated insulins have been assayed against the 4th International and European Standards for Insulin and the potencies compared with those obtained by the mouse blood glucose method. Results with bovine insulin were in full correspondence with the in vivo results. Porcine and human insulins were 15-20% more potent by the radioreceptor assay than by the in vivo method when the mixed bovine and porcine insulin 4th International and European Standards were used, but were equivalent when compared with like materials. Average 95% confidence limits for formulated insulins in two assays were +-6% of the mean. The coefficient of variation on repeated assay of the same sample was 3.8%. The three dose parallel line radioreceptor assay with appropriate species standards is a candidate biological test capable of international adoption as an alternative to in vivo animal testing of insulin. (author)

  18. Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

    Directory of Open Access Journals (Sweden)

    Kazutoyo Miura

    Full Text Available Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA. The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH Harmonised Tripartite Guideline Q2(R1 details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability. We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in

  19. Biological Oceanography

    Science.gov (United States)

    Abbott, M. R.

    1984-01-01

    Within the framework of global biogeochemical cycles and ocean productivity, there are two areas that will be of particular interest to biological oceanography in the 1990s. The first is the mapping in space time of the biomass and productivity of phytoplankton in the world ocean. The second area is the coupling of biological and physical processes as it affects the distribution and growth rate of phytoplankton biomass. Certainly other areas will be of interest to biological oceanographers, but these two areas are amenable to observations from satellites. Temporal and spatial variability is a regular feature of marine ecosystems. The temporal and spatial variability of phytoplankton biomass and productivity which is ubiquitous at all time and space scales in the ocean must be characterized. Remote sensing from satellites addresses these problems with global observations of mesocale (2 to 20 days, 10 to 200 km) features over a long period of time.

  20. Biological preconcentrator

    Science.gov (United States)

    Manginell, Ronald P.; Bunker, Bruce C.; Huber, Dale L.

    2008-09-09

    A biological preconcentrator comprises a stimulus-responsive active film on a stimulus-producing microfabricated platform. The active film can comprise a thermally switchable polymer film that can be used to selectively absorb and desorb proteins from a protein mixture. The biological microfabricated platform can comprise a thin membrane suspended on a substrate with an integral resistive heater and/or thermoelectric cooler for thermal switching of the active polymer film disposed on the membrane. The active polymer film can comprise hydrogel-like polymers, such as poly(ethylene oxide) or poly(n-isopropylacrylamide), that are tethered to the membrane. The biological preconcentrator can be fabricated with semiconductor materials and technologies.

  1. Marine Biology

    Science.gov (United States)

    Dewees, Christopher M.; Hooper, Jon K.

    1976-01-01

    A variety of informational material for a course in marine biology or oceanology at the secondary level is presented. Among the topics discussed are: food webs and pyramids, planktonic blooms, marine life, plankton nets, food chains, phytoplankton, zooplankton, larval plankton and filter feeders. (BT)

  2. Biology Notes.

    Science.gov (United States)

    School Science Review, 1983

    1983-01-01

    Describes laboratory procedures, demonstrations, and classroom activities/materials, including water relation exercise on auxin-treated artichoke tuber tissue; aerobic respiration in yeast; an improved potometer; use of mobiles in biological classification, and experiments on powdery mildews and banana polyphenol oxidase. Includes reading lists…

  3. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    OpenAIRE

    Wigdal, Susan S.; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V.; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily...

  4. Solid phase group specific absorbants in assays for glycoproteins

    International Nuclear Information System (INIS)

    The focus of this paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by use of the solid phase carbohydrate specific adsorbant concanavalin-A. Puriffication of glycoprotein radioligand after labelling by the chloramine-T method is readily accomplished using a small column of agarose bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose bound concanavalin-A is used to extract and concentrate the glycoproteins from various biologic samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5 fold by using concanavalin-A concentrates of 1.5 ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biologic samples, and for separation of glycoproteins from various interfering factors contained in biologic samples prior to radioligand or radioenzyme assay. (orig.)

  5. The comet assay – from toy to tool

    Directory of Open Access Journals (Sweden)

    Guenter Speit

    2015-04-01

    Full Text Available The comet assay is nowadays the most common method for measuring DNA damage and repair in single cells. It is based on the microelectrophoretic study published by Ostling and Johanson (1984 and was developed by Singh and coworkers (1988 to a versatile technique for quantitation of low levels of DNA damage in individual cells. This alkaline version still is the basis for the triumphant success of the comet assay in basic research into mechanisms of DNA damage and DNA repair, genotoxicity testing, ecotoxicology and human biomonitoring. Important technical improvements (e.g., the use of precoated slides, introduction of image analysis, high throughput methods, automated scoring systems made the assay more robust and more efficient. Modifications of the standard protocol provide more specific information on the type and biological significance of the damage studied. The introduction of lesion-specific endonucleases allowed the characterization of oxidative base damage, alkylation damage and UV-induced pyrimidine dimers. The combination with fluorescence in situ hybridization (FISH made it possible to identify DNA of particular chromosome regions and measure effects of damage and repair in particular genes. The comet assay is being increasingly used in genotoxicity testing. In particular, the in vivo comet assay has become a component of some genotoxicity test strategies and generally accepted test protocols have evolved over the years. A large international collaborative trial sponsored by the Japanese Center for the Validation of Alternative Methods (JaCVAM was recently completed and an OECD test guideline was approved. The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. However, there are still problems in comparing results from different laboratories and there is need for reducing inter-laboratory variation and identification of standard

  6. 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.

  7. Bacterial mutagenicity assays: test methods.

    Science.gov (United States)

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  8. Assays for B lymphocyte function.

    Science.gov (United States)

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  9. Literature classification for semi-automated updating of biological knowledgebases

    OpenAIRE

    Olsen, Lars Rønn; Kudahl, Ulrich Johan; Winther, Ole; Brusic, Vladimir

    2013-01-01

    Background: As the output of biological assays increase in resolution and volume, the body of specialized biological data, such as functional annotations of gene and protein sequences, enables extraction of higher-level knowledge needed for practical application in bioinformatics. Whereas common types of biological data, such as sequence data, are extensively stored in biological databases, functional annotations, such as immunological epitopes, are found primarily in semi-structured formats ...

  10. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S

    OpenAIRE

    Pais De Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-01-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and ...

  11. Mesoscopic biology

    Indian Academy of Sciences (India)

    G V Shivashankar

    2002-02-01

    In this paper we present a qualitative outlook of mesoscopic biology where the typical length scale is of the order of nanometers and the energy scales comparable to thermal energy. Novel biomolecular machines, governed by coded information at the level of DNA and proteins, operate at these length scales in biological systems. In recent years advances in technology have led to the study of some of the design principles of these machines; in particular at the level of an individual molecule. For example, the forces that operate in molecular interactions, the stochasticity involved in these interactions and their spatio-temporal dynamics are beginning to be explored. Understanding such design principles is opening new possibilities in mesoscopic physics with potential applications.

  12. The Dark Matter of Biology.

    Science.gov (United States)

    Ross, Jennifer L

    2016-09-01

    The inside of the cell is full of important, yet invisible species of molecules and proteins that interact weakly but couple together to have huge and important effects in many biological processes. Such "dark matter" inside cells remains mostly hidden, because our tools were developed to investigate strongly interacting species and folded proteins. Example dark-matter species include intrinsically disordered proteins, posttranslational states, ion species, and rare, transient, and weak interactions undetectable by biochemical assays. The dark matter of biology is likely to have multiple, vital roles to regulate signaling, rates of reactions, water structure and viscosity, crowding, and other cellular activities. We need to create new tools to image, detect, and understand these dark-matter species if we are to truly understand fundamental physical principles of biology. PMID:27602719

  13. Biological programming

    OpenAIRE

    Ramsden, Jeremy J.; Bándi, Gergely

    2010-01-01

    Biology offers a tremendous set of concepts that are potentially very powerfully usable for the software engineer, but they have been barely exploited hitherto. In this position paper we propose a fresh attempt to create the building blocks of a programming technology that could be as successful as life. A key guiding principle is to develop and make use of unambiguous definitions of the essential features of life.

  14. Biological radioprotector

    International Nuclear Information System (INIS)

    According to the patent description, the biological radioprotector is deuterium depleted water, DDW, produced by vacuum distillation with an isotopic content lower than natural value. It appears as such or in a mixture with natural water and carbon dioxide. It can be used for preventing and reducing the ionizing radiation effects upon humans or animal organisms, exposed therapeutically, professionally or accidentally to radiation. The most significant advantage of using DDW as biological radioprotector results from its way of administration. Indeed no one of the radioprotectors currently used today can be orally administrated, what reduces the patients' compliance to prophylactic administrations. The biological radioprotector is an unnoxious product obtained from natural water, which can be administrated as food additive instead of drinking water. Dose modification factor is according to initial estimates around 1.9, what is a remarkable feature when one takes into account that the product is toxicity-free and side effect-free and can be administrated prophylactically as a food additive. A net radioprotective action of the deuterium depletion was evidenced experimentally in laboratory animals (rats) hydrated with DDW of 30 ppm D/(D+H) concentration as compared with normally hydrated control animals. Knowing the effects of irradiation and mechanisms of the acute radiation disease as well as the effects of administration of radiomimetic chemicals upon cellular lines of fast cell division, it appears that the effects of administrating DDW result from stimulation of the immunity system. In conclusion, the biological radioprotector DDW presents the following advantages: - it is obtained from natural products without toxicity; - it is easy to be administrated as a food additive, replacing the drinking water; - besides radioprotective effects, the product has also immunostimulative and antitumoral effects

  15. Marine biology

    International Nuclear Information System (INIS)

    This book discusses both taxonomic and ecological topics on marine biology. Full coverage of marine organisms of all five kingdoms is provided, along with interesting and thorough discussion of all major marine habitats. Organization into six major parts allows flexibility. It also provides insight into important topics such as disposal of nuclear waste at sea, the idea that life began on the ocean floor, and how whales, krill, and people interact. A full-color photo chapter reviews questions, and exercises. The contents are: an overview marine biology: fundamental concepts/investigating life in the ocean; the physical ocean, the ocean floor, the nature of water, the nature and motion of ocean water; general ecology, conditions for life in the sea, biological productivity and energy transfer; marine organisms; monera, protista, mycota and metaphyta; the smaller marine animals, the large animals marine habitats, the intertidal zone/benthos of the continental shelf, the photic zone, the deep ocean, the ocean under stress, marine pollution, appendix a: the metric system and conversion factors/ appendix b: prefixes and suffixes/ appendix c: taxonomic classification of common marine organisms, and glossary, and index

  16. Marine biology

    Energy Technology Data Exchange (ETDEWEB)

    Thurman, H.V.; Webber, H.H.

    1984-01-01

    This book discusses both taxonomic and ecological topics on marine biology. Full coverage of marine organisms of all five kingdoms is provided, along with interesting and thorough discussion of all major marine habitats. Organization into six major parts allows flexibility. It also provides insight into important topics such as disposal of nuclear waste at sea, the idea that life began on the ocean floor, and how whales, krill, and people interact. A full-color photo chapter reviews questions, and exercises. The contents are: an overview marine biology: fundamental concepts/investigating life in the ocean; the physical ocean, the ocean floor, the nature of water, the nature and motion of ocean water; general ecology, conditions for life in the sea, biological productivity and energy transfer; marine organisms; monera, protista, mycota and metaphyta; the smaller marine animals, the large animals marine habitats, the intertidal zone/benthos of the continental shelf, the photic zone, the deep ocean, the ocean under stress, marine pollution, appendix a: the metric system and conversion factors/ appendix b: prefixes and suffixes/ appendix c: taxonomic classification of common marine organisms, and glossary, and index.

  17. Further approaches to biological indicators of radiation injury

    International Nuclear Information System (INIS)

    Despite of the decades-long investigations, the search for proper biological indicator of radiation injuries did not result in techniques fulfilling all the requirements. So far, the most reliable assay is the dicentric chromosome aberration analysis. New developments have been made recently on a cytogenetic technique, the micronucleus assay, and for local injuries on the application of thermography

  18. 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. PMID:26608293

  19. Nondestructive assay of sale materials

    International Nuclear Information System (INIS)

    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

  20. Formalization, annotation and analysis of diverse drug and probe screening assay datasets using the BioAssay Ontology (BAO.

    Directory of Open Access Journals (Sweden)

    Uma D Vempati

    Full Text Available Huge amounts of high-throughput screening (HTS data for probe and drug development projects are being generated in the pharmaceutical industry and more recently in the public sector. The resulting experimental datasets are increasingly being disseminated via publically accessible repositories. However, existing repositories lack sufficient metadata to describe the experiments and are often difficult to navigate by non-experts. The lack of standardized descriptions and semantics of biological assays and screening results hinder targeted data retrieval, integration, aggregation, and analyses across different HTS datasets, for example to infer mechanisms of action of small molecule perturbagens. To address these limitations, we created the BioAssay Ontology (BAO. BAO has been developed with a focus on data integration and analysis enabling the classification of assays and screening results by concepts that relate to format, assay design, technology, target, and endpoint. Previously, we reported on the higher-level design of BAO and on the semantic querying capabilities offered by the ontology-indexed triple store of HTS data. Here, we report on our detailed design, annotation pipeline, substantially enlarged annotation knowledgebase, and analysis results. We used BAO to annotate assays from the largest public HTS data repository, PubChem, and demonstrate its utility to categorize and analyze diverse HTS results from numerous experiments. BAO is publically available from the NCBO BioPortal at http://bioportal.bioontology.org/ontologies/1533. BAO provides controlled terminology and uniform scope to report probe and drug discovery screening assays and results. BAO leverages description logic to formalize the domain knowledge and facilitate the semantic integration with diverse other resources. As a consequence, BAO offers the potential to infer new knowledge from a corpus of assay results, for example molecular mechanisms of action of perturbagens.

  1. Assay of vitamin B12

    International Nuclear Information System (INIS)

    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)

  2. Biological Databases

    Directory of Open Access Journals (Sweden)

    Kaviena Baskaran

    2013-12-01

    Full Text Available Biology has entered a new era in distributing information based on database and this collection of database become primary in publishing information. This data publishing is done through Internet Gopher where information resources easy and affordable offered by powerful research tools. The more important thing now is the development of high quality and professionally operated electronic data publishing sites. To enhance the service and appropriate editorial and policies for electronic data publishing has been established and editors of article shoulder the responsibility.

  3. Localized irradiations, Evaluation through ''comet assay''

    International Nuclear Information System (INIS)

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources Co60, Cs137 or Ir192 at workplaces for industrial gammagraphy. Severe skin reaction may develop at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models ''Contaminated Poisson'' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. This give an indication of the proportion of haemopoietic stem cell compartment involved in the overexposure. There are also different biophysical techniques that can give responses in biological dosimetry. The ''Comet Assay'' (single cell gel electrophoresis) is a sensitive and rapid method for DNA strand break detection in individual cells. The advantages of the technique include: collection of data at the level of individual cell; the need for small numbers of cells per sample; its sensitivity for detecting DNA damage and that virtually any eukaryote cell population is amenable to analysis. The objective of this work is to apply ''Comet Assay'' method to evaluate the effect of radiation on skin and subcutaneous tissues, differentiating irradiated from unirradiated body areas. It could provide a useful tool to estimate the extension and the dose in the irradiated region, contributing with the current techniques. In this first study, we evaluate the alkaline comet assay as a method for detection of DNA radiation induced damage in keratinocytes from primary culture obtained from full thickness skin biopsies of patients requiring grafts. Skin and, particularly, keratinocytes were selected as an appropriate cellular system due to: Skin, the first barrier against

  4. Arrays in biological and chemical analysis

    DEFF Research Database (Denmark)

    Christensen, Claus Bo Vöge

    2002-01-01

    Recently a dramatic change has happened for biological and biochemical analysis. Originally developed as an academic massive parallel screening tool, industry has caught the idea as well of performing all kinds of assays in the new format of microarrays. From food manufacturers over water supply...

  5. Biological biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Jorge-Herrero, E. [Servicio de Cirugia Experimental. Clinica Puerta de Hierro, Madrid (Spain)

    1997-05-01

    There are a number of situations in which substances of biological origin are employed as biomaterials. Most of them are macromolecules derived from isolated connective tissue or the connective tissue itself in membrane form, in both cases, the tissue can be used in its natural form or be chemically treated. In other cases, certain blood vessels can be chemically pretreated and used as vascular prostheses. Proteins such as albumin, collagen and fibrinogen are employed to coat vascular prostheses. Certain polysaccharides have also been tested for use in controlled drug release systems. Likewise, a number of tissues, such as dura mater, bovine pericardium, procine valves and human valves, are used in the preparation of cardiac prostheses. We also use veins from animals or humans in arterial replacement. In none of these cases are the tissues employed dissimilar to the native tissues as they have been chemically modified, becoming a new bio material with different physical and biochemical properties. In short, we find that natural products are being utilized as biomaterials and must be considered as such; thus, it is necessary to study both their chemicobiological and physicomechanical properties. In the present report, we review the current applications, problems and future prospects of some of these biological biomaterials. (Author) 84 refs.

  6. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    OpenAIRE

    Carina Ladeira; Susana Viegas; Gomes, Manuel C.

    2015-01-01

    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  7. Development and evaluation of real-time PCR assays for bloodmeal identification in Culicoides midges.

    Science.gov (United States)

    VAN DER Saag, M R; Gu, X; Ward, M P; Kirkland, P D

    2016-06-01

    Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host-feeding preferences, is limited. Identification of host-feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi-quantitative real-time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan-reactive species group and seven species-specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood-fed midges of various species from several geographic locations in Australia. PMID:26854008

  8. Comet assay in higher plants

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš

    Katowice, 2003, s. 123-130. [Bioassays in Plant Cells. Katowice (PL), 15.05.2003] R&D Projects: GA ČR GA521/02/0400; GA MŠk LN00B030 Institutional research plan: CEZ:AV0Z5038910 Keywords : DNA damage * genotoxicity Subject RIV: EB - Genetics ; Molecular Biology

  9. The comet assay in testing the potential genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Amaya Azqueta

    2015-06-01

    Full Text Available In the last two decades the production and use of nanomaterials (NMs has impressively increased. Their small size, given a mass equal to that of the corresponding bulk material, implies an increase in the surface area and consequently in the number of atoms that can be reactive. They possess different physical, chemical and biological properties compared to bulk materials of the same composition, which makes them very interesting and valuable for many different applications in technology, energy, construction, electronics, agriculture, optics, paints, textiles, food, cosmetics, medicine... Toxicological assessment of NMs is crucial; the same properties that make them interesting also make them potentially harmful for health and the environment. However, the term NM covers many different kinds of particle , and so there is no simple, standard approach to assessing their toxicity. NMs can enter the cell, interact with cell components and even penetrate the nucleus and interfere with the genetic material. Among the different branches of toxicology, genotoxicity is a main area of concern since it is closely related with the carcinogenic potential of compounds. The Organisation for Economic Co-operation and Development (OECD has published internationally agreed in vitro and in vivo validated test methods to evaluate different genotoxic endpoints of chemicals, including chromosome and gene mutations, and DNA breaks. However not all the assays are suitable to study the genotoxic potential of NMs as has been shown by the OECD Working Party on Manufactured Nanomaterials (WPMN. Moreover, alterations to DNA bases, which are precursors to mutations and of great importance in elucidating the mechanism of action of NMs, are not covered by the OECD guidelines. The in vivo standard comet assay (which measures DNA breaks and alkali-labile sites was included in the OECD assays battery in September 2014 while the in vitro standard comet assay is currently under

  10. A protein-protein binding assay using coated microtitre plates: increased throughput, reproducibility and speed compared to bead-based assays.

    Science.gov (United States)

    Craig, Tim J; Ciufo, Leonora F; Morgan, Alan

    2004-07-30

    Protein-protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein-protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein-protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research. PMID:15236910

  11. Automated assay optimization with integrated statistics and smart robotics.

    Science.gov (United States)

    Taylor, P B; Stewart, F P; Dunnington, D J; Quinn, S T; Schulz, C K; Vaidya, K S; Kurali, E; Lane, T R; Xiong, W C; Sherrill, T P; Snider, J S; Terpstra, N D; Hertzberg, R P

    2000-08-01

    The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands. PMID:10992042

  12. Biological effects

    International Nuclear Information System (INIS)

    Following an introduction into the field of cellular radiation effect considering the most important experimental results, the biological significance of the colony formation ability is brought out. The inactivation concept of stem cells does not only prove to be good, according to the present results, in the interpretation of the pathogenesis of acute radiation effects on moult tissue, it also enables chronicle radiation injuries to be interpreted through changes in the fibrous part of the organs. Radiation therapy of tumours can also be explained to a large extent by the radiation effect on the unlimited reproductiveness of tumour cells. The more or less similar dose effect curves for healthy and tumour tissue in practice lead to intermittent irradiation. The dependence of the intermittent doses and intervals on factors such as Elkind recovery, synchronisation, redistribution, reoxygenation, repopulation and regeneration are reviewed. (ORU/LH)

  13. Biosensors and Bio-Bar Code Assays Based on Biofunctionalized Magnetic Microbeads

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Cloarec

    2007-04-01

    Full Text Available This review paper reports the applications of magnetic microbeads in biosensors and bio-bar code assays. Affinity biosensors are presented through different types of transducing systems: electrochemical, piezo electric or magnetic ones, applied to immunodetection and genodetection. Enzymatic biosensors are based on biofunctionalization through magnetic microbeads of a transducer, more often amperometric, potentiometric or conductimetric. The bio-bar code assays relie on a sandwich structure based on specific biological interaction of a magnetic microbead and a nanoparticle with a defined biological molecule. The magnetic particle allows the separation of the reacted target molecules from unreacted ones. The nanoparticles aim at the amplification and the detection of the target molecule. The bio-bar code assays allow the detection at very low concentration of biological molecules, similar to PCR sensitivity.

  14. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

    Full Text Available Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

  15. Radiosotopic assay and binder therefor

    International Nuclear Information System (INIS)

    A rapid and less costly radioisotopic assay for measuring the concentration of folate in blood serum is described. This procedure utilizes 3H-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. Creating biological nanomaterials using synthetic biology

    International Nuclear Information System (INIS)

    Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems. (review)

  17. Creating biological nanomaterials using synthetic biology

    Directory of Open Access Journals (Sweden)

    MaryJoe K Rice

    2014-01-01

    Full Text Available Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems.

  18. A nanostructure-initiator mass spectrometry-based enzyme activity assay

    OpenAIRE

    Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren; Yannone, Steven M; Wong, Chi-Huey; Siuzdak, Gary

    2008-01-01

    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This “soft” immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitiv...

  19. New approach in multipurpose optical diagnostics: fluorescence based assay for simultaneous determination of physicochemical parameters

    OpenAIRE

    Moczko, Ewa

    2009-01-01

    The development of sensors assays for comprehensive characterisation of biological samples and effective minimal-invasive diagnostics is highly prioritised. Last decade this research area has been actively developing due to possibility of simultaneous, real- time, in vivo detection and monitoring of diverse physicochemical parameters and analytes. The new approach which has been introduced in this thesis was to develop and examine an optical diagnostic assay consisting of a ...

  20. Indirect conductimetric assay of antibacterial activities.

    Science.gov (United States)

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  1. Structural Biology Fact Sheet

    Science.gov (United States)

    ... Home > Science Education > Structural Biology Fact Sheet Structural Biology Fact Sheet Tagline (Optional) Middle/Main Content Area What is structural biology? Structural biology is a field of science focused ...

  2. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  3. Cytotoxicity assays to evaluate tannery effluents treated by photoelectrooxidation.

    Science.gov (United States)

    Jaeger, N; Moraes, J P; Klauck, C R; Gehlen, G; Rodrigues, M A S; Ziulkoski, A L

    2015-12-01

    The advanced oxidation process (AOP) is used to increase the treatment efficiency of effluents however, it is necessary to compare the toxicity of treated and untreated effluents to evaluate if the decontamination process does not cause any biological harm. Cultured cells have been previously used to assess the genotoxic and cytotoxic potential of various compounds. Hence, the aim of this work was to assess the applicability of cytotoxicity assays to evaluate the toxicity related to the AOP treatment. Samples of an industrial effluent were collected after their treatment by a conventional method. Cytotoxicity of standard and AOP treated effluents was assessed in CRIB and HEp-2 cell line using the MTT and neutral red assays. We observed decrease at cell viability in the both assays (50% MTT and 13% NRU) when cells were exposed to the AOP treatment in the highest concentration. Thus, cytotoxic assays in cultured cells can be explored as an useful method to evaluate toxicity as well as to optimize effluents treatment process. PMID:26628242

  4. Serum indices: managing assay interference.

    Science.gov (United States)

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  5. SNO+ Scintillator Purification and Assay

    International Nuclear Information System (INIS)

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  6. Proteasome Assay in Cell Lysates

    Science.gov (United States)

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  7. SNO+ Scintillator Purification and Assay

    Science.gov (United States)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  8. 21 CFR 864.7525 - Heparin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  9. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... assay. (a) Identification. A sulfhemoglobin assay is a device consisting of the reagents,...

  10. 21 CFR 864.7250 - Erythropoietin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  11. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  12. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a) Identification. An endotoxin assay is a device that uses serological techniques in whole blood. The device...

  13. 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...

  14. Simulating Biological and Non-Biological Motion

    Science.gov (United States)

    Bruzzo, Angela; Gesierich, Benno; Wohlschlager, Andreas

    2008-01-01

    It is widely accepted that the brain processes biological and non-biological movements in distinct neural circuits. Biological motion, in contrast to non-biological motion, refers to active movements of living beings. Aim of our experiment was to investigate the mechanisms underlying mental simulation of these two movement types. Subjects had to…

  15. A Brief Introduction to Chinese Biological Biological

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Chinese Biological Abstracts sponsored by the Library, the Shanghai Institutes for Biological Sciences, the Biological Documentation and Information Network, all of the Chinese Academy of Sciences, commenced publication in 1987 and was initiated to provide access to the Chinese information in the field of biology.

  16. Design and Implementation of High-Throughput Screening Assays.

    Science.gov (United States)

    Powell, David J; Hertzberg, Robert P; Macarrόn, Ricardo

    2016-01-01

    HTS remains at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful consideration of many options and variables, starting with the choice of screening strategy and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:27316985

  17. Peroxynitrite scavenging by different antioxidants. Part I: convenient assay.

    Science.gov (United States)

    Balavoine, G G; Geletii, Y V

    1999-01-01

    A convenient "tube" assay to quantify relative antioxidant activities in aqueous solutions has been developed. Peroxynitrite was employed as a biologically relevant source of radicals with Pyrogallol Red as a detecting molecule. A variety of compounds have been examined, namely polyphenols, uric acid, glutathione, and ascorbic acid. Competition kinetics were observed for the majority of examined compounds, except thymol and ascorbic acid. Pyrogallol Red was fully protected by ascorbic acid against the bleaching by peroxynitrite until its total consumption. The deviation from competition kinetics in the case of thymol was due to the formation of radicals from thymol and their subsequent reaction with Pyrogallol Red. Quercetin was the most efficient scavenger of free radicals. The measurements of relative antioxidant activities using Pyrogallol Red and other detecting molecules, such as gallocyanine and carminic acid, were in fair agreement. The assay was successfully used for a screening of antioxidant activity of plant extracts of unknown composition. PMID:10355895

  18. Assaying nonspecific phospholipase C activity

    Czech Academy of Sciences Publication Activity Database

    Pejchar, Přemysl; Günther, F.E.S.; Martinec, Jan

    Vol. 1009. New York City: Humana Press, 2013 - (Munnik, T.; Heilmann, I.), s. 193-203. (Methods in Molecular Biology). ISBN 978-1-62703-400-5 R&D Projects: GA ČR(CZ) GAP501/12/1942; GA ČR(CZ) GPP501/12/P950 Institutional research plan: CEZ:AV0Z50380511 Keywords : Bodipy * Diacylglycerol * Nonspecific phospholipase C Subject RIV: ED - Physiology

  19. Membrane Feeding Assay to Determine the Infectiousness of Plasmodium vivax Gametocytes.

    Science.gov (United States)

    Sattabongkot, Jetsumon; Kumpitak, Chalermpon; Kiattibutr, Kirakorn

    2015-01-01

    The evaluation of Plasmodium vivax gametocyte infectiousness by the membrane feeding assay is herein described. While P. vivax cannot be cultured and different parasite isolates may infect mosquitoes at different rates, the protocol described in this chapter identifies critical parameters to be considered when performing the assay such as methods for the preparation of the mosquitoes, the size of the blood cup, and the blood volume used. In previous studies the data have shown that the membrane feeding assay is useful for studies of parasite biology, and the effects of transmission blocking drugs and vaccines. PMID:26450382

  20. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    Science.gov (United States)

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. PMID:24674072

  1. Creating biological nanomaterials using synthetic biology

    OpenAIRE

    MaryJoe K Rice; Ruder, Warren C.

    2014-01-01

    Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic bi...

  2. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    Science.gov (United States)

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

  3. Fluorescence polarization assays in high-throughput screening and drug discovery: a review

    Science.gov (United States)

    Hall, Matthew D.; Yasgar, Adam; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

    2016-06-01

    The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.

  4. Establishment of Immunoradiometric Assay for Carcinoembryonic Antigen

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two anti-CEA monoclonal antibodies are used, one is coated on the microtiter plate, the other is labeled to make 125I-CEAMcAb. The one-step assay is established based on immunoradiometric assay(IRMA). The sensitivity of the assay is 0.5 μ g/L. The intra-assay CVs and the inter-assay CVs are lower than 10.0% and 15.0%, respectively. The analytical recoveries are ranged from 97.4% to 107.8%. The reference cut-out value of 35 normal serum is lower than

  5. Making transuranic assay measurements using modern controllers

    International Nuclear Information System (INIS)

    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

  6. Activity assay of membrane transport proteins

    Institute of Scientific and Technical Information of China (English)

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  7. Non destructive assay (NDA) techniques

    International Nuclear Information System (INIS)

    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)

  8. Predictive Assay For Cancer Targets

    Energy Technology Data Exchange (ETDEWEB)

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  9. Comet assay as a predictive assay for radiosensitivity of two human brain tumor cell lines

    International Nuclear Information System (INIS)

    Micronucleus assay and comet assay were compared as a predictive assay for radiosensitivity of tumors. Two human brain tumor cell lines, Becker (derived from astrocytoma) and ONS76 (derived from medulloblastoma) were used. Colony methods as the gold standard showed ONS76 as radiosensitive and Becker as radioresistant cell lines. Micronucleus assay revealed no different radiosensitivity between them. With comet assay, Becker cells received irradiation showed less damage to the DNA and faster repair of the damage than ONS76 cells did. The results correlate with those from colony methods. Comet assay is simple and rapid method for clinical use and it has an advantage not to establish the primary culture. Moreover, the results of comet assay showed not only DNA damage but also repair from the damage. It is concluded that comet assay is a superior method than micronucleus assay and has a potent candidate for clinical predictive assay. (author)

  10. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    OpenAIRE

    Mahajan Supriya D.; Schwartz Stanley A; Nair Madhavan P.N.

    2003-01-01

    Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene arra...

  11. Microfluidic multiculture assay to analyze biomolecular signaling in angiogenesis.

    Science.gov (United States)

    Theberge, Ashleigh B; Yu, Jiaquan; Young, Edmond W K; Ricke, William A; Bushman, Wade; Beebe, David J

    2015-03-17

    Angiogenesis (the formation of blood vessels from existing blood vessels) plays a critical role in many diseases such as cancer, benign tumors, and macular degeneration. There is a need for cell culture methods capable of dissecting the intricate regulation of angiogenesis within the microenvironment of the vasculature. We have developed a microscale cell-based assay that responds to complex pro- and antiangiogenic soluble factors with an in vitro readout for vessel formation. The power of this system over traditional techniques is that we can incorporate the whole milieu of soluble factors produced by cells in situ into one biological readout (vessel formation), even if the identity of the factors is unknown. We have currently incorporated macrophages, endothelial cells, and fibroblasts into the assay, with the potential to include additional cell types in the future. Importantly, the microfluidic platform is simple to operate and multiplex to test drugs targeting angiogenesis in a more physiologically relevant context. As a proof of concept, we tested the effect of an enzyme inhibitor (targeting matrix metalloproteinase 12) on vessel formation; the triculture microfluidic assay enabled us to capture a dose-dependent effect entirely missed in a simplified coculture assay (p < 0.0001). This result underscores the importance of cell-based assays that capture chemical cross-talk occurring between cell types. The microscale dimensions significantly reduce cell consumption compared to conventional well plate platforms, enabling the use of limited primary cells from patients in future investigations and offering the potential to screen therapeutic approaches for individual patients in vitro. PMID:25719435

  12. Developed technique for rapid assay of radium isotopes in water

    International Nuclear Information System (INIS)

    Most biological systems are about 80% water. Hence, most primary ionizations in biological materials occur in water molecules. For this reason an improved analytical method for the rapid assay of radium isotopes in water is suggested. After removal of alkaline earths and any '234U by using a cation exchange column, radium is electrodeposited onto stainless steel discs and alpha-counted to determine 222Ra. The plating efficiency has been improved over previous methods by adding platinum in microgram amounts and ammonium oxalate to the plating solutions. Radium measurements at or near maximum permissible concentrations in water can be made on 10-50 ml of water sample within one day.(author)

  13. Expert system for transuranic waste assay

    International Nuclear Information System (INIS)

    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

  14. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    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

  15. Rapid classification of biological components

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Vicki S. (Idaho Falls, ID); Barrett, Karen B. (Meridian, ID); Key, Diane E. (Idaho Falls, ID)

    2010-03-23

    A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens of the surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array, thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to a subject's identity.

  16. Rapid classification of biological components

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Vicki S. (Idaho Falls, ID); Barrett, Karen B. (Meridian, ID); Key, Diane E. (Idaho Falls, ID)

    2010-03-23

    A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array, thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to a subject's identity.

  17. Rapid classification of biological components

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

    2013-10-15

    A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array, thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to a subject's identity.

  18. Rapid classification of biological components

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

    2006-01-24

    A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine, methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to the surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array, thereby forming immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  19. Radiobiological and PK assays at advance Centre for Training Research and Education in Cancer (ACTREC)

    International Nuclear Information System (INIS)

    Radiobiological, pharmacokinetic and biodistribution studies are of paramount importance for drug development and more so in the development of newer radiation modulators. Radiobiological studies have now graduated from simple cell survival and viability assays to more complex molecular and imaging studies to study radiation modulation both in in-vitro and in-vivo models. Tata Memorial Centre and its research centre (ACTREC) is a premiere cancer centre in India dedicated to cancer research. The Department of Radiation Oncology treats approximately 7000 new patients in a year and is uniquely placed to do both translational radiation and clinical research in the field of drug development. The Clinical Biology Lab of the Department of Radiation Oncology at ACTREC in collaboration with other labs at ACTREC has standardized cell survival assays, DNA damage assays such as Gamma H2AX assay (by flow as well as confocal microscopy), Micronuclei assay and COMET assays using CASP software for quantification. We have also done apoptotic assays. These assays have been conducted for development newer drug formulations (for e.g liposomal radiosensitizers). We also have a strong imaging division having sophisticated microscopes (confocal and single molecule super resolution microscopes) for in-vitro optical imaging and a dedicated preclinical PET/CT/SPECT for in-vivo imaging. The clinical 3T MRI and PET/CT is being used to study the effect of hypoxia in various cancers

  20. Biological effects of radiation

    International Nuclear Information System (INIS)

    This fourth chapter presents: cell structure and metabolism; radiation interaction with biological tissues; steps of the production of biological effect of radiation; radiosensitivity of tissues; classification of biological effects; reversibility, transmissivity and influence factors; pre-natal biological effects; biological effects in therapy and syndrome of acute irradiation

  1. An ultrafiltration assay for lysyl oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Shackleton, D.R.; Hulmes, D.J. (Univ. of Edinburgh Medical School (England))

    1990-03-01

    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.

  2. Assay development status report for total cyanide

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, B.C. [Westinghouse Hanford Co., Richland, WA (United States); Jones, T.E.; Pool, K.H. [Pacific Northwest Lab., Richland, WA (United States)

    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{sup {minus}} 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.

  3. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    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

  4. Thyroglobulin measurement by highly sensitive assays

    DEFF Research Database (Denmark)

    Giovanella, Luca; Feldt-Rasmussen, Ulla; Verburg, Frederik A;

    2015-01-01

    thyroglobulin (Tg), circulating Tg serves as a biochemical marker of persistent or recurrent disease in the follow-up of DTC. Due to the suboptimal clinical detection rate of older Tg assays endogenous or exogenous thyrotropin (TSH) stimulations are recommended for unmasking occult disease. However, the...... development of new Tg assays with improved analytical sensitivity and precision at low concentrations now allows detection of very low Tg concentrations, reflecting minimal amounts of thyroid tissue, even without the need for TSH stimulation. Even if the use of these assays still has not found its way in...... current clinical guidelines, such assays are now increasingly used in clinical practice. As serum Tg measurement is a technically challenging assay and criteria to define a 'highly sensitive' assay may be different, a good knowledge of the technical difficulties and interpretation criteria is of paramount...

  5. Automated amperometric plutonium assay system

    International Nuclear Information System (INIS)

    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

  6. Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.

    Science.gov (United States)

    Rösslein, Matthias; Elliott, John T; Salit, Marc; Petersen, Elijah J; Hirsch, Cordula; Krug, Harald F; Wick, Peter

    2015-01-20

    An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications. PMID:25473822

  7. Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

    International Nuclear Information System (INIS)

    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 239Pu 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

  8. Analysis of image-based phenotypic parameters for high throughput gene perturbation assays.

    Science.gov (United States)

    Song, Mee; Jeong, Euna; Lee, Tae-Kyu; Tsoy, Yury; Kwon, Yong-Jun; Yoon, Sukjoon

    2015-10-01

    Although image-based phenotypic assays are considered a powerful tool for siRNA library screening, the reproducibility and biological implications of various image-based assays are not well-characterized in a systematic manner. Here, we compared the resolution of high throughput assays of image-based cell count and typical cell viability measures for cancer samples. It was found that the optimal plating density of cells was important to obtain maximal resolution in both types of assays. In general, cell counting provided better resolution than the cell viability measure in diverse batches of siRNAs. In addition to cell count, diverse image-based measures were simultaneously collected from a single screening and showed good reproducibility in repetitions. They were classified into a few functional categories according to biological process, based on the differential patterns of hit (i.e., siRNAs) prioritization from the same screening data. The presented systematic analyses of image-based parameters provide new insight to a multitude of applications and better biological interpretation of high content cell-based assays. PMID:26256799

  9. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    International Nuclear Information System (INIS)

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems

  10. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu

    2007-10-15

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  11. Immunofluorescence Assay for Serologic Diagnosis of SARS

    OpenAIRE

    Chan, Paul K. S.; Ng, King-Cheung; Chan, Rickjason C. W.; Lam, Rebecca K. Y.; Chow, Viola C. Y.; Hui, Mamie; Wu, Alan; Lee, Nelson; Yap, Florence H.Y.; Cheng, Frankie W.T.; Joseph J. Y. Sung; Tam, John S

    2004-01-01

    We evaluated a virus-infected cell-based indirect immunofluorescence assay for detecting anti–severe acute respiratory syndrome-associated coronavirus (SARS-CoV) immunoglobulin (Ig) G antibody. All confirmed SARS cases demonstrated seroconversion or fourfold rise in IgG antibody titer; no control was positive. Sensitivity and specificity of this assay were both 100%. Immunofluorescence assay can ascertain the status of SARS-CoV infection.

  12. Trypan blue exclusion assay by flow cytometry

    OpenAIRE

    Avelar-Freitas, B.A.; V.G. Almeida; Pinto, M.C.X.; F.A.G. Mourão; A.R. Massensini; O.A. Martins-Filho; E. Rocha-Vieira; G.E.A. Brito-Melo

    2014-01-01

    Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating ce...

  13. Safeguards and Non-destructive Assay

    International Nuclear Information System (INIS)

    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

  14. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Science.gov (United States)

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  15. Supporting read-across using biological data.

    Science.gov (United States)

    Zhu, Hao; Bouhifd, Mounir; Donley, Elizabeth; Egnash, Laura; Kleinstreuer, Nicole; Kroese, E Dinant; Liu, Zhichao; Luechtefeld, Thomas; Palmer, Jessica; Pamies, David; Shen, Jie; Strauss, Volker; Wu, Shengde; Hartung, Thomas

    2016-01-01

    Read-across, i.e. filling toxicological data gaps by relating to similar chemicals, for which test data are available, is usually done based on chemical similarity. Besides structure and physico-chemical properties, however, biological similarity based on biological data adds extra strength to this process. In the context of developing Good Read-Across Practice guidance, a number of case studies were evaluated to demonstrate the use of biological data to enrich read-across. In the simplest case, chemically similar substances also show similar test results in relevant in vitro assays. This is a well-established method for the read-across of e.g. genotoxicity assays. Larger datasets of biological and toxicological properties of hundreds and thousands of substances become increasingly available enabling big data approaches in read-across studies. Several case studies using various big data sources are described in this paper. An example is given for the US EPA's ToxCast dataset allowing read-across for high quality uterotrophic assays for estrogenic endocrine disruption. Similarly, an example for REACH registration data enhancing read-across for acute toxicity studies is given. A different approach is taken using omics data to establish biological similarity: Examples are given for stem cell models in vitro and short-term repeated dose studies in rats in vivo to support read-across and category formation. These preliminary biological data-driven read-across studies highlight the road to the new generation of read-across approaches that can be applied in chemical safety assessment. PMID:26863516

  16. Recommended Immunological Assays to Screen for Ricin-Containing Samples

    Directory of Open Access Journals (Sweden)

    Stéphanie Simon

    2015-11-01

    Full Text Available Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120. Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests. Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

  17. Reconfigurable microfluidic dilution for high-throughput quantitative assays.

    Science.gov (United States)

    Fan, Jinzhen; Li, Baoqing; Xing, Siyuan; Pan, Tingrui

    2015-06-21

    This paper reports a reconfigurable microfluidic dilution device for high-throughput quantitative assays, which can easily produce discrete logarithmic/binary concentration profiles ranging from 1 to 100-fold dilution in parallel from a fixed sample volume (e.g., 10 μL) without any assistance of continuous fluidic pump or robotic automation. The integrated dilution generation chip consists of switchable distribution and collection channels, metering reservoirs, reaction chambers, and pressure-activatable Laplace valves. Following the sequential loading of a sample, a diluent, and a detection reagent into their individual metering chambers, the top microfluidic layer can be reconfigured to collect the metered chemicals into the reaction chambers in parallel, where detection will be conducted. To facilitate mixing and reaction in the microchambers, two acoustic microstreaming actuation mechanisms have been investigated for easy integrability and accessibility. Furthermore, the microfluidic dilution generator has been characterized by both colorimetric and fluorescent means. A further demonstration of the generic usage of the quantitative dilution chip has utilized the commonly available bicinchoninic acid (BCA) assay to analyse the protein concentrations of human tissue extracts. In brief, the microfluidic dilution generator offers a high-throughput high-efficiency quantitative analytical alternative to conventional quantitative assay platforms, by simple manipulation of a minute amount of chemicals in a compact microfluidic device with minimal equipment requirement, which can serve as a facile tool for biochemical and biological analyses in regular laboratories, point-of-care settings and low-resource environments. PMID:25994379

  18. Characterization of mesenchymal stromal cells: potency assay development.

    Science.gov (United States)

    Hematti, Peiman

    2016-04-01

    Based on their many different mechanisms of action, presumed immune-privileged status, and relative ease of production, mesenchymal stromal cells (MSCs) are under intensive clinical investigation for treating a wide range of degenerative, inflammatory, and immunologic disorders. Identification of relevant and robust potency assays is not only a regulatory requirement, but it is also the basis for producing and delivering a product that is consistent, safe, and ultimately an effective therapy. Although development of an appropriate potency assay is one of the most challenging issues in cell-based therapies, it is of paramount importance in the process of developing and testing cellular products. Regardless of the many different tissue sources and methods used in culture expansion of MSCs, they possess many of the same morphologic, cell surface markers, and differentiation characteristics. However, MSC products with similar phenotypic characteristics could still have major differences in their biologic and functional attributes. Understanding the different mechanisms of action and establishment of relevant potency assays is of pivotal importance in allowing investigators and regulatory agencies to compare MSCs used in different clinical trials. PMID:27079322

  19. LT-HSC Methylcellulose Assay

    Science.gov (United States)

    Kerenyi, Marc A.

    2016-01-01

    Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12–16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

  20. Radio-analysis. Applications: biological dosimetry

    International Nuclear Information System (INIS)

    Radioisotopes have revolutionized the medical biology. Radio-immunology remains the reference measurement of the infinitely small in biology. Constant efforts have been performed to improve the simpleness, detectability and fastness of the method thanks to an increasing automation. This paper presents: 1 - the advantages of compounds labelling and the isotopic dilution; 2 - the antigen-antibody system: properties, determination of the affinity constant using the Scatchard method; 3 - radio-immunologic dosimetry: competitive dosimetry (radioimmunoassay), calibration curve and mathematical data processing, application to the free thyroxine dosimetry, immunoradiometric dosimetry (immunoradiometric assay), evaluation of the analytical efficiency of a radioimmunoassay; 4 - detection of the radioactive signal (solid and liquid scintillation). (J.S.)

  1. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine

    Science.gov (United States)

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-01-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. PMID:26951077

  2. Statistical Aspects of Using Biologic Markers

    OpenAIRE

    Margolin, Barry H.

    1988-01-01

    This expository paper surveys a variety of statistical issues pertinent to the design and analysis of studies involving biologic markers of human genotoxic exposure. Examples with cytogenetic and mutagenic end points are presented. One principal theme is the valuable interplay of ideas among statistical analyses for in vitro, in vivo and human assays for genetic toxicity; e.g., statistical analysis of sister chromatid exchanges in vitro is suggestive of an analytical approach to the study of ...

  3. Acellular comet assay: A tool for assessing variables influencing the alkaline comet assay

    International Nuclear Information System (INIS)

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of 60Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from 60Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified. (authors)

  4. Development of an upconverting chelate assay

    Science.gov (United States)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  5. A bioluminescent assay for measuring glucose uptake.

    Science.gov (United States)

    Valley, Michael P; Karassina, Natasha; Aoyama, Natsuyo; Carlson, Coby; Cali, James J; Vidugiriene, Jolanta

    2016-07-15

    Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts. PMID:27130501

  6. Radioreceptor assay: theory and applications to pharmacology

    International Nuclear Information System (INIS)

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising

  7. Simple UV spectrophotometric assay of Clarithromycin

    Directory of Open Access Journals (Sweden)

    Dr. Safila Naveed

    2014-09-01

    Full Text Available Clarithromycin belongs to semi-synthetic macrolide antibiotic class of drugs that inhibits bacterial protein synthesis.. Our aim of study is to develop a efficient least time consuming and simple spectrophotometric method for the assay of clarithromycin. Comparision of assay of five different brands of clarithromycin (klaricid,klaribact,rithmo,clariteck,E-clark available in public medical store of Karachi, Pakistan has also been done. The assay is based on the ultraviolet UV absorbance maxima at about 210nm wavelength of mefenamic acid, water is used as solvent. A sample of drug was dissolved in water to produce a solution containing mefenamic acid. Similarly, a sample of ground tablets of different brand were dissolved in water and various dilutions were made. The absorbance of sample preparation was measured at 210nm against the solvent blank and the assay was determined by comparing with the absorbance of available brand. Our results reveals that among all the five brands of clarithromycin (klaricid,klaribact,rithmo,clariteck,E-clark Klaribact shows highest percentage assay i.e 115.3846%. Klaricid and Claritecl shows percentage assay of 107.693%. Rithmo shows a percent assay of 92.307% while E-clark shows lowest value for percentage assay 84.6153%

  8. Practical radiochromatographic assay for cholesterol epoxide hydrase

    International Nuclear Information System (INIS)

    A method for the assay of cholesterol epoxide hydrase activity is described. The assay involves the thin-layer chromatographic separation and quantitation of radiolabeled cholestan-3β,5α,6α-epoxide and its major hydration product, cholestan-3β,5α,6β-triol. Radiochromatographic scanning is employed to quantitate the reaction. The procedure is sensitive, rapid, and nondestructive

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

    International Nuclear Information System (INIS)

    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 E2) and common interfering substances (thymidine at 14 M) have been tested. The latter substance produces false positive results in the standard [3H] 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

  10. Emerging frontiers in radiation biology

    International Nuclear Information System (INIS)

    Radiation biology owes its origin to the spectacular success in the treatment of human diseases by x-rays and radium, just after their respective discoveries in 1895-96. From the very inception it has attracted researchers from all disciplines of science. The target and hit theory developed by physicists, dominated the scene till the advent of radiation chemistry concepts which offered an entirely different perspective to the mechanisms involved in biological effects of radiations and their modification by endogenous and exogenous agents like radioprotectors and radiosensitisers including hyperthermia. The applied aspect of radiation biology mainly relates to radiation therapy of cancer which, in spite of its long existence, is still to achieve scientific perfection. Nevertheless, it did not wait -and fortunately so-, for its radiobiological rationality but continued its development to be the main modality for cancer treatment today. Several approaches are now being attempted to improve its efficacy by selectively damaging the cancerous cells while sparing the normal tissues and also by devising suitable predictive assays for radioresponse of different tumours to enable individualisation of treatment schedules. (author). 99 refs., 1 fig., 2 tabs

  11. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman

    2015-12-14

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  12. Assays for Determination of Protein Concentration.

    Science.gov (United States)

    Olson, Bradley J S C

    2016-01-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. PMID:27248579

  13. Probabilistic interpretation of radioactive waste assay

    International Nuclear Information System (INIS)

    The non-destructive assay of radioactive wastes from nuclear power plants and fuel cycle facilities is required for assessing the disposal risks. Such assay is influenced by two distinct facts: the statistical uncertainty of the measurement and the spatial uncertainty due to the random or at least unknown spatial distribution of the assayed material in a waste container. In this paper a probabilistic interpretation procedure is presented for a single-detector assay system of nuclear waste by one-shot passive gamma technique. The key parameter for this study, the average escape probability for photons, has been obtained by a specific-purpose Monte Carlo code, MCRW. The code has been developed to simulate the entire pulse height spectral responses for point sources located within the compartment which is the basic idea describing the degree of spatial homogeneity of the nuclear and matrix materials. The methodology presented here can be extended to actual radioactive waste assay

  14. The haemolytic antibody isotope release (HAIR) assay; an efficient alternative technique to conventional plaque assays

    International Nuclear Information System (INIS)

    The haemolytic antibody isotope release (HAIR) assay quantitates antibody production by splenic antibody-producing cells by lysis of chromium-51-labelled sheep red blood cells. The amount of antibody quantitated by the HAIR assay directly correlates with the number of antibody-producing cells measured by a conventional plaque assay. The HAIR assay is an easy, sensitive, and reproducible technique that is especially useful when large numbers of animals are required for testing. (author)

  15. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    OpenAIRE

    Buch Karl; Peters Tanja; Nawroth Thomas; Sänger Markus; Schmidberger Heinz; Langguth Peter

    2012-01-01

    Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calc...

  16. Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

    International Nuclear Information System (INIS)

    Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

  17. Improved benzodiazepine radioreceptor assay using the MultiScreen (R) Assay System

    NARCIS (Netherlands)

    Janssen, MJ; Ensing, K; de Zeeuw, RA

    1999-01-01

    In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time, The filtration in this method was performed by using the MultiScreen(R) Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom,

  18. Data quality in drug discovery: the role of analytical performance in ligand binding assays.

    Science.gov (United States)

    Wätzig, Hermann; Oltmann-Norden, Imke; Steinicke, Franziska; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Albishri, Hassan M; Baumann, Knut; Exner, Thomas; Böckler, Frank M; El Deeb, Sami

    2015-09-01

    Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development. PMID:26070362

  19. Biological conversion system

    Science.gov (United States)

    Scott, C.D.

    A system for bioconversion of organic material comprises a primary bioreactor column wherein a biological active agent (zymomonas mobilis) converts the organic material (sugar) to a product (alcohol), a rejuvenator column wherein the biological activity of said biological active agent is enhanced, and means for circulating said biological active agent between said primary bioreactor column and said rejuvenator column.

  20. Synthetic biology: insights into biological computation.

    Science.gov (United States)

    Manzoni, Romilde; Urrios, Arturo; Velazquez-Garcia, Silvia; de Nadal, Eulàlia; Posas, Francesc

    2016-04-18

    Organisms have evolved a broad array of complex signaling mechanisms that allow them to survive in a wide range of environmental conditions. They are able to sense external inputs and produce an output response by computing the information. Synthetic biology attempts to rationally engineer biological systems in order to perform desired functions. Our increasing understanding of biological systems guides this rational design, while the huge background in electronics for building circuits defines the methodology. In this context, biocomputation is the branch of synthetic biology aimed at implementing artificial computational devices using engineered biological motifs as building blocks. Biocomputational devices are defined as biological systems that are able to integrate inputs and return outputs following pre-determined rules. Over the last decade the number of available synthetic engineered devices has increased exponentially; simple and complex circuits have been built in bacteria, yeast and mammalian cells. These devices can manage and store information, take decisions based on past and present inputs, and even convert a transient signal into a sustained response. The field is experiencing a fast growth and every day it is easier to implement more complex biological functions. This is mainly due to advances in in vitro DNA synthesis, new genome editing tools, novel molecular cloning techniques, continuously growing part libraries as well as other technological advances. This allows that digital computation can now be engineered and implemented in biological systems. Simple logic gates can be implemented and connected to perform novel desired functions or to better understand and redesign biological processes. Synthetic biological digital circuits could lead to new therapeutic approaches, as well as new and efficient ways to produce complex molecules such as antibiotics, bioplastics or biofuels. Biological computation not only provides possible biomedical and

  1. Biological and electrical properties of biosynthesized silver nanoparticles

    Indian Academy of Sciences (India)

    Madhulika Bhagat; Shayana Rajput; Sandeep Arya; Saleem Khan; Parveen Lehana

    2015-09-01

    In this work, silver nanoparticles (AgNPs) were synthesized biochemically at room temperature using aqueous extract of rhizome of Rheum australe plant. The as-synthesized AgNPs were further studied for their morphological, biological and electrical characterization. The morphological studies, such as scanning electron microscopy, X-ray diffraction and UV–vis spectrum confirmed their successful synthesis. Biological analysis revealed their antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Electrical characterization showed that the conductivity of the mixture of AgNPs with DPPH assay is more than the AgNPs dispersed in distilled water. The obtained results may have potential applications as sensors.

  2. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    Science.gov (United States)

    Müller, Francis; Fattinger, Christof

    Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfill these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future. Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal

  3. Computational Systems Chemical Biology

    OpenAIRE

    Oprea, Tudor I.; May, Elebeoba E.; Leitão, Andrei; Tropsha, Alexander

    2011-01-01

    There is a critical need for improving the level of chemistry awareness in systems biology. The data and information related to modulation of genes and proteins by small molecules continue to accumulate at the same time as simulation tools in systems biology and whole body physiologically-based pharmacokinetics (PBPK) continue to evolve. We called this emerging area at the interface between chemical biology and systems biology systems chemical biology, SCB (Oprea et al., 2007).

  4. Pictures of Synthetic Biology

    OpenAIRE

    Cserer, Amelie; Seiringer, Alexandra

    2009-01-01

    This article is concerned with the representation of Synthetic Biology in the media and by biotechnology experts. An analysis was made of German-language media articles published between 2004 and 2008, and interviews with biotechnology-experts at the Synthetic Biology conference SB 3.0 in Zurich 2007. The results have been reflected in terms of the definition of Synthetic Biology, applications of Synthetic Biology and the perspectives of opportunities and risks. In the media, Synthetic Biolog...

  5. Radioactive wastes assay technique and equipment

    International Nuclear Information System (INIS)

    The waste inventory records such as the activities and radio- nuclides contained in the waste packages are to be submitted with the radioactive wastes packages for the final disposal. The nearly around 10,000 drums of waste stocked in KAERI now should be assayed for the preparation of the waste inventory records too. For the successive execution of the waste assay, the investigation into the present waste assay techniques and equipment are to be taken first. Also the installation of the waste assay equipment through the comprehensive design, manufacturing and procurement should be proceeded timely. As the characteristics of the KAERI-stocked wastes are very different from that of the nuclear power plant and those have no regular waste streams, the application of the in-direct waste assay method using the scaling factors are not effective for the KAERI-generated wastes. Considering for the versal conveniency including the accuracy over the wide range of waste forms and the combination of assay time and sensitivity, the TGS(Tomographic Gamma Scanner) is appropriate as for the KAERI -generated radioactive waste assay equipment

  6. Mobile non-destructive assay system

    International Nuclear Information System (INIS)

    A mobile system for non-destructive assay (NDA), developed at the Los Alamos National Laboratory, provides accurate and sensitive measurements for transuranic (TRU) isotopes contained in 208-iota drums of miscellaneous nuclear wastes. The NDA unit consists of four major subsystems: an assay chamber, counting and digital electronics, data acquisition, and a neutron generator. It performs both active and passive neutron waste measurements. The former determines the amount of fissile isotopes at a sensitivity level of 1 mg plutonium. The latter determines spontaneous fission and α,n) isotopes at a comparable level. A complete assay consists of sequential active and passive measurements. The assay measurement and other supporting data are incorporated in a commercial spreadsheet program (Lotus 1,2,3) for further analysis, which includes various matrix corrections and a determination of whether or not the drum exceeds the 100-nCi/g threshold for TRU wastes. Field tests have been performed on three separate occasions, accomplishing more than 1800 waste drum assays. These waste drum assays are discussed, especially those comparing passive and active neutron measurements with independent segmented gamma scan assays. Results obtained with a set of 15 drums containing plutonium prepared from standards and actual hot waste matrices are also reviewed

  7. Simple UV spectrophotometric assay of Mefenamic acid

    Directory of Open Access Journals (Sweden)

    Dr. Safila Naveed

    2014-07-01

    Full Text Available Mefenamic acid belongs to non-steroidal antiinflammatory drugs (NSAID.. It is being used widely for the treatment of analgesia. It is also used as antirheumatic and antipyretic drug. Our aim of study is to develop a efficient least time consuming and simple spectrophotometric method for the assay of mefenamic acid. Comparision of assay of three different brands of mefenamic acid (mefnac,ponstan,dolar available in public medical store of Karachi, Pakistan has also been done. The assay is based on the ultraviolet UV absorbance maxima at about 288nm wavelength of mefenamic acid, water is used as solvent. A sample of drug was dissolved in water to produce a solution containing mefenamic acid. Similarly, a sample of ground tablets of different brand were dissolved in water and various dilutions were made. The absorbance of sample preparation was measured at 288nm against the solvent blank and the assay was determined by comparing with the absorbance of available brand. Our results reveals that among all the three brands of mefenamic acid (mefnac,ponstan,dolar rosulin and rovista shows highest percentage assay 107.5%. xplended and rosubar shows percent assay of 106.25% and 103.75% while rovactor shows lowest value for percentage assay 98.75%.

  8. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    International Nuclear Information System (INIS)

    For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98) were included in the experiment to study its principal and general applicability

  9. Environmental herbicides and mycotoxin by inmunoraciochemical assay

    International Nuclear Information System (INIS)

    Immunochemical assays based on antigen antibody recognition, are at present very attractive analytical tools for determination of molecules present in different matrixes.Due to its specificity, sensitivity and easy application, the Immuno radiochemical assays have been adopted by international agencies for control procedures of environmental impact analytes. Optimization conditions for two contaminants, aflatoxin-B1 (AfB1, bacterial myco toxin from Aspergillus flavus) in dry food and atrazine (Atr, chloro-derivative triazine herbicides) in milk and water, by immunoradiometric assays based on the use of polyclonal antibodies for the mycotoxins and specific monoclonal antibody for the triazine derivatives, are presented.Liquid Chromatography is used as reference

  10. Radioreceptor assay of human growth hormone

    International Nuclear Information System (INIS)

    A radioreceptor assay for human growth hormone (hGH) was developed. The receptor preparation was 25,000g pellet from the livers of pregnant rabbits. Iodination of GH with 125I was preformed by the methods of Lactoperoxidase and Iodogen. The sensitivity of assay was 0.67 ± 0.11 ng/ml serum. Serum hGH levels in 72 cases of normal subjects, 102 cases of acromegaly were measured by radioreceptor assay (RRA), and the results were compared with those obtained by radioimmunoassay (RIA)

  11. Passive neutron assay of irradiated nuclear fuels

    International Nuclear Information System (INIS)

    Passive neutron assay of irradiated nuclear fuel has been investigated by calculations and experiments as a simple, complementary technique to the gamma assay. From the calculations it is found that the neutron emission arises mainly from the curium isotopes, the neutrons exhibit very good penetrability of the assemblies, and the neutron multiplication is not affected by the burnup. From the experiments on BWR and PWR assemblies, it is found that the neutron emission rate is proportional to burnup raised to 3.4 power. Recent investigations indicate that the passive neutron assay is a simple and useful technique to determine the consistency of burnups between assemblies. 10 refs

  12. Passive neutron assay of irradiated nuclear fuels

    International Nuclear Information System (INIS)

    Passive neutron assay of irradiated nuclear fuel has been investigated by calculations and experiments as a simple, complementary technique to the gamma assay. From the calculations it was found that the neutron emission arises mainly from the curium isotopes, the neutrons exhibit very good penetrability of the assemblies, and the neutron multiplication is not affected by the burnup. From the experiments on BWR and PWR assemblies, the neutron emission rate is proportional to burnup raised to 3.4 power. The investigations indicate that the passive neutron assay is a simple and useful technique to determine the consistency of burnups between assemblies

  13. Nondestructive assay measurements applied to reprocessing plants

    International Nuclear Information System (INIS)

    Nondestructive assay for reprocessing plants relies on passive gamma-ray spectrometry for plutonium isotopic and plutonium mass values of medium-to-low-density samples and holdup deposits; on active x-ray fluorescence and densitometry techniques for uranium and plutonium concentrations in solutions; on calorimetry for plutonium mass in product; and passive neutron techniques for plutonium mass in spent fuel, product, and waste. This paper will describe the radiation-based nondestructive assay techniques used to perform materials accounting measurements. The paper will also discuss nondestructive assay measurements used in inspections of reprocessing plants

  14. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    OpenAIRE

    M Anthony Moody; Mattia Bonsignori

    2012-01-01

    The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities ha...

  15. Live-cell luciferase assay of drug resistant cells

    OpenAIRE

    sprotocols

    2015-01-01

    To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes u...

  16. BGRT: Biologically guided radiation therapy - The future is fast approaching!

    International Nuclear Information System (INIS)

    Rapid advances in functional and biological imaging, predictive assays, and our understanding of the molecular and cellular responses underpinning treatment outcomes herald the coming of the long-sought goal of implementing patient-specific biologically guided radiation therapy (BGRT) in the clinic. Biological imaging and predictive assays have the potential to provide patient-specific, three-dimensional information to characterize the radiation response characteristics of tumor and normal structures. Within the next decade, it will be possible to combine such information with advanced delivery technologies to design and deliver biologically conformed, individualized therapies in the clinic. The full implementation of BGRT in the clinic will require new technologies and additional research. However, even the partial implementation of BGRT treatment planning may have the potential to substantially impact clinical outcomes

  17. Biological research for radiation protection

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In Gyu; Kim, Kug Chan; Shim, Hae Won; Oh, Tae Jeong; Park, Seon Young; Lee, Kang Suk

    2000-04-01

    The work scope of Biological research for the radiation protection had contained the search of biological microanalytic methods for assessing the health effect by {gamma}-radiation and toxic agents, the standardization of human T-lymphocyte cell culture and polymerase chain reaction, T-cell clonal assay, and the quantification of mutation frequency in the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene locus by single exposure or combined exposure. Especially, the polymerase chain reaction methods using reverse transcriptase has been developed to analyze the mutant gene induced by {gamma}-radiation and chemical (pentachlorophenol) agent exposure, and to investigate the point mutations in the HPRT gene locus of T-lymphocytes. The HPRT T-cell clonal assay revealed that it could not differentiate {gamma}-irradiation from pentachlorophenol, because the frequency of somatic mutations induced by both damaging agents increased in a dose-dependent manner. The analysis of DNA sequence alterations of HPRT mutant clones clearly showed that both damaging agents induced different mutational spectra in the HPRT locus of T-cells. The large deletions, which account for 75 percent of the analyzed mutants, are characteristic mutations induced by {gamma}-irradiation. By contrast, point mutations such as base substitutions and insertion, come up to 97 percent in the case of pentachlorophenol-treated cells. The point mutation frequencies at 190 base pair and 444 base pair positions are 3-6 folds as high as in those at other mutation positions. It may be that these mutation sites are hot spots induced by pentachlorophenol. These results suggest that the HPRT mutation spectrum can be used as a potential bio marker for assessing a specific environmental risk. (author)

  18. Biological research for radiation protection

    International Nuclear Information System (INIS)

    The work scope of Biological research for the radiation protection had contained the search of biological microanalytic methods for assessing the health effect by γ-radiation and toxic agents, the standardization of human T-lymphocyte cell culture and polymerase chain reaction, T-cell clonal assay, and the quantification of mutation frequency in the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene locus by single exposure or combined exposure. Especially, the polymerase chain reaction methods using reverse transcriptase has been developed to analyze the mutant gene induced by γ-radiation and chemical (pentachlorophenol) agent exposure, and to investigate the point mutations in the HPRT gene locus of T-lymphocytes. The HPRT T-cell clonal assay revealed that it could not differentiate γ-irradiation from pentachlorophenol, because the frequency of somatic mutations induced by both damaging agents increased in a dose-dependent manner. The analysis of DNA sequence alterations of HPRT mutant clones clearly showed that both damaging agents induced different mutational spectra in the HPRT locus of T-cells. The large deletions, which account for 75 percent of the analyzed mutants, are characteristic mutations induced by γ-irradiation. By contrast, point mutations such as base substitutions and insertion, come up to 97 percent in the case of pentachlorophenol-treated cells. The point mutation frequencies at 190 base pair and 444 base pair positions are 3-6 folds as high as in those at other mutation positions. It may be that these mutation sites are hot spots induced by pentachlorophenol. These results suggest that the HPRT mutation spectrum can be used as a potential bio marker for assessing a specific environmental risk. (author)

  19. Stochastic modeling for the COMET-assay

    OpenAIRE

    Boulesteix, Anne-Laure; Hösel, Volker; Liebscher, Volkmar

    2003-01-01

    We present a stochastic model for single cell gel electrophoresis (COMET-assay) data. Essential is the use of point process structures, renewal theory and reduction to intensity histograms for further data analysis.

  20. ORNL TRU-waste-assay system

    International Nuclear Information System (INIS)

    ORNL has been selected as the demonstration site for a new transuranic neutron assay system developed at the Los Alamos National Laboratory. The major objectives of the cooperative interlaboratory program is to field test, calibrate, and evaluate the neutron interrogation system, to provide a demonstration and training facility for DOE of this sophisticated technology, to reduce the volume of TRU waste stored at ORNL, and to provide positive identification of the radionuclide content of the ORNL TRU waste. In order to meet these objectives, a two-tier assay system utilizing the LANL neutron interrogation assay system and an upgraded gamma-ray drum scanner has been employed. Each assay technique and the results obtained from the examination of authentic ORNL TRU waste are described

  1. Proximity assays for sensitive quantification of proteins

    Directory of Open Access Journals (Sweden)

    Christina Greenwood

    2015-06-01

    Full Text Available Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression.

  2. Safeguards and Non-destructive Assay

    Energy Technology Data Exchange (ETDEWEB)

    Carchon, R.; Bruggeman, M

    2001-04-01

    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.

  3. Hyaluronidase treatment of synovial fluid to improve assay precision for biomarker research using multiplex immunoassay platforms.

    Science.gov (United States)

    Jayadev, Chethan; Rout, Raj; Price, Andrew; Hulley, Philippa; Mahoney, David

    2012-12-14

    Synovial fluid (SF) is a difficult biological matrix to analyse due to its complex non-Newtonian nature. This can result in poor assay repeatability and potentially inefficient use of precious samples. This study assessed the impact of SF treatment by hyaluronidase and/or dilution on intra-assay precision using the Luminex and Meso Scale Discovery (MSD) multiplex platforms. SF was obtained from patients with knee osteoarthritis at the time of joint replacement surgery. Aliquots derived from the same sample were left untreated (neat), 2-fold diluted, 4-fold diluted or treated with 2mg/ml testicular hyaluronidase (with 2-fold dilution). Preparation methods were compared in a polysterene-bead Luminex 10-plex (N=16), magnetic-bead Luminex singleplex (N=7) and MSD 4-plex (N=7). Each method was assessed for coefficient of variation (CV) of replicate measurements, number of bead events (for Luminex assays) and dilution-adjusted analyte concentration. Percentage recovery was calculated for dilutions and HAse treatment. Hyaluronidase treatment significantly increased the number of wells with satisfactory bead events/region (95%) compared to neat (48%, pmagnetic-bead Luminex assay achieved ≥50 bead events irrespective of treatment method. Hyaluronidase treatment resulted in lower intra-assay CVs for detectable ligands (group average CVmagnetic-bead Luminex assays. In addition, measured sample concentrations were higher and recovery was poor (elevated) after hyaluronidase treatment. In the MSD 4-plex, within-group comparison of the intra-assay CV or concentration was not conclusively influenced by SF preparation. However, only hyaluronidase treatment resulted in CV<25% for all samples for TNF-α. There was no effect on analyte concentrations or recovery. Hyaluronidase treatment can improve intra-assay precision and assay signal of SF analysis by multiplex immunoassays and should be recommended for SF biomarker research, particularly using the Luminex platform. PMID:22955210

  4. A novel parameter in comet assay measurements

    OpenAIRE

    Kirilova Milena; Ivanov Rumen; Miloshev George

    2005-01-01

    Single Cell Gel Electrophoresis (SCGE) or Comet assay is a very sensitive method for assessing damages in DNA on a single cell level. It has found many applications in fields where genotoxic activity could be an issue. In environmental monitoring, health care, food industry Comet assay is used with increasing popularity. For verifying the results obtained by this method many parameters could be monitored. To that end several software packages exist. In the conditions that we are suggesting on...

  5. The comet assay in nanotoxicology research.

    OpenAIRE

    Karlsson, Hanna L

    2010-01-01

    Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The ar...

  6. Automated optical sensing system for biochemical assays

    Science.gov (United States)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  7. Passive nondestructive assay of nuclear materials

    International Nuclear Information System (INIS)

    The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

  8. A New Stem Cell Biology: The Continuum and Microvesicles

    OpenAIRE

    Quesenberry, Peter J.; Dooner, Mark S.; Goldberg, Laura R.; Aliotta, Jason M.; Pereira, Mandy; Amaral, Ashley; Del Tatto, Michael M.; Hixson, Douglas C.; Ramratnam, Bharat

    2012-01-01

    The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of “stem cells” using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells wi...

  9. Current laboratory methods for biological threat agent identification.

    Science.gov (United States)

    Henchal, E A; Teska, J D; Ludwig, G V; Shoemaker, D R; Ezzell, J W

    2001-09-01

    The authors present an integrated approach for the identification of biological threat agents. The methods used have been used extensively in field exercises and during response to incidents of biological terrorism. A diagnostic system, which integrates the clinical diagnosis or medical intelligence with immunodiagnostic tests, rapid gene amplification assays, and standard culture, provides results of the highest quality and confidence. In the future, selected reagents and technologies will be distributed through a network of civilian and military laboratories. PMID:11572145

  10. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [125I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [125I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 109 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  11. An ultrafiltration assay for lysyl oxidase.

    Science.gov (United States)

    Shackleton, D R; Hulmes, D J

    1990-03-01

    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. PMID:1971160

  12. Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins

    OpenAIRE

    Nicklisch, Sascha C.T.; Herbert Waite, J.

    2014-01-01

    © 2014 The Authors. (Graph Presented) The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pH...

  13. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    Science.gov (United States)

    Zhao, Yun; Xia, Qingyan; Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; Pvalue of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  14. Label-acquired magnetorotation for biosensing: An asynchronous rotation assay

    Energy Technology Data Exchange (ETDEWEB)

    Hecht, Ariel, E-mail: hecht@umich.ed [University of Michigan, Department of Biomedical Engineering, 2200 Bonisteel, Ann Arbor, MI 48109-2099 (United States); University of Michigan, Department of Chemistry, 930 North University, Ann Arbor, MI 48109-1055 (United States); Kinnunen, Paivo, E-mail: pkkinn@umich.ed [University of Michigan, Department of Chemistry, 930 North University, Ann Arbor, MI 48109-1055 (United States); University of Michigan, Applied Physics Program, 2477 Randall Laboratory, Ann Arbor, MI 48109-1120 (United States); McNaughton, Brandon, E-mail: bmcnaugh@umich.ed [University of Michigan, Department of Biomedical Engineering, 2200 Bonisteel, Ann Arbor, MI 48109-2099 (United States); University of Michigan, Department of Chemistry, 930 North University, Ann Arbor, MI 48109-1055 (United States); University of Michigan, Applied Physics Program, 2477 Randall Laboratory, Ann Arbor, MI 48109-1120 (United States); Kopelman, Raoul, E-mail: kopelman@umich.ed [University of Michigan, Department of Biomedical Engineering, 2200 Bonisteel, Ann Arbor, MI 48109-2099 (United States); University of Michigan, Department of Chemistry, 930 North University, Ann Arbor, MI 48109-1055 (United States); University of Michigan, Applied Physics Program, 2477 Randall Laboratory, Ann Arbor, MI 48109-1120 (United States)

    2011-02-15

    This paper presents a novel application of magnetic particles for biosensing, called label-acquired magnetorotation (LAM). This method is based on a combination of the traditional sandwich assay format with the asynchronous magnetic bead rotation (AMBR) method. In label-acquired magnetorotation, an analyte facilitates the binding of a magnetic label bead to a nonmagnetic solid phase sphere, forming a sandwich complex. The sandwich complex is then placed in a rotating magnetic field, where the rotational frequency of the sandwich complex is a function of the amount of analyte attached to the surface of the sphere. Here, we use streptavidin-coated beads and biotin-coated particles as analyte mimics, to be replaced by proteins and other biological targets in future work. We show this sensing method to have a dynamic range of two orders of magnitude.

  15. Detection of garlic gamma-irradiated by assay comet

    Energy Technology Data Exchange (ETDEWEB)

    Moreno Alvarez, Damaris L.; Miranda, Enrique F. Prieto; Carro, Sandra; Iglesias Enrique, Isora; Matos, Wilberto [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear (CEADEN), Ciudad de La Habana (Cuba)], e-mail: damaris@ceaden.edu.cu

    2009-07-01

    The garlic samples were irradiated in a facility with {sup 60}Co sources, at absorbed dose values of 0-0,15 kGy. The detection method utilized for the identification of the irradiated garlic was biological comet assay. The samples were classified post-irradiation several times. The irradiated samples showed high strand breaks of DNA exhibiting comets of several forms, while the not irradiated and lower dose samples showed a behavior like round shape and light comets. Significant differences were found for higher absorbed dose values at 0.06 kGy, this absorbed dose value is corresponding with the applied dose value at this food in order to avoid the germination. (author)

  16. Detection of garlic gamma-irradiated by assay comet

    International Nuclear Information System (INIS)

    The garlic samples were irradiated in a facility with 60Co sources, at absorbed dose values of 0-0,15 kGy. The detection method utilized for the identification of the irradiated garlic was biological comet assay. The samples were classified post-irradiation several times. The irradiated samples showed high strand breaks of DNA exhibiting comets of several forms, while the not irradiated and lower dose samples showed a behavior like round shape and light comets. Significant differences were found for higher absorbed dose values at 0.06 kGy, this absorbed dose value is corresponding with the applied dose value at this food in order to avoid the germination. (author)

  17. Hybridization assay based on evanescent fluorescence excitation and collection

    Science.gov (United States)

    Sumner, James J.; Mmerole, Robert U.; Stratis-Cullum, Dimitra N.; Yi, Hyunmin; Bentley, William E.; Gillespie, James B.

    2003-08-01

    There is a great need for high throughput and sensitive sensors for genetic analysis. These sensors can be used for varied purposes from monitoring gene expression in organims to speciation of possible pathogens. Consequently, an instrument capable of these tasks would be a great benefit for food and water safety, medical diagnostics and defense of military and civilian populations from biological threats. This work examines the development of a hybridization-based biosensor using a novel tapered fiber optic rpobe. The immobilization of single-stranded, synthetic ologinucleotides utilizing aminoproplytriethoxysilane and glutaraldehyde was implemented on the fiber optic sensor. Hybridization takes place with a complementary analyte sequence followed by a fluorescent, labeled signaling probe to form a sandwich assay. Following hybridization, the fiber is interrogated with a diode laser source and the resulting fluorescence signal is detected using a miniature spectrometer.

  18. Advances in Biological Science.

    Science.gov (United States)

    Oppenheimer, Steven B.; And Others

    1988-01-01

    Reviews major developments in areas that are at the cutting edge of biological research. Areas include: human anti-cancer gene, recombinant DNA techniques for the detection of Huntington disease carriers, and marine biology. (CW)

  19. Biology of Blood

    Science.gov (United States)

    ... Mail Facebook TwitterTitle Google+ LinkedIn Home Blood Disorders Biology of Blood Overview of Blood Medical Dictionary Also ... Version. DOCTORS: Click here for the Professional Version Biology of Blood Overview of Blood Components of Blood ...

  20. Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry.

    Science.gov (United States)

    Bravery, Christopher A; Carmen, Jessica; Fong, Timothy; Oprea, Wanda; Hoogendoorn, Karin H; Woda, Juliana; Burger, Scott R; Rowley, Jon A; Bonyhadi, Mark L; Van't Hof, Wouter

    2013-01-01

    The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development. PMID:23260082

  1. Engineering scalable biological systems

    OpenAIRE

    Lu, Timothy K.

    2010-01-01

    Synthetic biology is focused on engineering biological organisms to study natural systems and to provide new solutions for pressing medical, industrial, and environmental problems. At the core of engineered organisms are synthetic biological circuits that execute the tasks of sensing inputs, processing logic, and performing output functions. In the last decade, significant progress has been made in developing basic designs for a wide range of biological circuits in bacteria, yeast, and mammal...

  2. Systems interface biology

    OpenAIRE

    Francis J Doyle; Stelling, Jörg

    2006-01-01

    The field of systems biology has attracted the attention of biologists, engineers, mathematicians, physicists, chemists and others in an endeavour to create systems-level understanding of complex biological networks. In particular, systems engineering methods are finding unique opportunities in characterizing the rich behaviour exhibited by biological systems. In the same manner, these new classes of biological problems are motivating novel developments in theoretical systems approaches. Henc...

  3. Biological Races in Humans

    OpenAIRE

    Templeton, Alan R.

    2013-01-01

    Races may exist in humans in a cultural sense, but biological concepts of race are needed to access their reality in a non-species-specific manner and to see if cultural categories correspond to biological categories within humans. Modern biological concepts of race can be implemented objectively with molecular genetic data through hypothesis-testing. Genetic data sets are used to see if biological races exist in humans and in our closest evolutionary relative, the chimpanzee. Using the two m...

  4. Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals.

    Science.gov (United States)

    McCall, K A; Fierke, C A

    2000-09-10

    Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes. PMID:10964414

  5. Tango assay for ligand-induced GPCR-β-arrestin2 interaction: Application in drug discovery.

    Science.gov (United States)

    Dogra, Shalini; Sona, Chandan; Kumar, Ajeet; Yadav, Prem N

    2016-01-01

    G protein-coupled receptors (GPCRs) are widely known to modulate almost all physiological functions and have been demonstrated over the time as therapeutic targets for wide gamut of diseases. The design and implementation of high-throughput GPCR-based assays that permit the efficient screening of large compound libraries to discover novel drug candidates are essential for a successful drug discovery endeavor. Usually, GPCR-based functional assays depend primarily on the measurement of G protein-mediated second messenger generation. However, with advent of advanced molecular biology tools and increased understanding of GPCR signal transduction, many G protein-independent pathways such as β-arrestin translocation are being utilized to detect the activity of GPCRs. These assays provide additional information on functional selectivity (also known as biased agonism) of compounds that could be harnessed to develop pathway-selective drug candidates to reduce the adverse effects associated with given GPCR target. In this chapter, we describe the basic principle, detailed methodologies and assay setup, result analysis and data interpretations of the β-arrestin2 Tango assay, and its comparison with cell-based G protein-dependent GPCR assays, which could be employed in a simple academic setup to facilitate GPCR-based drug discovery. PMID:26928547

  6. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.

    Science.gov (United States)

    Dorgan, Kathleen M; Wooderchak, Whitney L; Wynn, Donraphael P; Karschner, Erin L; Alfaro, Joshua F; Cui, Yinqiu; Zhou, Zhaohui Sunny; Hevel, Joan M

    2006-03-15

    Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase. PMID:16460659

  7. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Directory of Open Access Journals (Sweden)

    Bas-jan Van Der Leede

    2015-08-01

    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  8. Evaluation of irradiation in foods using DNA comet assay

    International Nuclear Information System (INIS)

    Comet assay is a rapid, inexpensive and sensitive biological technique to detect DNA damage in food stuffs by irradiation. In this study the Comet assay is applied on foods of plant and animal origins. Samples were irradiated by using 60Co gamma-radiation source. The applied doses were 2, 6 and 10 kGy for food of plant origin and 0.5, 1 and 2 kGy for meat items. The un-irradiated and irradiated samples were clearly differentiated on the basis of DNA fragmentation. During the electrophoresis study, it was found that in un-irradiated cells DNA remained intact and appeared as Comets without tail whereas in irradiated cells Comets with tails were visible due to stretching of fragmented DNA. Moreover, it was also revealed that the DNA tail length was dose dependent. Dry food stuffs (seeds) showed good results as compared to moist foods (meat, fruits and vegetables) due to the absence of background damage. (author)

  9. Upgrading Undergraduate Biology Education

    Science.gov (United States)

    Musante, Susan

    2011-01-01

    On many campuses throughout the country, undergraduate biology education is in serious need of an upgrade. During the past few decades, the body of biological knowledge has grown exponentially, and as a research endeavor, the practice of biology has evolved. Education research has also made great strides, revealing many new insights into how…

  10. Biology Myth-Killers

    Science.gov (United States)

    Lampert, Evan

    2014-01-01

    "Biology Myth-Killers" is an activity designed to identify and correct common misconceptions for high school and college introductory biology courses. Students identify common myths, which double as biology misconceptions, and use appropriate sources to share the "truth" about the myths. This learner-centered activity is a fun…

  11. Designing synthetic biology.

    Science.gov (United States)

    Agapakis, Christina M

    2014-03-21

    Synthetic biology is frequently defined as the application of engineering design principles to biology. Such principles are intended to streamline the practice of biological engineering, to shorten the time required to design, build, and test synthetic gene networks. This streamlining of iterative design cycles can facilitate the future construction of biological systems for a range of applications in the production of fuels, foods, materials, and medicines. The promise of these potential applications as well as the emphasis on design has prompted critical reflection on synthetic biology from design theorists and practicing designers from many fields, who can bring valuable perspectives to the discipline. While interdisciplinary connections between biologists and engineers have built synthetic biology via the science and the technology of biology, interdisciplinary collaboration with artists, designers, and social theorists can provide insight on the connections between technology and society. Such collaborations can open up new avenues and new principles for research and design, as well as shed new light on the challenging context-dependence-both biological and social-that face living technologies at many scales. This review is inspired by the session titled "Design and Synthetic Biology: Connecting People and Technology" at Synthetic Biology 6.0 and covers a range of literature on design practice in synthetic biology and beyond. Critical engagement with how design is used to shape the discipline opens up new possibilities for how we might design the future of synthetic biology. PMID:24156739

  12. The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines

    International Nuclear Information System (INIS)

    Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells. The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation. The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types. Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells

  13. Biological Water or Rather Water in Biology?

    Czech Academy of Sciences Publication Activity Database

    Jungwirth, Pavel

    2015-01-01

    Roč. 6, č. 13 (2015), s. 2449-2451. ISSN 1948-7185 Institutional support: RVO:61388963 Keywords : biological water * protein * interface Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 7.458, year: 2014

  14. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  15. The micronucleus assay in radiation accidents

    International Nuclear Information System (INIS)

    The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is a standardised and validated technique for bio dosimetry. Automated scoring of micronuclei allows large scale applications as in population triage in case of radiation accidents or malevolent use of radioactive sources. The dose detection limit (95% confidence) of the micronucleus assay for individual dose assessment is restricted to 0.2 Gy but can be decreased to 0.1 Gy by scoring centromeres in micronuclei using fluorescence in situ hybridization (FISH). In the past the micronucleus assay was applied for a number of large scale bio monitoring studies of nuclear power plant workers and hospital workers. Baseline micronucleus frequencies depend strongly on age and gender. The assay was also already used for bio dosimetry of radiation accidents. In a multiple endpoint bio dosimetry study for dose assessment of a worker exposed accidentally in 2003 to X-rays, a good agreement was obtained between dose estimates resulting from the micronucleus assay, the scoring of dicentrics and translocations. Automated scoring of micronuclei in combination with centromere signals, allowing systematic bio dosimetry of exposed populations, remains a challenge for the future.

  16. Potentiometric assay for acid and alkaline phosphatase

    International Nuclear Information System (INIS)

    Simple potentiometric kinetic assay for evaluation of acid and alkaline phosphatase activity has been developed. Enzymatically catalyzed hydrolysis of monofluorophosphate, the simplest inorganic compound containing P-F bond, has been investigated as the basis of the assays. Fluoride ions formed in the course of the hydrolysis of this specific substrate have been detected using conventional fluoride ion-selective electrode based on membrane made of lanthanum fluoride. The key analytical parameters necessary for sensitive and selective detection of both enzymes have been assessed. Maximal sensitivity of the assays was observed at monofluorophosphate concentration near 10-3 M. Maximal sensitivity of acid phosphatase assay was found at pH 6.0, but pH of 4.8 is recommended to eliminate effects from alkaline phosphatase. Optimal pH for alkaline phosphatase assay is 9.0. The utility of the developed substrate-sensor system for determination of acid and alkaline phosphatase activity in human serum has been demonstrated

  17. Fundamental Approaches in Molecular Biology for Communication Sciences and Disorders

    Science.gov (United States)

    Bartlett, Rebecca S.; Jette, Marie E.; King, Suzanne N.; Schaser, Allison; Thibeault, Susan L.

    2012-01-01

    Purpose: This contemporary tutorial will introduce general principles of molecular biology, common deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein assays and their relevance in the field of communication sciences and disorders. Method: Over the past 2 decades, knowledge of the molecular pathophysiology of human disease has…

  18. Synthetic biological networks

    International Nuclear Information System (INIS)

    Despite their obvious relationship and overlap, the field of physics is blessed with many insightful laws, while such laws are sadly absent in biology. Here we aim to discuss how the rise of a more recent field known as synthetic biology may allow us to more directly test hypotheses regarding the possible design principles of natural biological networks and systems. In particular, this review focuses on synthetic gene regulatory networks engineered to perform specific functions or exhibit particular dynamic behaviors. Advances in synthetic biology may set the stage to uncover the relationship of potential biological principles to those developed in physics. (review article)

  19. Quantum biological information theory

    CERN Document Server

    Djordjevic, Ivan B

    2016-01-01

    This book is a self-contained, tutorial-based introduction to quantum information theory and quantum biology. It serves as a single-source reference to the topic for researchers in bioengineering, communications engineering, electrical engineering, applied mathematics, biology, computer science, and physics. The book provides all the essential principles of the quantum biological information theory required to describe the quantum information transfer from DNA to proteins, the sources of genetic noise and genetic errors as well as their effects. Integrates quantum information and quantum biology concepts; Assumes only knowledge of basic concepts of vector algebra at undergraduate level; Provides a thorough introduction to basic concepts of quantum information processing, quantum information theory, and quantum biology; Includes in-depth discussion of the quantum biological channel modelling, quantum biological channel capacity calculation, quantum models of aging, quantum models of evolution, quantum models o...

  20. Development of an integrated assay facility

    International Nuclear Information System (INIS)

    The I.R.I.S. concept proposed the use of passive examination and active interrogation techniques in an integrated assay facility. A linac would generate the interrogating gamma and neutron beams. Insufficiently detailed knowledge about active neutron and gamma interrogation of 500 litre drums of cement immobilised intermediate level waste led to a research programme which is now in its main experimental stage. Measurements of interrogation responses are being made using simulated waste drums containing actinide samples and calibration sources, in an experimental assay assembly. Results show that responses are generally consistent with theory, but that improvements are needed in some areas. A preliminary appraisal of the engineering and economic aspects of integrated assay shows that correct operational sequencing is required to achieve the short cycle time needed for high throughput. The main engineering features of a facility have been identified

  1. Nuclear Resonance Fluorescence for Materials Assay

    Energy Technology Data Exchange (ETDEWEB)

    Quiter, Brian J.; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-29

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  2. Nuclear Resonance Fluorescence for Materials Assay

    Energy Technology Data Exchange (ETDEWEB)

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  3. Monitoring environmental exposures with semen assays

    International Nuclear Information System (INIS)

    Semen studies in humans and animals have yielded extensive and compelling evidence that sperm can be used to assess reproductive potential and diagnose pathology. More recent studies on mutagens and carcinogens both at this and other laboratories suggest that a combination of mouse and human assays can be an efficient, effective approach to monitoring for reproductive hazards in the environment. We are investigating the potential of using variability in sperm morphology and DNA content to quantify and monitor the effects of environmental agents on the human testes. Here we review the status of human and mouse assays for environmental surveillance, discuss the genetic and fertility implications of chemically induced semen changes, and describe the high-speed flow methods being developed to automate sperm assays

  4. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall...... causing significant DNA damage was 20 μM for H2O2 and 200 mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA...

  5. Nuclear Resonance Fluorescence for Materials Assay

    International Nuclear Information System (INIS)

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has been performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  6. Nanoparticles for Use in Enzyme Assays.

    Science.gov (United States)

    Kim, Young-Pil; Kim, Hak-Sung

    2016-02-01

    Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology. Here we review recent advances in NP-based biosensors and their use in enzyme assays. With fast and specific responses to enzyme-mediated reactions, NPs transduce and amplify the initial responses into various types of signals, such as electrochemical, optical and magnetic ones. Translation of their potential should lead to functionalized NPs finding wide applications in diagnostics, drug development and biotechnology. PMID:26662229

  7. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

    Directory of Open Access Journals (Sweden)

    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  8. Recent applications of synthetic biology tools for yeast metabolic engineering

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Keasling, Jay

    2015-01-01

    engineer microbial chemical factories has steadily decreased, improvement is still needed. Through the development of synthetic biology tools for key microbial hosts, it should be possible to further decrease the development times and improve the reliability of the resulting microorganism. Together with...... continuous decreases in price and improvements in DNA synthesis, assembly and sequencing, synthetic biology tools will rationalize time-consuming strain engineering, improve control of metabolic fluxes, and diversify screening assays for cellular metabolism. This review outlines some recently developed...... synthetic biology tools and their application to improve production of chemicals and fuels in yeast. Finally, we provide a perspective for the challenges that lie ahead....

  9. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    Science.gov (United States)

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  10. Characterization of bioactivity in treatment wetlands utilising an enzymatic assay

    International Nuclear Information System (INIS)

    Microbial activity is a critical aspect of biological wastewater treatment which is not being routinely monitored as part of treatment wetland research and development. The level of microbial activity is a reference from which observed and variable treatment performance needs to be evaluated with respect to design and operating conditions. The purpose of the present study was to assess enzyme hydrolysis kinetics of the model substrate fluorescein diacetate (FDA) using activated sludge and to begin to relate these findings to wetland mesocosm in-situ enzyme activity measurements. In activated sludge samples, the FDA hydrolysis rate was found to correlate with microbial abundance measured as mixed liquor volatile suspended solids (MLVSS). The ratio of biomass to substrate concentration was also found to influence the extent of FDA consumption and a critical saturation loading for activated sludge on FDA was estimated. Of the numerous empirical enzyme reaction models available in the literature, the Tessier model was determined to most closely fit the experimental data. FDA hydrolysis experiments conducted on activated sludge samples and laboratory wetland mesocosms at the same initial substrate concentration indicate that the enzyme assay is sensitive enough to exhibit characteristic reaction kinetics that can be used to quantify biomass concentrations present within laboratory treatment wetland mesocosms. In continued investigation, changes in mesocosm biomass levels as the wetland vegetation matures will be related to an equivalent MLVSS concentration and the biological treatment system performance. (author)

  11. The Comet Assay: Tails of the (Un)expected. Use of the comet assay in pharmaceutical development.

    OpenAIRE

    Bas-jan Van Der Leede

    2015-01-01

    In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occu...

  12. Method validation and clinical utility of chromogenic factor VIII assay compared to one-stage assay.

    Science.gov (United States)

    Gouws, Wilmare; Botha, Elsabie; Visser, Adele

    2014-01-01

    The chromogenic FVIII assay is currently considered the gold standard for quantitation of factor VIII levels in both haemophilia A patients and as part of screening for thrombophilia. A method validation and evaluation of clinical utility within a routine diagnostic laboratory was undertaken by comparing the currently used one-stage assay to a commercially available chromogenic assay (Siemens, Johannesburg, South Africa). In total, 60 samples were included in this study to encompass the whole diagnostic range of the assay. Both low and high values showed very good correlation on linear regression analysis with correlation coeffients of 0.949 and 0.888 respectively. However, the lower detection limit of the Siemens Chromogenic assay was 1.5 IU/dL rendering it impossible to utilize in the setting of classifying a haemophilia A patient in terms of disease severity. Although the Siemens FVIII chromogenic assay shows excellent correlation to the currently used one-stage assay, the relatively high detection limit restrict implementation as a stand-alone assay in a routine diagnostic laboratory. PMID:23504571

  13. Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

    International Nuclear Information System (INIS)

    KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay (51Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

  14. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    Science.gov (United States)

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page: https://assays.cancer.gov/support/

  15. Bicinchoninic acid (BCA) assay in low volume.

    Science.gov (United States)

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format. PMID:21078286

  16. A sensitive assay for Staphylococcus aureus nucleases

    International Nuclear Information System (INIS)

    A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured [3H] thymidine labelled DNA from E.coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate. (author). 26 refs., 3 figs ., 3 tabs

  17. Sensitive radioenzymatic assay for catechol drugs

    International Nuclear Information System (INIS)

    This assay measures picogram quantities of catechol drugs and endogenous catecholamines in body tissues and fluids. The catechols are converted to their 3H-O-methyl metabolites during incubation with 3H-S-adenosylmethionine then separated by solvent extraction and thin-layer chromatography. Most drugs containing the catechol structure can be radiolabeled and separated from norepinephrine and epinephrine by this technique to provide simultaneous measurement of endogenous and exogenously administered catechols. The disposition of isoproterenol in tissues and fluids of man and experimental animals is measured to illustrate the utility of this assay. The reactivity of several commonly administered catechol drugs with COMT is described and the possible implications discussed

  18. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  19. Elements of nondestructive assay (NDA) technology

    International Nuclear Information System (INIS)

    A thorough introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards is presented. The general principles and major applications of NDA are discussed and situations in which NDA is particularly useful for nuclear safeguards purposes are described. Various passive and active γ-ray and neutron methods are examined and assay situations particularly suited to γ-ray techniques, or to neutron techniques are identified. The role of calorimetry in the NDA of plutonium-bearing materials is also discussed. The advantages and disadvantages of various NDA methods for different types of nuclear materials are mentioned

  20. A Soil-free System for Assaying Nematicidal Activity of Chemicals

    OpenAIRE

    Preiser, F. A.; Babu, J. R.; Haidri, A. A.

    1981-01-01

    A biological assay system for studying the nematicidal activity of chemicals has been devised using a model consisting of cucumber (Cucumis sativus L. cv. Long Marketer) seedlings growing in the diSPo® growth-pouch apparatus. Meloidogyne incognita was used as the test organism. The response was quantified in terms of the numbers of galls produced. Statistical procedures were applied to estimate the ED50 values of currently available nematicides. This system permits accurate quantification of ...

  1. The Comet Assay for the Evaluation of Genotoxic Potential of Landfill Leachate

    OpenAIRE

    Kamila Widziewicz; Joanna Kalka; Magdalena Skonieczna; Paweł Madej

    2012-01-01

    Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) wer...

  2. Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum

    OpenAIRE

    Park, D J; Drobniewski, F. A.; Meyer, A.; Wilson, S M

    2003-01-01

    The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplificati...

  3. Application of single cell gel electrophoresis assay in radiation biodose estimation and environment monitoring

    International Nuclear Information System (INIS)

    Single cell gel electrophoresis(SCGE) or comet assay(CA) is a rapid, simple, sensitive,reliable and inexpensive method for detecting DNA damage in eukaryotic individual cells. The methodology and principles of SCGE are first introduced, such as analytical parameters, automatic analysis of images, advantages of SCGE etc., and then its application is investigated and discussed, including radiation protection and environment monitoring, organism monitoring, biological dose estimation. (authors)

  4. Design of a fluorescence polarization assay platform for the study of human Hsp70

    OpenAIRE

    Kang, Yanlong; Taldone, Tony; Clement, Cristina C.; Fewell, Sheara W.; Aguirre, Julia; Brodsky, Jeffrey L.; Chiosis, Gabriela

    2008-01-01

    The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regu...

  5. Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

    OpenAIRE

    White, L O; Scammell, L. M.; Reeves, D S

    1981-01-01

    Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.

  6. Detection of Dientamoeba fragilis in animal faeces using species specific real time PCR assay.

    Science.gov (United States)

    Chan, Douglas; Barratt, Joel; Roberts, Tamalee; Phillips, Owen; Šlapeta, Jan; Ryan, Una; Marriott, Deborah; Harkness, John; Ellis, John; Stark, Damien

    2016-08-30

    Dientamoeba fragilis is a potentially pathogenic, enteric, protozoan parasite with a worldwide distribution. While clinical case reports and prevalence studies appear regularly in the scientific literature, little attention has been paid to this parasite's biology, life cycle, host range, and possible transmission routes. Overall, these aspects of Dientamoeba biology remain poorly understood at best. In this study, a total of 420 animal samples, collected from Australia, were surveyed for the presence of Dientamoeba fragilis using PCR. Several PCR assays were evaluated for sensitivity and specificity. Two previously published PCR methods demonstrated cross reactivity with other trichomonads commonly found in animal samples. Only one assay exhibited excellent specificity. Using this assay D. fragilis was detected from one dog and one cat sample. This is the first report of D. fragilis from these animals and highlights the role companion animals may play in D. fragilis transmission. This study demonstrated that some published D. fragilis molecular assays cross react with other closely related trichomonads and consequently are not suitable for animal prevalence studies. PMID:27523936

  7. Assays of residual antibiotics after treatment of {gamma}-ray and UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ji Hye; Nam, Ji Hyun; Lee, Dong Hun [Chungbuk National University, Cheongju (Korea, Republic of); Yu, Seung Ho; Lee, Myun Joo [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2010-03-15

    The pollution of antibiotics is a major cause of spreading antibiotics resistant bacteria in the environment. Applications of ozonation, UV, and {gamma}-ray irradiations have been introduced to remove antibiotics in the effluents from wastewater treatment system. In this study, we compared the chemical (HPLC) and biological (antimicrobial susceptibility test, AMS) assays in measuring of the concentrations of residual antibiotics after {gamma}-ray and UV irradiation. Most samples were degraded by {gamma}-ray irradiation (1 {approx} 2 kGy). However, lincomycin and tetracycline were not degraded by UV irradiation. The concentration of residual antibiotics, that was treated with {gamma}-ray and UV irradiation, measuring by bioassay was similar to HPLC. The concentrations of {gamma}-ray irradiated cephradine measured by AMS test were 2 times higher than of HPLC assay, indicating AMS test is more sensitive than HPLC assay. These results indicate that {gamma}-ray irradiation technique is more useful than UV irradiation, and biological assay is more useful to detect the antibiotics and toxic intermediates in antibiotics degradation.

  8. Cell-Based Assay Design for High-Content Screening of Drug Candidates.

    Science.gov (United States)

    Nierode, Gregory; Kwon, Paul S; Dordick, Jonathan S; Kwon, Seok-Joon

    2016-02-01

    To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner. PMID:26428732

  9. Biological efficiency of interaction between various radiation and chemicals

    International Nuclear Information System (INIS)

    This research project has been carried out jointly with INP (Poland) to develop technologies to assess the biological efficiency of interaction between radiation and chemicals. Through the cooperative project, KAERI and INP have established wide variety of bioassay techniques applicable to radiation bioscience, human monitoring, molecular epidemiology and environmental science. The joint experiment, in special, made it possible to utilize the merits of both institutes and to upgrade and verify KAERI's current technology level. All results of the cooperative research will be jointly published in high standard scientific journals listed in the Science Citation Index (SCI), which can make the role of fundamental basis for improving relationship between Korea and Poland. Research skills such as Trad-MCN assay, SCGE assay, immunohistochemical assay and molecular assay developed through joint research will be further elaborated and will be continuously used for the collaboration between two institutes

  10. Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies.

    Science.gov (United States)

    Wang, Lan; Xu, Gang-Ling; Gao, Kai; Wilkinson, Jennifer; Zhang, Feng; Yu, Lei; Liu, Chun-Yu; Yu, Chuan-Fei; Wang, Wen-Bo; Li, Meng; Chen, Wei; Fan, Frank; Cong, Mei; Wang, Jun-Zhi

    2016-06-01

    Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics. PMID:27042807

  11. 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

    2013-09-01

    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. PMID:23346868

  12. Solid-phase group-specific adsorbants in assays for glycoproteins

    International Nuclear Information System (INIS)

    The focus of the paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by the use of the solid-phase cabohydrate-specific adsorbant concanavalin-A. Purification of glycoprotein radioligand after labelling by the Chloramine-T method is readily accomplished using a small column of agarose-bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose-bound concanavalin-A is used to extract and concentrate the glycoproteins from various biological samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5-fold by using concanavalin-A concentrates of 1.5ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose-bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biological samples and for separation of glycoproteins from various interfering factors contained in biological samples before radioligand or radioenzyme assay. (author)

  13. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  14. Genotoxicity screening via the γH2AX by flow assay.

    Science.gov (United States)

    Smart, D J; Ahmedi, K P; Harvey, J S; Lynch, A M

    2011-10-01

    The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on

  15. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    Science.gov (United States)

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  16. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero

    2013-12-01

    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  17. A novel fluorescent assay for sucrose transporters

    Directory of Open Access Journals (Sweden)

    Gora Peter J

    2012-04-01

    Full Text Available Abstract Background We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae is routinely used for the heterologous expression of SUTs and does not take up esculin. Results When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS. Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant and BY4742. Conclusions The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

  18. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida;

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of thes...

  19. Homogeneous screening assay for human tankyrase.

    Science.gov (United States)

    Narwal, Mohit; Fallarero, Adyary; Vuorela, Pia; Lehtiö, Lari

    2012-06-01

    Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors. PMID:22357873

  20. Verification of prior measurements by nondestructive assay

    International Nuclear Information System (INIS)

    Emphasis in this report is placed on the factors affecting nondestructive assay (NDA) measurement accuracies and the selection of the appropriate NDA techniques for certain measurements based on these factors. Factors affecting measurement accuracies are operational characteristics, isotopic composition of SNM sample, response dependence on the SNM and matrix materials within sample, and composition and dimension of the container

  1. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    A radioligand assay for biotin in liver tissue is described. 3H-biotin is used as tracer and avidin as binder. The biotin-loaded avidin is separated from free biotin on dextran-coated charcoal, which leaves the avidin-biotin complex in the supernatant liquid. Thus, the avidin-biotin complex can easily be utilized for determination of the radioactivity. Calibration with known additions of biotin in the range 0.25-8.0 ng per assay sample yields a linear logit-log plot. The biotin is extracted from liver tissues by enzymatic proteolysis with papain. This treatment is optimized to liberate the bound forms of the vitamin. Microbiological parallel assays with Lactobacillus plantarum were in good agreement with the radioligand assay giving a regression coefficient of 0.974(n=44). The coefficient of variation was found to be 4.2% in the range 500-1200 ng of biotin per g of liver tissue (n=46). The method is simple and reliable and allows the simultaneous analysis of a considerable number of samples. (Auth.)

  2. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...

  3. Antibacterial effect of protamine assayed by impedimetry

    DEFF Research Database (Denmark)

    Johansen, Charlotte; Gill, T.; Gram, Lone

    1995-01-01

    Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained (r = 0 . 99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when...

  4. Advanced analysis techniques for uranium assay

    International Nuclear Information System (INIS)

    Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples count rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.

  5. A radiometric assay for HIV-1 protease

    International Nuclear Information System (INIS)

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources

  6. Nondestructive assay of boxed radioactive waste

    International Nuclear Information System (INIS)

    This paper describes the problems related to the nondestructive assay (NDA) of boxed radioactive waste at the Hanford Site and how Westinghouse Hanford company (WHC) is solving the problems. The waste form and radionuclide content are described. The characteristics of the combined neutron and gamma-based measurement system are described

  7. Branching processes in biology

    CERN Document Server

    Kimmel, Marek

    2015-01-01

    This book provides a theoretical background of branching processes and discusses their biological applications. Branching processes are a well-developed and powerful set of tools in the field of applied probability. The range of applications considered includes molecular biology, cellular biology, human evolution and medicine. The branching processes discussed include Galton-Watson, Markov, Bellman-Harris, Multitype, and General Processes. As an aid to understanding specific examples, two introductory chapters, and two glossaries are included that provide background material in mathematics and in biology. The book will be of interest to scientists who work in quantitative modeling of biological systems, particularly probabilists, mathematical biologists, biostatisticians, cell biologists, molecular biologists, and bioinformaticians. The authors are a mathematician and cell biologist who have collaborated for more than a decade in the field of branching processes in biology for this new edition. This second ex...

  8. Application of the parallel line assay to assessment of biosimilar products based on binary endpoints.

    Science.gov (United States)

    Lin, Jr-Rung; Chow, Shein-Chung; Chang, Chih-Hsi; Lin, Ya-Ching; Liu, Jen-pei

    2013-02-10

    Biological drug products are therapeutic moieties manufactured by a living system or organisms. These are important life-saving drug products for patients with unmet medical needs. Because of expensive cost, only a few patients have access to life-saving biological products. Most of the early biological products will lose their patent in the next few years. This provides the opportunity for generic versions of the biological products, referred to as biosimilar drug products. The US Biologic Price Competition and Innovation Act passed in 2009 and the draft guidance issued in 2012 provide an approval pathway for biological products shown to be biosimilar to, or interchangeable with, a Food and Drug Administration-licensed reference biological product. Hence, cost reduction and affordability of the biosimilar products to the average patients may become possible. However, the complexity and heterogeneity of the molecular structures, complicated manufacturing processes, different analytical methods, and possibility of severe immunogenicity reactions make evaluation of equivalence between the biosimilar products and their corresponding reference product a great challenge for statisticians and regulatory agencies. To accommodate the stepwise approach and totality of evidence, we propose to apply a parallel assay to evaluate the extrapolation of the similarity in product characteristics such as doses or pharmacokinetic responses to the similarity in binary efficacy endpoints. We also report the results of simulation studies to evaluate the performance, in terms of size and power, of our proposed methods. We present numerical examples to illustrate the suggested procedures. PMID:22911920

  9. Spectroscopy of biological nanocrystals

    OpenAIRE

    Ortac, Inanc; Severcan, Feride

    2007-01-01

    Nanocrystals have gained much interest in recent years, due to their unusual properties allowing interesting applications in physical and biological science. In this literature review, biological nanocrystals are discussed from the spectroscopic point of view. Firstly, the theory behind the outstanding abilities of the nanocrystals is described. Secondly, the spectroscopic properties of biological nanocrystals are mentioned. Lastly, the use of nanocrystals with various spectroscopic applicati...

  10. Biological detector and method

    Energy Technology Data Exchange (ETDEWEB)

    Sillerud, Laurel; Alam, Todd M; McDowell, Andrew F

    2014-04-15

    A biological detector includes a conduit for receiving a fluid containing one or more magnetic nanoparticle-labeled, biological objects to be detected and one or more permanent magnets or electromagnet for establishing a low magnetic field in which the conduit is disposed. A microcoil is disposed proximate the conduit for energization at a frequency that permits detection by NMR spectroscopy of whether the one or more magnetically-labeled biological objects is/are present in the fluid.

  11. Biological detector and method

    Energy Technology Data Exchange (ETDEWEB)

    Sillerud, Laurel; Alam, Todd M.; McDowell, Andrew F.

    2015-11-24

    A biological detector includes a conduit for receiving a fluid containing one or more magnetic nanoparticle-labeled, biological objects to be detected and one or more permanent magnets or electromagnet for establishing a low magnetic field in which the conduit is disposed. A microcoil is disposed proximate the conduit for energization at a frequency that permits detection by NMR spectroscopy of whether the one or more magnetically-labeled biological objects is/are present in the fluid.

  12. Introducing Aquatic Biology

    OpenAIRE

    Kinne, Otto; Browman, Howard I.; Seaman, Matthias

    2007-01-01

    The Inter-Research Science Center (IR) journals Marine Ecology Progress Series (MEPS) and Aquatic Microbial Ecology (AME) have been receiving increasing numbers of high-quality manuscripts that are principally biological, rather than ecological. With regret, we have had to turn these submissions away. Also, leading limnologists have for many years suggested that IR should provide an outlet for top quality articles on freshwater biology and ecology. Aquatic Biology (...

  13. Biological detector and method

    Science.gov (United States)

    Sillerud, Laurel; Alam, Todd M; McDowell, Andrew F

    2013-02-26

    A biological detector includes a conduit for receiving a fluid containing one or more magnetic nanoparticle-labeled, biological objects to be detected and one or more permanent magnets or electromagnet for establishing a low magnetic field in which the conduit is disposed. A microcoil is disposed proximate the conduit for energization at a frequency that permits detection by NMR spectroscopy of whether the one or more magnetically-labeled biological objects is/are present in the fluid.

  14. Glycobiology Current Molecular Biology

    OpenAIRE

    Sabire KARAÇALI

    2003-01-01

    Carbohydrate chemistry evolved into carbohydrate biochemistry and gradually into the biology of carbohydrates, or glycobiology, at the end of the last century. Glycobiology is the new research area of modern molecular biology, and it investigates the structure, biosynthesis and biological functions of glycans. The numbers, linkage types (a or b), positions, binding points and functional group differences of monosaccharides create microheterogeneity. Thus, numerous glycoforms with precise stru...

  15. Foundations of biology

    OpenAIRE

    Sikorav, Jean-Louis; Braslau, Alan; Goldar, Arach

    2014-01-01

    It is often stated that there are no laws in biology, where everything is contingent and could have been otherwise, being solely the result of historical accidents. Furthermore, the customary introduction of fundamental biological entities such as individual organisms, cells, genes, catalysts and motors remains largely descriptive; constructive approaches involving deductive reasoning appear, in comparison, almost absent. As a consequence, both the logical content and principles of biology ne...

  16. Non-separation assay for glycohemoglobin.

    Science.gov (United States)

    Blincko, S; Edwards, R

    1998-06-01

    The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD +/-0.03)%HbA1 +0.6(SD +/-0.3), S(y/x) = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD +/-0.04)%HbA1c + 1.8(SD +/-0.3), S(y/x) = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was <2% (intraassay, range 5-22% GHb) and <4.2% (interassay, range 4-16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 micromol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results. PMID:9625057

  17. Nondestructive waste assay capability evaluation project

    International Nuclear Information System (INIS)

    For most containerized waste forms, the most common and efficient means to determine the TRU material quantity is the use of nondestructive assay (NDA) techniques. Requirements and quality assurance objectives (QAOs) for NDA techniques employed to characterize TRU waste destined for the Waste Isolation Pilot Plant are delineated in the National TRU Program Transuranic Waste Characterization Quality Assurance Program Plan (QAPP). These QAOs represent minimum performance requirements consisting of allowable measurement parameter ranges such as bias and precision for which compliance must be demonstrated in accordance with the prescriptions of the QAPP. To establish actual capabilities of existing nondestructive waste assay systems relative to the requirements of the QAPP, a mechanism is required to acquire performance data for waste types typical of the DOE inventory and to objectively evaluate such data. In accordance with this need, the DOE mixed waste focus area operating in conjunction with the characterization monitoring and sensor technology crosscut area program has instituted an assay capability evaluation project (CEP). The project is focused on mobile NDA technologies but is applicable to most permanently installed systems operating the same NDA technology. The evaluation project is currently being conducted at the Idaho National Engineering and Environmental Laboratory (INEEL) Radioactive Waste Management Complex (RWMC) using actual Rocky Flats Plant-generated waste forms currently in storage and appropriate matrix/radioactive material surrogates. Six mobile waste assay system technologies are participating in the CEP: (1) segmented gamma scanner; (2) active and passive computed tomography; (3) high-efficiency neutron counter; (4) IQ3-gamma assay system; (5) tomographic gamma scanner; and (6) an imaging passive/active neutron system

  18. Assays for mammalian tyrosinase: a comparative study

    International Nuclear Information System (INIS)

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed

  19. Production and assay of forskolin antibodies

    International Nuclear Information System (INIS)

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

  20. Biological aerosol background characterization

    Science.gov (United States)

    Blatny, Janet; Fountain, Augustus W., III

    2011-05-01

    To provide useful information during military operations, or as part of other security situations, a biological aerosol detector has to respond within seconds or minutes to an attack by virulent biological agents, and with low false alarms. Within this time frame, measuring virulence of a known microorganism is extremely difficult, especially if the microorganism is of unknown antigenic or nucleic acid properties. Measuring "live" characteristics of an organism directly is not generally an option, yet only viable organisms are potentially infectious. Fluorescence based instruments have been designed to optically determine if aerosol particles have viability characteristics. Still, such commercially available biological aerosol detection equipment needs to be improved for their use in military and civil applications. Air has an endogenous population of microorganisms that may interfere with alarm software technologies. To design robust algorithms, a comprehensive knowledge of the airborne biological background content is essential. For this reason, there is a need to study ambient live bacterial populations in as many locations as possible. Doing so will permit collection of data to define diverse biological characteristics that in turn can be used to fine tune alarm algorithms. To avoid false alarms, improving software technologies for biological detectors is a crucial feature requiring considerations of various parameters that can be applied to suppress alarm triggers. This NATO Task Group will aim for developing reference methods for monitoring biological aerosol characteristics to improve alarm algorithms for biological detection. Additionally, they will focus on developing reference standard methodology for monitoring biological aerosol characteristics to reduce false alarm rates.

  1. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  2. 21 CFR 866.3305 - Herpes simplex virus serological assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices... herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus...

  3. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864... reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the... fluorescence and photometry. The results of this assay are used in the diagnosis of liver disease,...

  4. 21 CFR 864.7455 - Fetal hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fetal hemoglobin assay. 864.7455 Section 864.7455... assay. (a) Identification. A fetal hemoglobin assay is a device that is used to determine the presence... hemoglobin present. The assay may be used to detect fetal red cells in the maternal circulation or to...

  5. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  6. A quantitative comet infection assay for influenza virus

    OpenAIRE

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

    2011-01-01

    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 in...

  7. Assessment of Genotoxicity of Ionizing radiation using Tradescantia-Comet assay

    International Nuclear Information System (INIS)

    Over the last two decades, several new methodologies for the detection of DNA damage have been developed. The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, also called the single cell gel electrophoresis (SCGE) was first introduced by Ostling and Johanson as a microelectrophoretic technique for the direct visualization of DNA damage in individual cells. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Although the genotoxic effects detected by Tradescantia tests cannot be associated with mutagenesis or even carcinogenesis in humans, these bioassays are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay

  8. Complementary assays for monitoring susceptibility of varicella-zoster virus resistance to antivirals.

    Science.gov (United States)

    Perrier, Marine; Désiré, Nathalie; Deback, Claire; Agut, Henri; Boutolleau, David; Burrel, Sonia

    2016-07-01

    The emergence of varicella-zoster virus (VZV) resistance to current antivirals as acyclovir (ACV) constitutes a hindrance to antiviral treatment effectiveness of VZV infections, especially in immunocompromised patients. The molecular mechanisms of VZV resistance reported so far rely on the presence of mutations within thymidine kinase (TK, ORF36) and DNA polymerase (ORF28) viral genes. The aim of this work was to develop reliable and complementary diagnostic methods to detect VZV antiviral resistance: (i) a genotypic assay based on TK and DNA polymerase genes sequencing, (ii) a plaque reduction assay to determine antiviral 50% effective concentrations, and (iii) a functional assay to evaluate in vitro phosphorylation activity of recombinant TKs. As a whole, this study included the analysis of 21 VZV clinical isolates and 62 biological samples from patients experiencing VZV infection. Genetic analysis revealed 3 and 9 new amino acid changes that have not been previously described within the highly conserved TK and DNA polymerase, respectively. Then, VZV isolates bearing newly identified mutations considered as natural polymorphisms were characterized as susceptible to ACV using plaque-reduction assay in MeWo cells. In parallel, the impact of TK changes on ACV phosphorylation activity was examined using a nonradioactive in vitro enzymatic assay. PMID:26994966

  9. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    Science.gov (United States)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  10. Novel bioluminescent binding assays for interaction studies of protein/peptide hormones with their receptors.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-05-01

    Protein/peptide hormones are the largest group of endogenous signaling molecules and exert various biological functions by binding to specific cell membrane receptors. To study the interactions between these hormones and their receptors, quantitative ligand-receptor binding assays have been widely used for decades. However, the assays conventionally relied on the use of radioligands, which have some major drawbacks and can only be used in laboratories with a radioactive material license. We recently developed novel bioluminescent binding assays for several protein/peptide hormones using the brightest bioluminescent reporter known to date, nanoluciferase (NanoLuc). The NanoLuc reporter can be either chemically conjugated to an appropriate position, or genetically fused at one terminus, of protein/peptide hormones. Compared to conventional radioligands, these bioluminescent ligands have higher sensitivity, better safety, and longer shelf lives, and thus, represent a novel class of non-radioactive tracers for quantitative receptor binding assays. In the present review, we provide some general considerations and specific examples for setting up the bioluminescent binding assays. Such techniques can be applied to other protein/peptide hormones in future to facilitate their interaction studies with their receptors. PMID:27020777

  11. Comparative titers of egg assay against immunofluorescent assay of Chlamydia psittaci.

    OpenAIRE

    Tessler, J

    1985-01-01

    A comparison of titers was made between an egg assay and a direct fluorescent antibody assay of three chlamydial strains propagated in Vero cells with and without cortisone plus cytochalasin B. The titer of NJ-1 strain was similar in the egg titration and the fluorescent antibody assay in the untreated sample and a little lower for the sample treated with cytochalasin B and cortisone. The SCT and CDC strains had approximately the same titers in the egg titration and the fluorescent antibody a...

  12. Test and standardization of a radiometric method for free testosterone assay in biological materials

    International Nuclear Information System (INIS)

    The principles of equilibrium dialysis technique using semi-permeable cellulose membranes were analysed and a modification was presented for the calculation of the unbound or free fraction of plasma steroids. The results were compared with those from the classical way of calculation used in the equilibrium dialysis technique in which it is necessary to include the change in weight of the dialysis bags. Technical aspects of the method were studied such as weight variation of the dialysis bags during dialysis, comparison of the results obtained using multiple plasma dilutions or one dilution only and comparing serum and plasma. The results obtained with the multiple dilutions technique were similar to those with non-diluted plasma while significantly different from those using one dilution only. The operational characteristics of the method were evaluated showing its specificity at least by the dilution test, besides being precise considering intra and interassay reproductibility. The mean + - s.d. of the percent free testosterone levels were 2.57 + - 0.45% and 1.67 + - 0.30% for males and females at the mid-follicular phase respectively. (Author)

  13. Assessment of biochar safety via its leachate characterization using physicochemical and biological assays

    Science.gov (United States)

    Dailianis, Stefanos; Tsouloufa, Argyro; Antonopoulou, Maria; Konstantinou, Ioannis; Karapanagioti, Hrissi K.; Manariotis, Ioannis D.

    2016-04-01

    The present study investigates the physicochemical composition of water aliquots derived from biochars produced from the pyrolysis of malt spent rootlets, in combination with the concomitant toxicological profile in each case. Specifically, physicochemical parameters and heavy metal ions were determined in aliquots of six (6) serial washes of biochar (1.5 g of solid was added in column and washed 6 times with 40 mL of distilled water per wash). The chemical analysis of each aliquot showed increased levels of PO4-3, Cl-, NO3-, SO4-2, F- and Br- in the first wash aliquot, followed by a significant decrease over washes. Non-detectable concentrations were observed after 3 washes in almost all cases. Similarly, the increased levels of Zn, Be, Cs, Mn, V and Se determined in the first wash aliquot were eliminated followed successive washes. In parallel, the toxic potency of each wash aliquot was recorded by (a) a multi-well test plate bioassay, using instars II-III larvae of the fairy shrimp Thamnocephalus platyurus, hatched from cysts derived from Screening Toxicity test supplied by MicroBio Tests Inc. (Thamnotoxkit FTM) and (b) the Microtox bioassay, using bioluminescent bacteria Vibrio fischeri. According to the results, first and second wash aliquots were toxic for T. platyurus (LC50 values of 22.12 and 68.28% v/v, respectively), followed by a significant elimination of toxicity after further washes in all cases. Similarly, the Microtox bioassay showed a significant inhibition of Vibrio luminescence after treatment for a period of 5-90 min (98-100% inhibition of luminescence) with the first wash aliquot (EC50 ≤ 0.01 % v/v), with no toxicity to be observed after successive washes. According to the results, at least one wash of biochar is prerequisite for improving its safety for further use. Moreover, the removal of both inorganic and organic, such as metal ions, substances commonly washed by the biochar, could be a crucial step for its sustainable use and final application, thus avoiding the induction of adverse effects on biota.

  14. Biological Assays and Chemical Composition of Volatile Oils of Bupleurum fruticosum L. (Apiaceae

    Directory of Open Access Journals (Sweden)

    Andrea Maxia

    2011-01-01

    Full Text Available The composition of supercritical CO 2 extracts and essential oils obtained by hydrodistillation of Bupleurum fruticosum L., growing spontaneously in Italy and Portugal, and its antifungal activity is reported. The collected extracts were analyzed by GC-FID and GC-MS methods. The minimal inhibitory concentration (MIC and the minimal lethal concentration (MLC were used to evaluate the antifungal activity of the oils against Candida albicans, C. tropicalis, C. krusei, C. guillermondii, C. parapsilosis, Cryptococcus neoformans, Trichophyton rubrum, T. mentagrophytes, Microsporum canis, M. gypseum, Epidermophyton floccosum, Aspergillus niger, A. fumigatus and A. flavus.

  15. Comparison of components of biological variation between 3 equine thromboelastography assays

    DEFF Research Database (Denmark)

    Laursen, Sigrid Hyldahl; Andersen, Pia Haubro; Kjelgaard-Hansen, Mads; Wiinberg, Bo

    2013-01-01

    There is a paucity of information about the analytical performance of thromboelastography (TEG) in horses, specifically concerning components of variation among different analytical methods. Such data may be obtained by nested analysis of repetitive standardized sampling of healthy individuals....

  16. Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays

    DEFF Research Database (Denmark)

    Molina, Lazaro; Rodriguez, Cayo Juan Ramos; Ronchel, Maria C.; Molin, Søren; Ramos, Juan L.

    1998-01-01

    fusion of the Pm promoter to the lad gene plus the positive regulator of the Pm promoter, the xylS gene. This mini-Tn5 transposon was transferred to the chromosome of Pseudomonas putida CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide...

  17. Assessment of pollution in road runoff using a Bufo viridis biological assay

    International Nuclear Information System (INIS)

    Road runoff is a major source of environmental pollution, significantly threatening nearby aquatic habitats. Chemical analyses indicate high pollutant concentrations in the road's 'first flush', but bioassays are more advantageous for addressing the cumulative effects of the numerous pollutants within the runoff. We used Bufo viridis embryos and larvae to assess the toxicity of road runoff from two major highways in Israel. We show, for the first time, that exposure to midseason runoff not only has an adverse effect on growth and development rates of B. viridis larvae but can also lead to increased rates of morphological deformations. Seasonal first flushes, despite having higher metal concentrations, did not adversely affect the toad larvae, apparently due to a counter effect of organic matter that potentially served as a supplementary energy resource. Road runoff can be a major cause for a qualitative decrease in the quality of aquatic habitats threatening amphibians in Israel. - Highway runoff has detrimental effects on the development of B. viridis larvae.

  18. Biological dosimetry at the tissue-barium sulphate interface: a jejunal crypt survival assay

    International Nuclear Information System (INIS)

    Fission Electric Cells (FEC) able to deliver significant power at high neutron fluxes and to operate without supply were tested in the nuclear reactor of the Central Institute of Physics in Bucharest, Romania, at a 3 x 109 neutrons/cm2s thermal neutron flux. The experimental results would make the design of efficient FEC for space reactors feasible. (author)

  19. Assessment of pollution in road runoff using a Bufo viridis biological assay

    Energy Technology Data Exchange (ETDEWEB)

    Dorchin, A., E-mail: adorchin@campus.haifa.ac.i [Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, Haifa 31905 (Israel); Shanas, U., E-mail: shanas@research.haifa.ac.i [Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, Haifa 31905 (Israel); Department of Biology, Faculty of Natural sciences, University of Haifa - Oranim, Tiv' on 36006 (Israel)

    2010-12-15

    Road runoff is a major source of environmental pollution, significantly threatening nearby aquatic habitats. Chemical analyses indicate high pollutant concentrations in the road's 'first flush', but bioassays are more advantageous for addressing the cumulative effects of the numerous pollutants within the runoff. We used Bufo viridis embryos and larvae to assess the toxicity of road runoff from two major highways in Israel. We show, for the first time, that exposure to midseason runoff not only has an adverse effect on growth and development rates of B. viridis larvae but can also lead to increased rates of morphological deformations. Seasonal first flushes, despite having higher metal concentrations, did not adversely affect the toad larvae, apparently due to a counter effect of organic matter that potentially served as a supplementary energy resource. Road runoff can be a major cause for a qualitative decrease in the quality of aquatic habitats threatening amphibians in Israel. - Highway runoff has detrimental effects on the development of B. viridis larvae.

  20. A Sensitive Cell-Based Assay to Measure the Doxycycline Concentration in Biological Samples

    NARCIS (Netherlands)

    W. Kleibeuker; X. Zhou; M. Centlivre; N. Legrand; M. Page; N. Almond; B. Berkhout; A.T. Das

    2009-01-01

    Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measu

  1. Extracellular Matrix Invasion in Metastases and Angiogenesis: Commentary on the Matrigel "Chemoinvasion Assay".

    Science.gov (United States)

    Albini, Adriana

    2016-08-15

    Invasive and metastatic cells must cross the basement membrane's extracellular matrix to disseminate to distant sites. Although in the eighties the concept was well established, no easy in vitro functional assay was available. Working in Hynda Kleinman's and George Martin's laboratory at NIH (Bethesda, MD), where the reconstituted basement membrane Matrigel was discovered, I had the intuition that Matrigel coating of migration filters could represent a valid tool to mimic in vitro biological matrix barriers. The "chemoinvasion assay" using Matrigel in Boyden blind-well chambers was developed in 1985-1986 and published in Cancer Research in 1987. It was a rapid and easy tool for studying invasion, a crucial step in cancer metastasis. Since its conception, the assay has been employed for studies on the metastatic process, angiogenesis, and for the screening of drugs that are potentially able to decrease cell invasion. It was adapted to be easily employed as a routine assay and commercialized. In that historical article, we also described the use of thick layers of Matrigel for the study of morphogenesis of invasive cells, a simple and visual assay, adaptable to reproduce collective cell migration in vitro To date, in its diverse optimized variants, the chemoinvasion assay is still widely used, contributing to novel data production. In the era of precision medicine and next-generation sequencing, the cheap, fast, and reproducible chemoinvasion assay may have further developments, including possible applications in the investigations on cancer stem cells, immunity and immune modulators, applications with siRNA silencing, selection of aggressive cell populations, and phenotypes and genetic evaluations. Cancer Res; 76(16); 4595-7. ©2016 AACR.See related article by Albini A et al., Cancer Res 1987;47:3239-45Visit the Cancer Research 75(th) Anniversary timeline. PMID:27528578

  2. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    OpenAIRE

    Sasaki, Yu F.; Satomi Kawaguchi; Takanori Nakamura; Gisho Honda; Ayumi Yamamoto

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 hum...

  3. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

    OpenAIRE

    F.J. Negrão; A.A. Alfieri; A.F. Alfieri

    2007-01-01

    Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV) by a reverse transcription-polymerase chain reaction (RT-PCR) assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and ...

  4. Biological Clocks & Circadian Rhythms

    Science.gov (United States)

    Robertson, Laura; Jones, M. Gail

    2009-01-01

    The study of biological clocks and circadian rhythms is an excellent way to address the inquiry strand in the National Science Education Standards (NSES) (NRC 1996). Students can study these everyday phenomena by designing experiments, gathering and analyzing data, and generating new experiments. As students explore biological clocks and circadian…

  5. Experimenting with Mathematical Biology

    Science.gov (United States)

    Sanft, Rebecca; Walter, Anne

    2016-01-01

    St. Olaf College recently added a Mathematical Biology concentration to its curriculum. The core course, Mathematics of Biology, was redesigned to include a wet laboratory. The lab classes required students to collect data and implement the essential modeling techniques of formulation, implementation, validation, and analysis. The four labs…

  6. Bioinformatics and School Biology

    Science.gov (United States)

    Dalpech, Roger

    2006-01-01

    The rapidly changing field of bioinformatics is fuelling the need for suitably trained personnel with skills in relevant biological "sub-disciplines" such as proteomics, transcriptomics and metabolomics, etc. But because of the complexity--and sheer weight of data--associated with these new areas of biology, many school teachers feel…

  7. Biological Macromolecule Crystallization Database

    Science.gov (United States)

    SRD 21 Biological Macromolecule Crystallization Database (Web, free access)   The Biological Macromolecule Crystallization Database and NASA Archive for Protein Crystal Growth Data (BMCD) contains the conditions reported for the crystallization of proteins and nucleic acids used in X-ray structure determinations and archives the results of microgravity macromolecule crystallization studies.

  8. Biological pretreatment sewages water

    OpenAIRE

    Veselý, Václav

    2009-01-01

    Bachelor's thesis deals with waste water purification at the stage of pre-inflow of water into the biological waste water treatment plants. It is divided into two parts, a theoretical and calculation. The theoretical part deals about sewage water and the method of biological treatment. Design proposal is part of the activation tank for quantity EO.

  9. Integrated Biological Control

    International Nuclear Information System (INIS)

    Biological control is any activity taken to prevent, limit, clean up, or remediate potential environmental, health and safety, or workplace quality impacts from plants, animals, or microorganisms. At Hanford the principal emphasis of biological control is to prevent the transport of radioactive contamination by biological vectors (plants, animals, or microorganisms), and where necessary, control and clean up resulting contamination. Other aspects of biological control at Hanford include industrial weed control (e.g.; tumbleweeds), noxious weed control (invasive, non-native plant species), and pest control (undesirable animals such as rodents and stinging insects; and microorganisms such as molds that adversely affect the quality of the workplace environment). Biological control activities may be either preventive (apriori) or in response to existing contamination spread (aposteriori). Surveillance activities, including ground, vegetation, flying insect, and other surveys, and apriori control actions, such as herbicide spraying and placing biological barriers, are important in preventing radioactive contamination spread. If surveillance discovers that biological vectors have spread radioactive contamination, aposteriori control measures, such as fixing contamination, followed by cleanup and removal of the contamination to an approved disposal location are typical response functions. In some cases remediation following the contamination cleanup and removal is necessary. Biological control activities for industrial weeds, noxious weeds and pests have similar modes of prevention and response

  10. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    Science.gov (United States)

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  11. Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion

    Energy Technology Data Exchange (ETDEWEB)

    Vuojola, Johanna, E-mail: johanna.vuojola@utu.fi [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland); Riuttamaeki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland)

    2012-05-06

    Highlights: Black-Right-Pointing-Pointer We demonstrate the use of photon upconversion in a caspase-3 activity assay. Black-Right-Pointing-Pointer The separation-free assay uses an internally quenched substrate peptide. Black-Right-Pointing-Pointer UCPs enable simple instrumentation and total elimination of autofluorescence. Black-Right-Pointing-Pointer A sensitive assay with high signal-to-background ratios was achieved. Black-Right-Pointing-Pointer Suitable for high-throughput screening through miniaturization and white plates. - Abstract: Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC{sub 50} values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an

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

    International Nuclear Information System (INIS)

    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

  13. Biological evaluation of RVNRL

    International Nuclear Information System (INIS)

    Cytotoxicity testing (cell growth inhibition, LDH-assay) has shown that RVNRL materials are considerably less cytotoxic than sulphur-vulcanised materials. Also the effect of applying different leaching times during the production process was investigated and gave interesting results. Protein testing (BCA - comparable to a Lowry assay ) has shown that some, but not all proteins are destroyed during irradiation. For the total protein content of the materials as determined in this way, the effect of leaching proved to be very important. This was also checked by; SDS-PAGE assays: After irradiation a smear is observed with SDS-PAGE; when the irradiated material has been leached, most of the smear has disappeared. An important protein (14 kD) is still present and seems hardly to be affected. The materials have also been evaluated clinically, using well characterised patient sera (Western blotting) in order to determine whether the remaining proteins are allergenic

  14. Biological sample collector

    Science.gov (United States)

    Murphy, Gloria A.

    2010-09-07

    A biological sample collector is adapted to a collect several biological samples in a plurality of filter wells. A biological sample collector may comprise a manifold plate for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the tube to deposit a biological sample onto a filter in the bottom of the selected filter well. The biological sample collector may be portable.

  15. Optics of Biological Particles

    CERN Document Server

    Hoekstra, Alfons; Videen, Gorden

    2007-01-01

    This book covers the optics of single biological particles, both theory and experiment, with emphasis on Elastic Light Scattering and Fluorescence. It deals with the optics of bacteria (bio-aerosols), marine particles (selected phytoplankton communities) and red and white blood cells. Moreover, there are dedicated chapters on a general theory for scattering by a cell, and modelling and simulation of scattering by inhomogeneous biological cells. Finally, one chapter is dedicated to astro-biological signatures, discussing the possibilities for detecting non-terrestrial biological material. The volume has up-to-date discussions on new experimental and numerical techniques, and many examples of applications of these techniques in real-life systems, as used to detect and characterize e.g. biological warfare agents or human blood cells.

  16. Frontiers in mathematical biology

    CERN Document Server

    1994-01-01

    Volume 100, which is the final volume of the LNBM series serves to commemorate the acievements in two decades of this influential collection of books in mathematical biology. The contributions, by the leading mathematical biologists, survey the state of the art in the subject, and offer speculative, philosophical and critical analyses of the key issues confronting the field. The papers address fundamental issues in cell and molecular biology, organismal biology, evolutionary biology, population ecology, community and ecosystem ecology, and applied biology, plus the explicit and implicit mathematical challenges. Cross-cuttting issues involve the problem of variation among units in nonlinear systems, and the related problems of the interactions among phenomena across scales of space, time and organizational complexity.

  17. Comparison of LC-MS Assay and HPLC Assay of Busulfan in Clinical Pharmacokinetics Studies

    OpenAIRE

    Hongxia Lin; Susan Goodin; Strair, Roger K.; Robert S. DiPaola; Gounder, Murugesan K.

    2012-01-01

    Busulfan is used in preparative regimens for bone marrow transplantation and timely busulfan plasma concentration reporting is critical for subsequent dose adjustment. We compared two sensitive methods for pharmacokinetics studies including LC-MS assay and HPLC precolumn derivatization assay. Chromatographic separation was performed on a Gemini C18 column. Liquid-liquid extraction with ethyl acetate was used for plasma sample preparation. Busulfan and internal standard ([2H8]-busulfan) were d...

  18. Identification of irradiated pepper with comet assay

    International Nuclear Information System (INIS)

    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  19. Identification of irradiated pepper with comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail: efprieto@ceaden.edu.cu; damaris@ceaden.edu.cu; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)

    2007-07-01

    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  20. Developments in plutonium waste assay at AWE.

    Science.gov (United States)

    Miller, T J

    2009-06-01

    In 2002 a paper was presented at the 43rd Annual Meeting of the Institute of Nuclear Materials Management (INMM) on the assay of low level plutonium (Pu) in soft drummed waste (Miller 2002 INMM Ann. Meeting (Orlando, FL, 23-27 July 2002)). The technique described enabled the Atomic Weapons Establishment (AWE), at Aldermaston in the UK, to meet the stringent Low Level Waste Repository at Drigg (LLWRD) conditions for acceptance for the first time. However, it was initially applied to only low density waste streams because it relied on measuring the relatively low energy (60 keV) photon yield from Am-241 during growth. This paper reviews the results achieved when using the technique to assay over 10,000 waste packages and presents the case for extending the range of application to denser waste streams. PMID:19454791

  1. Developments in plutonium waste assay at AWE

    Energy Technology Data Exchange (ETDEWEB)

    Miller, T J [AWE, Aldermaston, Reading, Berkshire RG7 4PR (United Kingdom)], E-mail: Timothy.Miller@awe.co.uk

    2009-06-15

    In 2002 a paper was presented at the 43rd Annual Meeting of the Institute of Nuclear Materials Management (INMM) on the assay of low level plutonium (Pu) in soft drummed waste (Miller 2002 INMM Ann. Meeting (Orlando, FL, 23-27 July 2002)). The technique described enabled the Atomic Weapons Establishment (AWE), at Aldermaston in the UK, to meet the stringent Low Level Waste Repository at Drigg (LLWRD) conditions for acceptance for the first time. However, it was initially applied to only low density waste streams because it relied on measuring the relatively low energy (60 keV) photon yield from Am-241 during growth. This paper reviews the results achieved when using the technique to assay over 10 000 waste packages and presents the case for extending the range of application to denser waste streams.

  2. High-Throughput Cell Toxicity Assays.

    Science.gov (United States)

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  3. Expert system technology for nondestructive waste assay

    International Nuclear Information System (INIS)

    Nondestructive assay waste characterization data generated for use in the National TRU Program must be of known and demonstrable quality. Each measurement is required to receive an independent technical review by a qualified expert. An expert system prototype has been developed to automate waste NDA data review of a passive/active neutron drum counter system. The expert system is designed to yield a confidence rating regarding measurement validity. Expert system rules are derived from data in a process involving data clustering, fuzzy logic, and genetic algorithms. Expert system performance is assessed against confidence assignments elicited from waste NDA domain experts. Performance levels varied for the active, passive shielded, and passive system assay modes of the drum counter system, ranging from 78% to 94% correct classifications

  4. Mobile nondestructive assay and examination instruments

    International Nuclear Information System (INIS)

    A compact system that evaluates radioactive materials can furnish a big savings to taxpayers by ensuring that only properly identified nuclear waste is sent to a Department of Energy (DOE) radioactive waste storage area. The Los Alamos National Laboratory's Advanced Nuclear Technology Group has developed and field tested two easily transportable, self-contained modules: one x-rays the contents of special 208-iota shipment containers, the other assays the contents. The assay and evaluation system is a simple, portable solution to a complex problem that ensures that only properly packaged transuranic (TRU) waste is shipped to the Department of Energy's Waste Isolation Pilot Plant (WIPP). Caustic chemicals, liquids, and other objects or materials that could cause a container leak during shipment are the objects of an x-ray and video camera used in the system

  5. Nondestructive assay methods for solids containing plutonium

    International Nuclear Information System (INIS)

    Specific nondestructive assay (NDA) methods, e.g. calorimetry, coincidence neutron counting, singles neutron counting, and gamma ray spectrometry, were studied to provide the Savannah River Plant with an NDA method to measure the plutonium content of solid scrap (slag and crucible) generated in the JB-Line plutonium metal production process. Results indicate that calorimetry can be used to measure the plutonium content to within about 3% in 4 to 6 hours by using computerized equilibrium sample power predictive models. Calorimetry results confirm that a bias exists in the present indirect measurement method used to estimate the plutonium content of slag and crucible. Singles neutron counting of slag and crucible can measure plutonium to only +-30%, but coincidence neutron counting methods improve measurement precision to better than +-10% in less than ten minutes. Only four portions of a single slag and crucible sample were assayed, and further study is recommended

  6. System and method for assaying radiation

    Science.gov (United States)

    DiPrete, David P; Whiteside, Tad; Pak, Donald J; DiPrete, Cecilia C

    2013-11-12

    A system for assaying radiation includes a sample holder configured to hold a liquid scintillation solution. A photomultiplier receives light from the liquid scintillation solution and generates a signal reflective of the light. A control circuit biases the photomultiplier and receives the signal from the photomultiplier reflective of the light. A light impermeable casing surrounds the sample holder, photomultiplier, and control circuit. A method for assaying radiation includes placing a sample in a liquid scintillation solution, placing the liquid scintillation solution in a sample holder, and placing the sample holder inside a light impermeable casing. The method further includes positioning a photomultiplier inside the light impermeable casing and supplying power to a control circuit inside the light impermeable casing.

  7. Delayed gamma technique for fissile material assay

    Energy Technology Data Exchange (ETDEWEB)

    Mozin, Vladimir [Los Alamos National Laboratory; Tobin, Stephen [Los Alamos National Laboratory; Vujie, Jasmina [UC BERKELEY; Hunt, Alan [IDAHO ACCELERATOR CENTER

    2010-01-01

    Research sponsored by the Next Generation Safeguards Initiative are investigating several non-destructive assay techniques for the quantification of fissile plutonium mass in spent nuclear fuel assemblies. AppHcation of the delayed gamma signatures is investigated in this context. The objective of the research is to assess whether the delayed gamma assay instrument can provide sufficient sensitivity, isotope specificity and accuracy as required in nuclear material safeguards. This effort includes theoretical and experimental components for the optimal combination of interrogation parameters. A new modeling algorithm offering a high level of detail was developed specifically for this purpose and was validated in series of benchmark experiments. Preliminary modeling of the delayed gamma response from spent fuel assemblies was accomplished offering a future direction for the design process.

  8. Depleted uranium waste assay at AWE

    International Nuclear Information System (INIS)

    The Atomic Weapons Establishment (AWE) at Aldermaston has recently conducted a Best Practical Means (BPM) study, for solid Depleted Uranium (DU) waste assay, in order to satisfy key stakeholders that AWE is applying best practice. This study has identified portable passive High Resolution Gamma Spectrometry (HRGS), combined with an analytical software package called Spectral Nondestructive Assay Platform (SNAP), as the preferred option with the best balance between performance and costs. HRGS/SNAP performance has been assessed by monitoring 200 l DU waste drum standards and also heterogeneous, high density drums from DU firing trials. Accuracy was usually within 30 % with Detection Limits (DL) in the region of 10 g DU for short count times. Monte Carlo N-Particle (MCNP) calculations have been used to confirm the shape of the calibration curve generated by the SNAP software procured from Eberline Services Inc. (authors)

  9. Assay of 25-OH vitamin D3

    International Nuclear Information System (INIS)

    A simplified version of the competitive protein-binding assay for 25-OH vitamin D3 (25-OH D3) derived from the method of Belsey et al. is presented. The procedure does not include a chromatographic step, and it is performed on an alcoholic extract of 0.1 ml plasma or serum. Normal rat serum (1:20000) was used as binding protein. No β-lipoproteins were added to the assay buffer. A 10% displacement of the tracer was observed at 0.04 ng/tube and a 50% displacement at 0.15 ng/tube, allowing for the measurement of 25-OH D3 concentrations between 2 ng/ml and 200 ng/ml. Mean values in a normal group were 23.1+-6.5 ng/ml (range 16-37 ng/ml, n=11). (author)

  10. Comet assay on mice testicular cells

    OpenAIRE

    Anoop Kumar Sharma

    2015-01-01

    Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS)” has published classification criteria for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cel...

  11. Quantitative microtiter cytotoxicity assay for Shigella toxin.

    OpenAIRE

    Gentry, M. K.; Dalrymple, J M

    1980-01-01

    The cytotoxic activity of Shigella dysenteriae 1 was assayed by exposing HeLa cells in microtiter cultures to dilutions of toxin. Exposure to toxin caused either failure of cells in suspension to attach or detachment of cells from established monolayers. Estimates of toxin potency were made by staining residual cells with crystal violet and visually inspecting the stained plates. Quantitation of the cytotoxic effect was made possible by eluting and spectrophotometrically measuring the stain. ...

  12. Recent advances in sulfotransferase enzyme activity assays

    OpenAIRE

    Paul, Priscilla; Suwan, Jiraporn; Liu, Jian; Dordick, Jonathan S.; Linhardt, Robert J.

    2012-01-01

    Sulfotransferases are enzymes that catalyze the transfer of sulfo groups from a donor, for example 3′-phosphoadenosine 5′-phosphosulfate, to an acceptor, for example the amino or hydroxyl groups of a small molecule, xenobiotic, carbohydrate, or peptide. These enzymes are important targets in the design of novel therapeutics for treatment of a variety of diseases. This review examines assays used for this important class of enzyme, paying particular attention to sulfotransferases acting on car...

  13. Assaying the reporter gene chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

    These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product

  14. Radioligand assay: current trends in automation

    International Nuclear Information System (INIS)

    Using automatic equipment to perform routine RIA procedures can save time and money, improve precision, and free technologists to work on more-complex, non-routine assays. This article examines some of the instruments available for semiautomated and fully automated RIA testing. The pros and cons of automated testing are explored, and a framework for judging the true value and limitations of systems is presented

  15. SWEPP assay system software: An update

    International Nuclear Information System (INIS)

    The development of a new software package to control data acquisition and perform data analysis for a Passive/Active Neutron Assay system was reported at this conference in 1994. The software has undergone additional development including improvements to the user interface, additional data integrity checks and support for a shift register coincidence analyzer. An overview of this additional work is presented in this report

  16. Transformation Assay for Identification of Psychrotrophic Achromobacters

    OpenAIRE

    Juni, Elliot; Heym, Gloria A.

    1980-01-01

    The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototro...

  17. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  18. Development of extremely sensitive radiometric enzyme assays

    Czech Academy of Sciences Publication Activity Database

    Pavelka, Stanislav; Soukup, Tomáš

    Bratislava: Slovenská technická univerzita, 2012, s. 231-235. ISBN 978-80-227-3722-7. [Priemyselná toxikológia 2012 /32./. Svit, Vysoké Tatry (SK), 20.06.2012-22.06.2012] R&D Projects: GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : enzyme activity * radiometric assay * thyroid hormones Subject RIV: ED - Physiology

  19. Two important Saudi crude assays updated

    International Nuclear Information System (INIS)

    This paper reports on updating two important Saudi Arabian crude oils-Arabian Heavy (Safaniya) and Arabian Light-which is part of a continuing series on world export crudes. Arabian Heavy (Safaniya) was reassayed in early 1990; Arabian Light in May 1990. These assays, therefore, replace those that appeared previously. However, as is frequently the case with crudes from major, single reservoirs, there is little, if any, change in their characteristics

  20. A Quantitative Assay for Aggrecanase Activity

    OpenAIRE

    Will, Horst; Dettloff, Matthias; Bendzkô, Peter; Sveshnikov, Peter

    2005-01-01

    Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N...

  1. Comparison of risks due to bisphenol A and radiation with trad-MCN assay

    International Nuclear Information System (INIS)

    Many kinds of synthetic chemicals have been being used for various purposes. Some of them are called 'environmental hormones' because they can disturb the endocrine system of organisms. Presently no technique is established for the quantitative assessment of biological risk of the environmental hormones. The pollen mother cells (PMC) of Tradescantia are very sensitive to chemical toxicants or ionizing radiation, and thus can be used as a biological end-point assessing their effect. Micronucleus frequencies in PMC showed a good dose- and concentration-response relationship for radiation and bisphenol A. From the dose-response relationship, it is possible to estimate the equivalent bisphenol A concentration, or vice versa. One μM/ml of bisphenol A is equivalent to 1.8 cGy of radiation in the induction of micronuclei. It is known from the result that Trad-MCN assay can be an excellent tool for detection of biological risk due to environmental toxicants or synthetic chemicals

  2. Design of radiation dose tumor response assays

    International Nuclear Information System (INIS)

    The efficient utilization of animals in a radiation dose response assay for tumor control requires a definition of the goal, e.g., TCD50 or slope. A series of computer modelled ''experiments'' have been performed for each of a number of allocations of dose levels (DL) and number of animals/DL. The authors stipulated that the assumed TCD50 was .85 of true value; assumed slope was correct. They stipulated a binominal distribution of observed tumor control results at each dose level. A pilot assay used 6 tumors at 7 DL (from TCD1-TCD97). The second assay used 30 tumors assigned to 2,3,5 or 9 DL and to selected tumor control probabilities (TCP derived from the pilot run. Results from 100 test runs were combined with the pilot run for each of the combination of DL and TCP values. Logit regression lines were fitted through these ''data'' and the 95% CL around the TCD50 and the TCD37 values and the variances of the slopes were computed. These experiments were repeated using the method suggested by Porter (1980). Results show that a different strategy is needed depending upon the goal, viz. TCD50 or TCD37 vs slope. The differences between the two approaches are discussed

  3. Mixture models for single-cell assays with applications to vaccine studies.

    Science.gov (United States)

    Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

    2014-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers (or combinations of biomarkers) that are differentially expressed between two biological conditions (e.g. before/after stimulation), where expression is defined as the proportion of cells expressing that biomarker (or biomarker combination) in the cell subset(s) of interest. Here, we present a Bayesian hierarchical framework based on a beta-binomial mixture model for testing for differential biomarker expression using single-cell assays. Our model allows the inference to be subject specific, as is typically required when assessing vaccine responses, while borrowing strength across subjects through common prior distributions. We propose two approaches for parameter estimation: an empirical-Bayes approach using an Expectation-Maximization algorithm and a fully Bayesian one based on a Markov chain Monte Carlo algorithm. We compare our method against classical approaches for single-cell assays including Fisher's exact test, a likelihood ratio test, and basic log-fold changes. Using several experimental assays measuring proteins or genes at single-cell level and simulations, we show that our method has higher sensitivity and specificity than alternative methods. Additional simulations show that our framework is also robust to model misspecification. Finally, we demonstrate how our approach can be extended to testing multivariate differential expression across multiple biomarker combinations using a Dirichlet-multinomial model and illustrate this approach using single-cell gene

  4. Biological and Chemical Security

    Energy Technology Data Exchange (ETDEWEB)

    Fitch, P J

    2002-12-19

    The LLNL Chemical & Biological National Security Program (CBNP) provides science, technology and integrated systems for chemical and biological security. Our approach is to develop and field advanced strategies that dramatically improve the nation's capabilities to prevent, prepare for, detect, and respond to terrorist use of chemical or biological weapons. Recent events show the importance of civilian defense against terrorism. The 1995 nerve gas attack in Tokyo's subway served to catalyze and focus the early LLNL program on civilian counter terrorism. In the same year, LLNL began CBNP using Laboratory-Directed R&D investments and a focus on biodetection. The Nunn-Lugar-Domenici Defense Against Weapons of Mass Destruction Act, passed in 1996, initiated a number of U.S. nonproliferation and counter-terrorism programs including the DOE (now NNSA) Chemical and Biological Nonproliferation Program (also known as CBNP). In 2002, the Department of Homeland Security was formed. The NNSA CBNP and many of the LLNL CBNP activities are being transferred as the new Department becomes operational. LLNL has a long history in national security including nonproliferation of weapons of mass destruction. In biology, LLNL had a key role in starting and implementing the Human Genome Project and, more recently, the Microbial Genome Program. LLNL has over 1,000 scientists and engineers with relevant expertise in biology, chemistry, decontamination, instrumentation, microtechnologies, atmospheric modeling, and field experimentation. Over 150 LLNL scientists and engineers work full time on chemical and biological national security projects.

  5. Biological activities of Morus celtidifolia leaf extracts.

    Science.gov (United States)

    Viveros-Valdez, Ezequiel; Oranday-Cárdenas, Azucena; Rivas-Morales, Catalina; Verde-Star, María Julia; Carranza-Rosales, Pilar

    2015-07-01

    The aims of this research were to examine the antibacterial, cytotoxic and antiradical/antioxidant activities of the organic extracts obtained from the leaves of the medicinal plant Morus celtidifolia (Family: Moraceae). To evaluate its antimicrobial properties, M. celtidifolia was tested against the bacteria of medical importance: Bacillus subtilis, Staphyloccocus aureus, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae and Enterobacter aerogenes. Cytotoxic activity was assessed by using the brine shrimp (Artemia salina) lethality assay and also by toxicity screening against human cancer cell lines: MCF-7 (human breast adenocarcinoma) and HeLa (cervix adenocarcinoma). The free radical-scavenging activity was determined by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Results revealed that the hexanic extract has antibacterial activity only against Gram positive strains, while the methanolic extract showed better cytotoxic and antioxidant activities than the non- polar extract with a median lethal dose (LD₅₀) of 125μg/ml, 90μg/ml and 75μg/ml against A. salina, MCF-7 and HeLa cells respectively, and median effective concentration (EC₅₀) of 152μg/ml on radical scavenging assay. This is the first study reporting the biological activities of leaves of Morus celtidifolia. PMID:26142508

  6. Radioimmunoassay of penicilloyl groups in biological fluids

    International Nuclear Information System (INIS)

    Penicilloyl-protein conjugates may be formed in man or animals after therapeutic treatment but cannot be detected by the classical microbiological assay methods used for penicillin. Allergies have been noted in consumers of products coming from animals treated with penicillin (milk particularly). Although these compounds present a risk to public health, they passed unnoticed through hygienic food inspection. Therefore a specific and sensitive method was devised for the assay of these derivatives in biological fluids where they can be present in trace amounts. An iodine-125 labelled conjugate has been prepared. Already usable for the detection of antipenicilloyl antibodies in sera of penicillin allergic patients, it has been used for the development of a radioimmunoassay of penicilloyl groups. Assay is done directly on milk, urine and serum without previous extraction and with a detection limit of a few ppb. It permits a rapid, specific and easy to handle determination in the sera of hospital patients as well as in the inspection of animal products at the slaughter house and at the dairy

  7. Neutron in biology

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1997-11-01

    Neutron in biology can provide an experimental method of directly locating relationship of proteins and DNA. However, there are relatively few experimental study of such objects since it takes a lot of time to collect a sufficient number of Bragg reflections and inelastic spectra due to the low flux of neutron illuminating the sample. Since a next generation neutron source of JAERI will be 5MW spallation neutron source and its effective neutron flux will be 10{sup 2} to 10{sup 3} times higher than the one of JRR-3M, neutron in biology will open a completely new world for structural biology. (author)

  8. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

  9. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  10. A Molecular Biology Database Digest

    OpenAIRE

    Bry, François; Kröger, Peer

    2000-01-01

    Computational Biology or Bioinformatics has been defined as the application of mathematical and Computer Science methods to solving problems in Molecular Biology that require large scale data, computation, and analysis [18]. As expected, Molecular Biology databases play an essential role in Computational Biology research and development. This paper introduces into current Molecular Biology databases, stressing data modeling, data acquisition, data retrieval, and the integration...

  11. SOIL BIOLOGY AND ECOLOGY

    Science.gov (United States)

    The term "Soil Biology", the study of organism groups living in soil, (plants, lichens, algae, moss, bacteria, fungi, protozoa, nematodes, and arthropods), predates "Soil Ecology", the study of interactions between soil organisms as mediated by the soil physical environment. oil ...

  12. Insecticides and Biological Control

    Science.gov (United States)

    Furness, G. O.

    1972-01-01

    Use of insecticides has been questioned due to their harmful effects on edible items. Biological control of insects along with other effective practices for checking spread of parasites on crops are discussed. (PS)

  13. Teaching evolutionary biology

    Directory of Open Access Journals (Sweden)

    Tidon Rosana

    2004-01-01

    Full Text Available Evolutionary Biology integrates several disciplines of Biology in a complex and interactive manner, where a deep understanding of the subject demands knowledge in diverse areas. Since this knowledge is often inaccessible to the majority of specialized professionals, including the teachers, we present some reflections in order to stimulate discussions aimed at the improvement of the conditions of education in this area. We examine the profile of evolutionary teaching in Brazil, based on questionnaires distributed to teachers in Secondary Education in the Federal District, on data provided by the "National Institute for Educational Studies and Research", and on information collected from teachers working in various regions of this country. Issues related to biological misconceptions, curriculum and didactic material are discussed, and some proposals are presented with the objective of aiding discussions aimed at the improvement of the teaching of evolutionary biology.

  14. Vibrations, Quanta and Biology

    CERN Document Server

    Huelga, S F

    2013-01-01

    Quantum biology is an emerging field of research that concerns itself with the experimental and theoretical exploration of non-trivial quantum phenomena in biological systems. In this tutorial overview we aim to bring out fundamental assumptions and questions in the field, identify basic design principles and develop a key underlying theme -- the dynamics of quantum dynamical networks in the presence of an environment and the fruitful interplay that the two may enter. At the hand of three biological phenomena whose understanding is held to require quantum mechanical processes, namely excitation and charge transfer in photosynthetic complexes, magneto-reception in birds and the olfactory sense, we demonstrate that this underlying theme encompasses them all, thus suggesting its wider relevance as an archetypical framework for quantum biology.

  15. Nutritional Systems Biology

    DEFF Research Database (Denmark)

    Jensen, Kasper

    and network biology has the potential to increase our understanding of how small molecules affect metabolic pathways and homeostasis, how this perturbation changes at the disease state, and to what extent individual genotypes contribute to this. A fruitful strategy in approaching and exploring the field...... biology research. The paper also shows as a proof-of-concept that a systems biology approach to diet is meaningful and demonstrates some basic principles on how to work with diet systematic. The second chapter of this thesis we developed the resource NutriChem v1.0. A foodchemical database linking...... sites of diet on the disease pathway. We propose a framework for interrogating the critical targets in colon cancer process and identifying plant-based dietary interventions as important modifiers using a systems chemical biology approach. The fifth chapter of the thesis is on discovering of novel anti...

  16. Mechanical Biological Treatment

    DEFF Research Database (Denmark)

    Bilitewski, B-; Oros, Christiane; Christensen, Thomas Højlund

    The basic processes and technologies of composting and anaerobic digestion, as described in the previous chapters, are usually used for specific or source-separated organic waste flows. However, in the 1990s mechanical biological waste treatment technologies (MBT) were developed for unsorted or...... residual waste (after some recyclables removed at the source). The concept was originally to reduce the amount of waste going to landfill, but MBT technologies are today also seen as plants recovering fuel as well as material fractions. As the name suggests the technology combines mechanical treatment...... technologies (screens, sieves, magnets, etc.) with biological technologies (composting, anaerobic digestion). Two main technologies are available: Mechanical biological pretreatment (MBP), which first removes an RDF fraction and then biologically treats the remaining waste before most of it is landfilled, and...

  17. Enhanced Biological Sampling Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This is a database of a variety of biological, reproductive, and energetic data collected from fish on the continental shelf in the northwest Atlantic Ocean....

  18. Hammond Bay Biological Station

    Data.gov (United States)

    Federal Laboratory Consortium — Hammond Bay Biological Station (HBBS), located near Millersburg, Michigan, is a field station of the USGS Great Lakes Science Center (GLSC). HBBS was established by...

  19. Mammalian cell biology

    International Nuclear Information System (INIS)

    This section contains summaries of research on mechanisms of lethality and radioinduced changes in mammalian cell properties, new cell systems for the study of the biology of mutation and neoplastic transformation, and comparative properties of ionizing radiations

  20. Mechanical Biological Treatment

    DEFF Research Database (Denmark)

    Bilitewski, B-; Oros, Christiane; Christensen, Thomas Højlund

    2011-01-01

    The basic processes and technologies of composting and anaerobic digestion, as described in the previous chapters, are usually used for specific or source-separated organic waste flows. However, in the 1990s mechanical biological waste treatment technologies (MBT) were developed for unsorted or...... residual waste (after some recyclables removed at the source). The concept was originally to reduce the amount of waste going to landfill, but MBT technologies are today also seen as plants recovering fuel as well as material fractions. As the name suggests the technology combines mechanical treatment...... technologies (screens, sieves, magnets, etc.) with biological technologies (composting, anaerobic digestion). Two main technologies are available: Mechanical biological pretreatment (MBP), which first removes an RDF fraction and then biologically treats the remaining waste before most of it is landfilled, and...

  1. Laboratory of Biological Modeling

    Data.gov (United States)

    Federal Laboratory Consortium — The Laboratory of Biological Modeling is defined by both its methodologies and its areas of application. We use mathematical modeling in many forms and apply it to...

  2. The Biology of Behaviour.

    Science.gov (United States)

    Broom, D. M.

    1981-01-01

    Discusses topics to aid in understanding animal behavior, including the value of the biological approach to psychology, functional systems, optimality and fitness, universality of environmental effects on behavior, and evolution of social behavior. (DS)

  3. Biological satellite Kosmos-936

    Science.gov (United States)

    Vedeshin, L. A.

    1978-01-01

    A description is given of physiological experiments performed on the biological satellite Kosmos-936. Other experiments to determine the electrostatic and dielectric responses to the effects of cosmic radiation are discussed.

  4. Ontologies for molecular biology.

    Science.gov (United States)

    Schulze-Kremer, S

    1998-01-01

    Molecular biology has a communication problem. There are many databases using their own labels and categories for storing data objects and some using identical labels and categories but with a different meaning. A prominent example is the concept "gene" which is used with different semantics by major international genomic databases. Ontologies are one means to provide a semantic repository to systematically order relevant concepts in molecular biology and to bridge the different notions in various databases by explicitly specifying the meaning of and relation between the fundamental concepts in an application domain. Here, the upper level and a database branch of a prospective ontology for molecular biology (OMB) is presented and compared to other ontologies with respect to suitability for molecular biology (http:/(/)igd.rz-berlin.mpg.de/approximately www/oe/mbo.html). PMID:9697223

  5. Fishery Biology Database (AGDBS)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Basic biological data are the foundation on which all assessments of fisheries resources are built. These include parameters such as the size and age composition of...

  6. Large Pelagics Biological Survey

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Large Pelagics Biological Survey (LPBS) collects additional length and weight information and body parts such as otoliths, caudal vertebrae, dorsal spines, and...

  7. The Biology of Aging.

    Science.gov (United States)

    Sprott, Richard L.; And Others

    1992-01-01

    Thirteen articles in this special issue discuss aging theories, biomarkers of aging, aging research, disease, cancer biology, Alzheimer's disease, stress, oxidation of proteins, gene therapy, service delivery, biogerontology, and ethics and aging research. (SK)

  8. Synthetic biology: A foundation for multi-scale molecular biology

    OpenAIRE

    Bower, Adam G; McClintock, Maria K; Stephen S. Fong

    2010-01-01

    The field of synthetic biology has made rapid progress in a number of areas including method development, novel applications and community building. In seeking to make biology “engineerable,” synthetic biology is increasing the accessibility of biological research to researchers of all experience levels and backgrounds. One of the underlying strengths of synthetic biology is that it may establish the framework for a rigorous bottom-up approach to studying biology starting at the DNA level. Bu...

  9. Coated tube for immunochemical and enzymatic assays

    International Nuclear Information System (INIS)

    Containers such as test tubes suitable for use in solid phase immunochemical, enzymatical and particularly radioimmunoassay procedures are described. The lower part of the tube is a polymer, coated with an inert protein to which a biologically active substance eg an antibody to triiodothyronine, thyroxine or digoxin, is attached. (U.K.)

  10. Computational Thinking in Biology

    OpenAIRE

    Priami, Corrado

    2007-01-01

    The paper presents a new approach based on process calculi to systems modeling suitable for biological systems. The main characteristic of process calculi is a linguistic description level to dene incrementally and compositionally executable models. The formalism is suitable to be exploited on the same systems at dierent levels of abstractions connected through well dened formal rules. The abstraction principle that represents biological entities as interacting computational units is the basi...

  11. Biological Sciences Building

    OpenAIRE

    Kumaraswamy, Mohan

    2002-01-01

    One element of the CIVCAL project Web-based resources containing images, tables, texts and associated data on the construction of the Biological Sciences Building. The HKU Kadoorie Biological Sciences Building is an 11-storey reinforced concrete framed building with a structural steel arch roof. The building dimensions are approximately 60 metres x 30 metres. The estimated project cost for the superstructure construction (excluding the bored pile foundations) was about HK$ 400 million....

  12. Biological treatment for sewage

    OpenAIRE

    Xintai, Wang; Luc Sanya, Eric

    2007-01-01

    The sewage treatment is by no means insignificant in our world, and for many sewage treatment plants, the biological treatment is the best choice to eliminate the nutrients and organic compounds in the waste water. Today, in most waste water treatment plants, there are two main kinds of biological waste water treatment – the active sludge method and the biofilm method. Each of these two methods have their own advantages and disadvantages. For different towns or cities, the waste water treatme...

  13. Synthetic biology and biosecurity.

    Science.gov (United States)

    Robienski, Jürgen; Simon, Jürgen

    2014-01-01

    This article discusses the conflict fields and legal questions of synthetic biology, esp. concerning biosecurity. A respective jurisprudential discussion has not taken place yet in Germany apart from few statements and recommendations. But in Germany, Europe and the USA, it is generally accepted that a broad discussion is necessary. This is esp. true for the question of biosecurity and the possible dangers arising from Synthetic Biology. PMID:25845204

  14. Biological sequence analysis

    OpenAIRE

    Speed, T. P.

    2003-01-01

    This talk will review a little over a decade's research on applying certain stochastic models to biological sequence analysis. The models themselves have a longer history, going back over 30 years, although many novel variants have arisen since that time. The function of the models in biological sequence analysis is to summarize the information concerning what is known as a motif or a domain in bioinformatics, and to provide a tool for discovering instances of that motif or domain in a separa...

  15. Noise in Biology

    OpenAIRE

    Tsimring, Lev S

    2014-01-01

    Noise permeates biology on all levels, from the most basic molecular, sub-cellular processes to the dynamics of tissues, organs, organisms, and populations. The functional roles of noise in biological processes can vary greatly. Along with standard, entropy-increasing effects of producing random mutations, diversifying phenotypes in isogenic populations, limiting information capacity of signaling relays, it occasionally plays more surprising constructive roles by accelerating the pace of evol...

  16. Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

    Science.gov (United States)

    Le Goff, Gaelle C; Desmet, Cloé; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-12-15

    We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures. PMID:20663657

  17. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Rapid and highly informative diagnostic assay for H5N1 influenza viruses.

    Directory of Open Access Journals (Sweden)

    Nader Pourmand

    Full Text Available A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

  19. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    Energy Technology Data Exchange (ETDEWEB)

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  20. Genotoxic effect of gamma radiation in vivo rats as evaluated by comet assay and micronucleus assay

    International Nuclear Information System (INIS)

    DNA is one of the most critical site of damage induced by UV, gamma radiation and other the environmental chemical pollutants. Genotoxic evaluation of environmental agents is generally carried out using cytogenetic markers like chromosomal aberrations, sister chromatid exchanges and micronuclei formation. During the recent years Single Cell Gel Electrophoresis/Comet Assay, has emerged as one of the most powerful assays to detect DNA damage at molecular level in any eukaryotic cell. Present studies were undertaken to investigate the effect of gamma radiation at DNA and chromosomal level using comet assay and micronucleus test. Female Wistar rats were exposed to gamma radiation using 60Co Teletherapy machine; 0.125, 0.25, 0.5 and 1.0 Gy at a dose rate of 0.33 Gy/min. Blood and bone marrow samples were processed for comet assay using the standard protocol. Bone marrow cells from femur bone were also processed for micronucleus assay. Results of these studies indicated a dose dependent increase in DNA damage as indicated by increase in tail length (TL), tail moment (TM) and % DNA in tail (% DNA- T). Similarly, a dose dependent increase in the frequency of micronucleated polychromatic erythrocytes (mn-PCEs) was also observed. A good correlation between, DNA damage and the induction of micronucleated erythrocytes was observed. (author)