WorldWideScience

Sample records for two-hybrid assays showed

  1. A two-hybrid assay to study protein interactions within the secretory pathway.

    Directory of Open Access Journals (Sweden)

    Danielle H Dube

    Full Text Available Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H, a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.

  2. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-09-10

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  3. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    International Nuclear Information System (INIS)

    Bloch, Donald B.; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-01-01

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: → A two-hybrid assay was developed to study interactions in macromolecular complexes. → The assay was applied to interactions between components of mRNA P-bodies. → The assay effectively and efficiently identified protein interaction domains. → P-body assembly in mammalian cells differs from that in other species.

  4. FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor

    Science.gov (United States)

    Du, Mengyan; Yang, Fangfang; Mai, Zihao; Qu, Wenfeng; Lin, Fangrui; Wei, Lichun; Chen, Tongsheng

    2018-04-01

    We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.

  5. Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo

    2010-12-01

    Full Text Available Abstract Background Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins. Results Using the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD, a glyceraldehyde-3-P dehydrogenase (GAPDH and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment. Conclusions This study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association

  6. [Identification of C(2)M interacting proteins by yeast two-hybrid screening].

    Science.gov (United States)

    Yue, Shan-shan; Xia, Lai-xin

    2015-11-01

    The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

  7. A urine midmolecule osteocalcin assay shows higher discriminatory power than a serum midmolecule osteocalcin assay during short-term alendronate treatment of osteoporotic patients.

    Science.gov (United States)

    Srivastava, A K; Mohan, S; Singer, F R; Baylink, D J

    2002-07-01

    We isolated and characterized a peptide fragment corresponding to amino acid sequence 14-28 of human osteocalcin in urine from Paget's disease, and developed a polyclonal antibody reactive to this peptide in urine. We used this antibody to measure urinary fragments of osteocalcin and compared to efficacy of the urinary osteocalcin assay with a serum osteocalcin (sOC) assay (ELISA-Osteo, Cis-Bio International) to monitor the short-term changes in bone turnover in response to alendronate treatment. The synthetic peptide-based urinary osteocalcin (uOC) radioimmunoassay (RIA) showed an analytical sensitivity of 6.25 ng/mL, standard curve range of 3.12-400 ng/mL, and mean intra- (n = 20) and interassay (n = 30) coefficient of variation (CV) of sALP) (Alkphose-B, Metra Biosystems) in serum samples. The percent change data obtained between baseline and 30 days (n = 18) posttreatment suggested a rapid decline in uOC concentration (-27%, p sALP (-3.4%, p = 0.689), two specific markers of bone formation. As expected, due to the coupling of bone formation and bone resorption, the concentration of all markers showed a 30%-45% decline compared with baseline values after 90 days (n = 16) of treatment. Correlation of markers after a 30 day treatment with alendronate revealed a higher correlation (r = 0.61, p sALP (r = -0.14, p = 0.295) with uNTx. Similarly, correlation coefficients with r values between 0.48 and 0.55 (p < 0.05) were observed between uOC, sNTx, and sCTx, whereas no significant correlation was observed between sOC and sNTx or sCTx. These results provide indirect evidence that fragments measured by the urine assay probably originated from bone resorption, and suggest that the uOC assay could be used to assess short-term changes in bone metabolism with regard to osteocalcin.

  8. Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis.

    Science.gov (United States)

    Huber, P A; Medhurst, A L; Youssoufian, H; Mathew, C G

    2000-02-05

    Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified. Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial. We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins. No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA. Copyright 2000 Academic Press.

  9. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    Science.gov (United States)

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE.

  10. Construction of gateway-compatible yeast two-hybrid vectors for ...

    African Journals Online (AJOL)

    Yeast two-hybrid system combined with the gateway technology will greatly facilitate the cloning of interested DNA fragment into yeast two-hybrid vectors and therefore increase the efficiency of yeast two-hybrid analysis. In this study, we constructed a pair of Gateway-compatible yeast two-hybrid vectors pBTM116GW and ...

  11. Novel protein interactions with an actin homolog (MreB) of Helicobacter pylori determined by bacterial two-hybrid system.

    Science.gov (United States)

    Zepeda Gurrola, Reyna Cristina; Fu, Yajuan; Rodríguez Luna, Isabel Cristina; Benítez Cardoza, Claudia Guadalupe; López López, María de Jesús; López Vidal, Yolanda; Gutíerrez, Germán Rubén Aguilar; Rodríguez Pérez, Mario A; Guo, Xianwu

    2017-08-01

    The bacterium Helicobacter pylori infects more than 50% of the world population and causes several gastroduodenal diseases, including gastric cancer. Nevertheless, we still need to explore some protein interactions that may be involved in pathogenesis. MreB, an actin homolog, showed some special characteristics in previous studies, indicating that it could have different functions. Protein functions could be realized via protein-protein interactions. In the present study, the MreB protein from H. pylori 26695 fused with two tags 10×His and GST in tandem was overexpressed and purified from Escherchia coli. The purified recombinant protein was used to perform a pull-down assay with H. pylori 26695 cell lysate. The pulled-down proteins were identified by mass spectrometry (MALDI-TOF), in which the known important proteins related to morphogenesis were absent but several proteins related to pathogenesis process were observed. The bacterial two-hybrid system was further used to evaluate the protein interactions and showed that new interactions of MreB respectively with VacA, UreB, HydB, HylB and AddA were confirmed but the interaction MreB-MreC was not validated. These results indicated that the protein MreB in H. pylori has a distinct interactome, does not participate in cell morphogenesis via MreB-MreC but could be related to pathogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Screening and identification of host proteins interacting with Theileria annulata cysteine proteinase (TaCP by yeast-two-hybrid system

    Directory of Open Access Journals (Sweden)

    Shuaiyang Zhao

    2017-10-01

    Full Text Available Abstract Background Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells. Methods The cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING. Results Two host proteins, CRBN (Bos taurus cereblon transcript variant X2 and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. Conclusions This study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are

  13. Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

    Directory of Open Access Journals (Sweden)

    Cataldi Angel A

    2009-01-01

    Full Text Available Abstract Background The exported repetitive protein (erp gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. Results In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC. The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. Conclusion We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.

  14. Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy

    NARCIS (Netherlands)

    Haer-Wigman, Lonneke; Ji, Yanli; Lodén, Martin; de Haas, Masja; van der Schoot, C. Ellen; Veldhuisen, Barbera

    2013-01-01

    In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation-dependent probe amplification (MLPA)

  15. Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

    Science.gov (United States)

    Nilles, Matthew L

    2017-01-01

    Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

  16. Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

    Directory of Open Access Journals (Sweden)

    Gonzalez-Mendez Ricardo

    2011-07-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

  17. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2.

    Science.gov (United States)

    Li, Qun; Laumonnier, Yves; Syrovets, Tatiana; Simmet, Thomas

    2011-11-01

    To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.

  18. A Special Family of LMM with Two Hybrid Points for Stiff ODEs ...

    African Journals Online (AJOL)

    Enright (1974) discussed the formulation of the second derivative LMM which was found to be stiffly stable for step number k £ 7 for the numerical solution of stiff Initial Value Problems (IVPs) in Ordinary Differential Equations (ODEs). In this paper some second derivative continuous linear multistep methods with two hybrid ...

  19. A Gateway((R)) -compatible bacterial adenylate cyclase-based two-hybrid system

    Czech Academy of Sciences Publication Activity Database

    Ouellette, S. P.; Gauliard, E.; Antošová, Zuzana; Ladant, D.

    2014-01-01

    Roč. 6, č. 3 (2014), s. 259-267 ISSN 1758-2229 Institutional support: RVO:67985823 Keywords : bacterial two-hybrid system * protein–protein interactions * cell division * Gateway((R))(GW) cloning system Subject RIV: EE - Microbiology, Virology Impact factor: 3.293, year: 2014

  20. Construction of gateway-compatible yeast two-hybrid vectors for ...

    African Journals Online (AJOL)

    USER

    2010-03-01

    Mar 1, 2010 ... vectors pBTM116GW and pVP16GW by introducing the gateway cassette ... Key words: Yeast two-hybrid, gateway cloning technology, protein interaction. .... cycling parameters were as follows: an initial denaturation step at.

  1. The bacterial two-hybrid system uncovers the involvement of acetylation in regulating of Lrp activity in Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ran Qin

    2016-11-01

    Full Text Available Nε-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat - or deacetylase CobB-mediated acetylation. Then, the in vitro (deacetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36 in helix-turn-helix (HTH DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

  2. Degradation of Acid Orange 7 Dye in Two Hybrid Plasma Discharge Reactors

    Science.gov (United States)

    Shen, Yongjun; Lei, Lecheng; Zhang, Xingwang; Ding, Jiandong

    2014-11-01

    To get an optimized pulsed electrical plasma discharge reactor and to increase the energy utilization efficiency in the removal of pollutants, two hybrid plasma discharge reactors were designed and optimized. The reactors were compared via the discharge characteristics, energy transfer efficiency, the yields of the active species and the energy utilization in dye wastewater degradation. The results showed that under the same AC input power, the characteristics of the discharge waveform of the point-to-plate reactor were better. Under the same AC input power, the two reactors both had almost the same peak voltage of 22 kV. The peak current of the point-to-plate reactor was 146 A, while that of the wire-to-cylinder reactor was only 48.8 A. The peak powers of the point-to-plate reactor and the wire-to-cylinder reactor were 1.38 MW and 1.01 MW, respectively. The energy per pulse of the point-to-plate reactor was 0.2221 J, which was about 29.4% higher than that of the wire-to-cylinder reactor (0.1716 J). To remove 50% Acid Orange 7 (AO7), the energy utilizations of the point-to-plate reactor and the wire-to-cylinder reactor were 1.02 × 10-9 mol/L and 0.61 × 10-9 mol/L, respectively. In the point-to-plate reactor, the concentration of hydrogen peroxide in pure water was 3.6 mmol/L after 40 min of discharge, which was higher than that of the wire-to-cylinder reactor (2.5 mmol/L). The concentration of liquid phase ozone in the point-to-plate reactor (5.7 × 10-2 mmol/L) was about 26.7% higher than that in the wire-to-cylinder reactor (4.5 × 10-2 mmol/L). The analysis results of the variance showed that the type of reactor and reaction time had significant impacts on the yields of the hydrogen peroxide and ozone. The main degradation intermediates of AO7 identified by gas chromatography and mass spectrometry (GCMS) were acetic acid, maleic anhydride, p-benzoquinone, phenol, benzoic acid, phthalic anhydride, coumarin and 2-naphthol. Proposed degradation pathways were

  3. Degradation of Acid Orange 7 Dye in Two Hybrid Plasma Discharge Reactors

    International Nuclear Information System (INIS)

    Shen Yongjun; Ding Jiandong; Lei Lecheng; Zhang Xingwang

    2014-01-01

    To get an optimized pulsed electrical plasma discharge reactor and to increase the energy utilization efficiency in the removal of pollutants, two hybrid plasma discharge reactors were designed and optimized. The reactors were compared via the discharge characteristics, energy transfer efficiency, the yields of the active species and the energy utilization in dye wastewater degradation. The results showed that under the same AC input power, the characteristics of the discharge waveform of the point-to-plate reactor were better. Under the same AC input power, the two reactors both had almost the same peak voltage of 22 kV. The peak current of the point-to-plate reactor was 146 A, while that of the wire-to-cylinder reactor was only 48.8 A. The peak powers of the point-to-plate reactor and the wire-to-cylinder reactor were 1.38 MW and 1.01 MW, respectively. The energy per pulse of the point-to-plate reactor was 0.2221 J, which was about 29.4% higher than that of the wire-to-cylinder reactor (0.1716 J). To remove 50% Acid Orange 7 (AO7), the energy utilizations of the point-to-plate reactor and the wire-to-cylinder reactor were 1.02 × 10 −9 mol/L and 0.61 × 10 −9 mol/L, respectively. In the point-to-plate reactor, the concentration of hydrogen peroxide in pure water was 3.6 mmol/L after 40 min of discharge, which was higher than that of the wire-to-cylinder reactor (2.5 mmol/L). The concentration of liquid phase ozone in the point-to-plate reactor (5.7 × 10 −2 mmol/L) was about 26.7% higher than that in the wire-to-cylinder reactor (4.5 × 10 −2 mmol/L). The analysis results of the variance showed that the type of reactor and reaction time had significant impacts on the yields of the hydrogen peroxide and ozone. The main degradation intermediates of AO7 identified by gas chromatography and mass spectrometry (GCMS) were acetic acid, maleic anhydride, p-benzoquinone, phenol, benzoic acid, phthalic anhydride, coumarin and 2-naphthol. Proposed degradation

  4. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    Directory of Open Access Journals (Sweden)

    Peter Kusch

    2011-01-01

    Full Text Available Balanites aegyptiaca (Balanitaceae is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 g/L±3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a of 2.35 g/L and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a of 4.8 g/L. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 M±3 and 47.82 M±2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed.

  5. Development of a Premature Stop Codon-detection method based on a bacterial two-hybrid system

    Directory of Open Access Journals (Sweden)

    Mayorga Luis S

    2006-09-01

    Full Text Available Abstract Background The detection of Premature Stop Codons (PSCs in human genes is very useful for the genetic diagnosis of different hereditary cancers, e.g. Familial Breast Cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC. The products of these PSCs are truncated proteins, detectable in vitro by the Protein Truncation Test and in vivo by using the living translation machinery of yeast or bacteria. These living strategies are based on the construction of recombinant plasmids where the human sequence of interest is inserted upstream of a reporter gene. Although simple, these assays have their limitations. The yeast system requires extensive work to enhance its specificity, and the bacterial systems yield many false results due to translation re-initiation events occurring post PSCs. Our aim was to design a recombinant plasmid useful for detecting PSCs in human genes and resistant to bacterial translation re-initiation interferences. Results A functional recombinant plasmid (pREAL was designed based on a bacterial two-hybrid system. In our design, the in vivo translation of fused fragments of the Bordetella pertussis adenylate cyclase triggers the production of cAMP giving rise to a selectable bacterial phenotype. When a gene of interest is inserted between the two fragments, any PSC inhibits the enzymatic activity of the product, and translation re-initiation events post-PSC yield separated inactive fragments. We demonstrated that the system can accurately detect PSCs in human genes by inserting mutated fragments of the brca1 and msh2 gene. Western Blot assays revealed translation re-initiation events in all the tested colonies, implying that a simpler plasmid would not be resistant to this source of false negative results. The application of the system to a HNPCC family with a nonsense mutation in the msh2 gene correctly diagnosed wild type homozygous and heterozygous patients. Conclusion The developed pREAL is applicable to the

  6. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  7. Histograms showing variations in oil yield, water yield, and specific gravity of oil from Fischer assay analyses of oil-shale drill cores and cuttings from the Piceance Basin, northwestern Colorado

    Science.gov (United States)

    Dietrich, John D.; Brownfield, Michael E.; Johnson, Ronald C.; Mercier, Tracey J.

    2014-01-01

    Recent studies indicate that the Piceance Basin in northwestern Colorado contains over 1.5 trillion barrels of oil in place, making the basin the largest known oil-shale deposit in the world. Previously published histograms display oil-yield variations with depth and widely correlate rich and lean oil-shale beds and zones throughout the basin. Histograms in this report display oil-yield data plotted alongside either water-yield or oil specific-gravity data. Fischer assay analyses of core and cutting samples collected from exploration drill holes penetrating the Eocene Green River Formation in the Piceance Basin can aid in determining the origins of those deposits, as well as estimating the amount of organic matter, halite, nahcolite, and water-bearing minerals. This report focuses only on the oil yield plotted against water yield and oil specific gravity.

  8. Comparison of Two Hybrid Models for Forecasting the Incidence of Hemorrhagic Fever with Renal Syndrome in Jiangsu Province, China.

    Directory of Open Access Journals (Sweden)

    Wei Wu

    Full Text Available Cases of hemorrhagic fever with renal syndrome (HFRS are widely distributed in eastern Asia, especially in China, Russia, and Korea. It is proved to be a difficult task to eliminate HFRS completely because of the diverse animal reservoirs and effects of global warming. Reliable forecasting is useful for the prevention and control of HFRS.Two hybrid models, one composed of nonlinear autoregressive neural network (NARNN and autoregressive integrated moving average (ARIMA the other composed of generalized regression neural network (GRNN and ARIMA were constructed to predict the incidence of HFRS in the future one year. Performances of the two hybrid models were compared with ARIMA model.The ARIMA, ARIMA-NARNN ARIMA-GRNN model fitted and predicted the seasonal fluctuation well. Among the three models, the mean square error (MSE, mean absolute error (MAE and mean absolute percentage error (MAPE of ARIMA-NARNN hybrid model was the lowest both in modeling stage and forecasting stage. As for the ARIMA-GRNN hybrid model, the MSE, MAE and MAPE of modeling performance and the MSE and MAE of forecasting performance were less than the ARIMA model, but the MAPE of forecasting performance did not improve.Developing and applying the ARIMA-NARNN hybrid model is an effective method to make us better understand the epidemic characteristics of HFRS and could be helpful to the prevention and control of HFRS.

  9. Interaction of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system.

    Directory of Open Access Journals (Sweden)

    Douglas P Gladue

    Full Text Available E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV and bovine viral diarrhea virus (BVDV. E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.

  10. Exploring Protein Interactions on a Minimal Type II Polyketide Synthase Using a Yeast Two-Hybrid System

    Directory of Open Access Journals (Sweden)

    Gaetano Castaldo

    2005-01-01

    Full Text Available Interactions between proteins that form the ’minimal’ type II polyketide synthase in the doxorubicin producing biosynthetic pathway from Streptomyces peucetius were investigated using a yeast two-hybrid system (Y2H. Proteins that function as the so called ’chain length factor’ (DpsB and putative transacylase (DpsD were found to interact with the ketosynthase subunit (DpsA, which can also interact with itself. On the basis of these results we propose a head-to-tail homodimeric structure, which is consistent with previously published in vivo mutagenesis studies. No interactions were found between the acyl-carrier protein (DpsG and any of the other constituents of the complex, however, transient interactions, not detectable using the Y2H system, cannot be discounted and warrant further investigation.

  11. Safety Assessment of Two Hybrid Instrumentation Techniques in a Dental Student Endodontic Clinic: A Retrospective Study.

    Science.gov (United States)

    Coelho, Marcelo Santos; Card, Steven John; Tawil, Peter Zahi

    2017-03-01

    The aim of this study was to retrospectively assess the safety potential of a hybrid technique combining nickel-titanium (NiTi) reciprocating and rotary instruments by third- and fourth-year dental students in the predoctoral endodontics clinic at one U.S. dental school. For the study, 3,194 root canal treatments performed by 317 dental students from 2012 through 2015 were evaluated for incidence of ledge creation and instrument separation. The hybrid reciprocating and rotary technique (RRT) consisted of a glide path creation with stainless steel hand files up to size 15/02, a crown down preparation with a NiTi reciprocating instrument, and an apical preparation with NiTi rotary instruments. The control was a traditional rotary and hand technique (RHT) that consisted of the same glide path procedure followed by a crown down preparation with NiTi rotary instruments and an apical preparation with NiTi hand instruments. The results showed that the RHT technique presented a rate of ledge creation of 1.4% per root and the RRT technique was 0.5% per root (protary technique for root canal instrumentation by these dental students provided good safety. This hybrid technique offered a low rate of ledge creation along with no NiTi instrument separation.

  12. Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

    Science.gov (United States)

    De Marchi, Fabiola; Seraglia, Roberta; Molin, Laura; Traldi, Pietro; De Rosso, Mirko; Panighel, Annarita; Dalla Vedova, Antonio; Gardiman, Massimo; Giust, Mirella; Flamini, Riccardo

    2012-09-01

    Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties. Copyright © 2012 John Wiley & Sons, Ltd.

  13. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

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

  14. Assay system

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  15. Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

    Science.gov (United States)

    Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

    2014-01-01

    Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

  16. Screening for proteins interacting with MCM7 in human lung cancer library using yeast two hybrid system

    Directory of Open Access Journals (Sweden)

    Yuchen HAN

    2008-08-01

    Full Text Available Background and objective MCM7 is a subunit of the MCM complex that plays a key role in DNA replication initiation. But little is known about its interaction proteins. In this study yeast two hybrid screening was used to identify the MCM7 interacting proteins. Methods Yeast expression vector containing human full length MCM7-pGBKT7 plasmid was constructed, and with a library of cDNAs from human lung cancer-pACT2 plasmid was transformed into yeast strain AH109, and was electively grew in X-a-gal auxotrophy medium SD/-Trp-Leu-His-Ade, and the blue colonies were picked up, the plasmid of the yeast colonies was extracted , and transformed into E. Coli to extract DNA and performed sequence analysis. Results Eleven proteins were identified which could specifically interact with MCM7 proteins, among these five were cytoskeleton proteins, six were enzymes, kinases and related receptors. Conclusion The investigation provides functional clues for further exploration of MCM7 gene.

  17. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

    Directory of Open Access Journals (Sweden)

    Canard Bruno

    2011-10-01

    Full Text Available Abstract Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4. Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

  18. Spatial variability of soil carbon and nitrogen in two hybrid poplar-hay crop systems in southern Quebec, Canada

    Science.gov (United States)

    Winans, K. S.

    2013-12-01

    Canadian agricultural operations contribute approximately 8% of national GHG emissions each year, mainly from fertilizers, enteric fermentation, and manure management (Environment Canada, 2010). With improved management of cropland and forests, it is possible to mitigate GHG emissions through carbon (C) sequestration while enhancing soil and crop productivity. Tree-based intercropped (TBI) systems, consisting of a fast-growing woody species such as poplar (Populus spp.) planted in widely-spaced rows with crops cultivated between tree rows, were one of the technologies prioritized for investigation by the Agreement for the Agricultural Greenhouse Gases Program (AAGGP), because fast growing trees can be a sink for atmospheric carbon-dioxide (CO2) as well as a long-term source of farm income (Montagnini and Nair, 2004). However, there are relatively few estimates of the C sequestration in the trees or due to tree inputs (e.g., fine root turnover, litterfall that gets incorporated into SOC), and hybrid poplars grow exponentially in the first 8-10 years after planting. With the current study, our objectives were (1) to evaluate spatial variation in soil C and nitrogen (N) storage, CO2 and nitrogen oxide (N20), and tree and crop productivity for two hybrid poplar-hay intercrop systems at year 9, comparing TBI vs. non-TBI systems, and (2) to evaluate TBI systems in the current context of C trading markets, which value C sequestration in trees, unharvested crop components, and soils of TBI systems. The study results will provide meaningful measures that indicate changes due to TBI systems in the short-term and in the long-term, in terms of GHG mitigation, enhanced soil and crop productivity, as well as the expected economic returns in TBI systems.

  19. Comparative analysis of two hybrid energy storage systems used in a two front wheel driven electric vehicle during extreme start-up and regenerative braking operations

    International Nuclear Information System (INIS)

    Itani, Khaled; De Bernardinis, Alexandre; Khatir, Zoubir; Jammal, Ahmad

    2017-01-01

    Highlights: • Comparison of HESS Ultracapacitor and Flywheel for maximizing EV energy recovery. • Energy recovery performed for extreme two front-wheel driven EV brake conditions. • Regenerative EV braking control strategies and constraints for HESS. • Comparative cost effectiveness for two HESS solutions Ultracapacitors and Flywheel. - Abstract: This paper presents the comparative study of two hybrid energy storage systems (HESS) of a two front wheel driven electric vehicle. The primary energy source of the HESS is a Li-Ion battery, whereas the secondary energy source is either an ultracapacitor (UC) or a flywheel energy system (FES). The main role of the secondary source is to deliver/recover energy during high peak power demand, but also to increase battery lifetime, considered among the most expensive items in the electric vehicle. As a first step, a techno-economic comparative study, supported by strong literature research, is performed between the UC and the FES. The design and sizing of each element will be presented. The comparison criteria and specifications are also described. The adopted approach in this paper is based on an academic non-oriented point of view. In a second step, each of the HESS will be integrated in a more global Simulink model which includes the vehicle model, the traction control system (TCS), the regenerative braking system and the vehicle actuators. Simulation tests are performed for an extreme braking and vehicle starting-up operations. Tests are realized on two different surface road types and conditions (high and low friction roads) and for different initial system states. In order to show the most appropriate storage system regarding compactness, weight and battery constraints minimization, deep comparative analysis is provided.

  20. The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration.

    Science.gov (United States)

    Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin

    2011-07-01

    Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration. 2011 Elsevier B.V. All rights reserved.

  1. Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

    Science.gov (United States)

    Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

    2018-01-16

    "A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

  2. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    Levin, G.V.; Straat, P.A.

    1981-01-01

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  3. Show-Bix &

    DEFF Research Database (Denmark)

    2014-01-01

    The anti-reenactment 'Show-Bix &' consists of 5 dias projectors, a dial phone, quintophonic sound, and interactive elements. A responsive interface will enable the Dias projectors to show copies of original dias slides from the Show-Bix piece ”March på Stedet”, 265 images in total. The copies are...

  4. Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

    DEFF Research Database (Denmark)

    Mansilla, Francisco; Friis, Irene; Jadidi, Mandana

    2002-01-01

    In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B...... of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first...... gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit...

  5. Genomic and transcriptomic analysis of aroma synthesis in two hybrids between Saccharomyces cerevisiae and S. kudriavzevii in winemaking conditions.

    Science.gov (United States)

    Gamero, Amparo; Belloch, Carmela; Querol, Amparo

    2015-09-04

    Aroma is one of the most important attributes defining wine quality in which yeasts play a crucial role, synthesizing aromatic compounds or releasing odourless conjugates. A present-day trend in winemaking consists of lowering fermentation temperature to achieve higher aroma production and retention. S. cerevisiae × S. kudriavzevii hybrids seem to have inherited beneficial traits from their parental species, like fermenting efficiently at low temperature or producing higher amounts of certain aromatic compounds. In this study, allelic composition and gene expression of the genes related to aroma synthesis in two genetically and phenotypically different S. cerevisiae × S. kudriavzevii hybrids, Lalvin W27 and VIN7, were compared and related to aroma production in microvinifications at 12 and 28 °C. In addition, the contribution of the allele coming from each parental to the overall expression was explored by RT-PCR. The results indicated large differences in allele composition, gene expression and the contribution of each parental to the overall expression at the fermentation temperatures tested. Results obtained by RT-PCR showed that in ARO1 and ATF2 genes the S. kudriavzevii allele was more expressed than that of S. cerevisiae particularly at 12 °C. This study revealed high differences regarding allele composition and gene expression in two S. cerevisiae × S. kudriavzevii hybrids, which may have led to different aroma profiles in winemaking conditions. The contribution of the alleles coming from each parental to the overall expression has proved to differently influence aroma synthesis. Besides, the quantitative contribution to the overall gene expression of the alleles coming from one parental strain or the other was clearly determined by the fermentation temperature for some genes.

  6. Lesion detection performance: comparative analysis of low-dose CT data of the chest on two hybrid imaging systems.

    Science.gov (United States)

    Jessop, Maryam; Thompson, John D; Coward, Joanne; Sanderud, Audun; Jorge, José; de Groot, Martijn; Lança, Luís; Hogg, Peter

    2015-03-01

    Incidental findings on low-dose CT images obtained during hybrid imaging are an increasing phenomenon as CT technology advances. Understanding the diagnostic value of incidental findings along with the technical limitations is important when reporting image results and recommending follow-up, which may result in an additional radiation dose from further diagnostic imaging and an increase in patient anxiety. This study assessed lesions incidentally detected on CT images acquired for attenuation correction on two SPECT/CT systems. An anthropomorphic chest phantom containing simulated lesions of varying size and density was imaged on an Infinia Hawkeye 4 and a Symbia T6 using the low-dose CT settings applied for attenuation correction acquisitions in myocardial perfusion imaging. Twenty-two interpreters assessed 46 images from each SPECT/CT system (15 normal images and 31 abnormal images; 41 lesions). Data were evaluated using a jackknife alternative free-response receiver-operating-characteristic analysis (JAFROC). JAFROC analysis showed a significant difference (P detection, with the figures of merit being 0.599 (95% confidence interval, 0.568, 0.631) and 0.810 (95% confidence interval, 0.781, 0.839) for the Infinia Hawkeye 4 and Symbia T6, respectively. Lesion detection on the Infinia Hawkeye 4 was generally limited to larger, higher-density lesions. The Symbia T6 allowed improved detection rates for midsized lesions and some lower-density lesions. However, interpreters struggled to detect small (5 mm) lesions on both image sets, irrespective of density. Lesion detection is more reliable on low-dose CT images from the Symbia T6 than from the Infinia Hawkeye 4. This phantom-based study gives an indication of potential lesion detection in the clinical context as shown by two commonly used SPECT/CT systems, which may assist the clinician in determining whether further diagnostic imaging is justified. © 2015 by the Society of Nuclear Medicine and Molecular Imaging

  7. Talking with TV shows

    DEFF Research Database (Denmark)

    Sandvik, Kjetil; Laursen, Ditte

    2014-01-01

    User interaction with radio and television programmes is not a new thing. However, with new cross-media production concepts such as X Factor and Voice, this is changing dramatically. The second-screen logic of these productions encourages viewers, along with TV’s traditional one-way communication...... mode, to communicate on interactive (dialogue-enabling) devices such as laptops, smartphones and tablets. Using the TV show Voice as our example, this article shows how the technological and situational set-up of the production invites viewers to engage in new ways of interaction and communication...

  8. Talk Show Science.

    Science.gov (United States)

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  9. Obesity in show cats.

    Science.gov (United States)

    Corbee, R J

    2014-12-01

    Obesity is an important disease with a high prevalence in cats. Because obesity is related to several other diseases, it is important to identify the population at risk. Several risk factors for obesity have been described in the literature. A higher incidence of obesity in certain cat breeds has been suggested. The aim of this study was to determine whether obesity occurs more often in certain breeds. The second aim was to relate the increased prevalence of obesity in certain breeds to the official standards of that breed. To this end, 268 cats of 22 different breeds investigated by determining their body condition score (BCS) on a nine-point scale by inspection and palpation, at two different cat shows. Overall, 45.5% of the show cats had a BCS > 5, and 4.5% of the show cats had a BCS > 7. There were significant differences between breeds, which could be related to the breed standards. Most overweight and obese cats were in the neutered group. It warrants firm discussions with breeders and cat show judges to come to different interpretations of the standards in order to prevent overweight conditions in certain breeds from being the standard of beauty. Neutering predisposes for obesity and requires early nutritional intervention to prevent obese conditions. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  10. Honored Teacher Shows Commitment.

    Science.gov (United States)

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  11. The energy show

    International Nuclear Information System (INIS)

    1988-01-01

    The Energy Show is a new look at the problems of world energy, where our supplies come from, now and in the future. The programme looks at how we need energy to maintain our standards of living. Energy supply is shown as the complicated set of problems it is - that Fossil Fuels are both raw materials and energy sources, that some 'alternatives' so readily suggested as practical options are in reality a long way from being effective. (author)

  12. 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.

  13. Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

    Science.gov (United States)

    Reuter, Tanja Y; Medhurst, Annette L; Waisfisz, Quinten; Zhi, Yu; Herterich, Sabine; Hoehn, Holger; Gross, Hans J; Joenje, Hans; Hoatlin, Maureen E; Mathew, Christopher G; Huber, Pia A J

    2003-10-01

    Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

  14. Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.

    Science.gov (United States)

    Urcuqui-Inchima, S; Walter, J; Drugeon, G; German-Retana, S; Haenni, A L; Candresse, T; Bernardi, F; Le Gall, O

    1999-05-25

    Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro. Copyright 1999 Academic Press.

  15. Showing Value (Editorial

    Directory of Open Access Journals (Sweden)

    Denise Koufogiannakis

    2009-06-01

    Full Text Available When Su Cleyle and I first decided to start Evidence Based Library and Information Practice, one of the things we agreed upon immediately was that the journal be open access. We knew that a major obstacle to librarians using the research literature was that they did not have access to the research literature. Although Su and I are both academic librarians who can access a wide variety of library and information literature from our institutions, we belong to a profession where not everyone has equal access to the research in our field. Without such access to our own body of literature, how can we ever hope for practitioners to use research evidence in their decision making? It would have been contradictory to the principles of evidence based library and information practice to do otherwise.One of the specific groups we thought could use such an open access venue for discovering research literature was school librarians. School librarians are often isolated and lacking access to the research literature that may help them prove to stakeholders the importance of their libraries and their role within schools. Certainly, school libraries have been in decline and the use of evidence to show value is needed. As Ken Haycock noted in his 2003 report, The Crisis in Canada’s School Libraries: The Case for Reform and Reinvestment, “Across the country, teacher-librarians are losing their jobs or being reassigned. Collections are becoming depleted owing to budget cuts. Some principals believe that in the age of the Internet and the classroom workstation, the school library is an artifact” (9. Within this context, school librarians are looking to our research literature for evidence of the impact that school library programs have on learning outcomes and student success. They are integrating that evidence into their practice, and reflecting upon what can be improved locally. They are focusing on students and showing the impact of school libraries and

  16. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  17. Absolute nuclear material assay

    Science.gov (United States)

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

    2010-07-13

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

  18. Endogenous Locus Reporter Assays.

    Science.gov (United States)

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

    2018-01-01

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

  19. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    Shackleton, D.R.; Hulmes, D.J.

    1990-01-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

  20. Solid phase assays

    International Nuclear Information System (INIS)

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

    1980-01-01

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

  1. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul [Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul (Korea, Republic of); Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo [Seoul National University college of Medicine, Seoul (Korea, Republic of)

    2009-08-15

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+-1.7%, 3.9+-2.1%, 7.1+-6.2%, 11.2+-7.2%. The CVs by random assay were 2.1+-1.7%, 4.8+-3.1%, 3.6+-4.8%, and 7.4+-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  2. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    International Nuclear Information System (INIS)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul; Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo

    2009-01-01

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2±1.7%, 3.9±2.1%, 7.1±6.2%, 11.2±7.2%. The CVs by random assay were 2.1±1.7%, 4.8±3.1%, 3.6±4.8%, and 7.4±6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  3. Factor IX assay

    Science.gov (United States)

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

  4. Factor VIII assay

    Science.gov (United States)

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

  5. Factor II assay

    Science.gov (United States)

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

  6. Factor VII assay

    Science.gov (United States)

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

  7. Microbead agglutination based assays

    KAUST Repository

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

    2013-01-01

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

  8. Construction of high quality Gateway™ entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Kumimoto Roderick W

    2011-05-01

    Full Text Available Abstract Background Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings, developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. Results We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark and long day (LD, 20/4-h light/dark grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. Conclusions Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user

  9. Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Taddei Kevin

    2010-01-01

    Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

  10. 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.

  11. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

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

  12. Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

    Science.gov (United States)

    Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

    2005-01-01

    We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

  13. Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

    Science.gov (United States)

    Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

    2014-09-01

    Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Nondestructive assay of sale materials

    International Nuclear Information System (INIS)

    Rodenburg, W.W.; Fleissner, J.G.

    1981-01-01

    This paper covers three primary areas: (1) reasons for performing nondestructive assay on SALE materials; (2) techniques used; and (3) discussion of investigators' revised results. The study shows that nondestructive calorimetric assay of plutonium offers a viable alternative to traditional wet chemical techniques. For these samples, the precision ranged from 0.4 to 0.6% with biases less than 0.2%. Thus, for those materials where sampling errors are the predominant source of uncertainty, this technique can provide improved accuracy and precision while saving time and money as well as reducing the amount of liquid wastes to be handled. In addition, high resolution gamma-ray spectroscopy measurements of solids can provide isotopic analysis data in a cost effective and timely manner. The timeliness of the method can be especially useful to the plant operator for production control and quality control measurements

  15. Radioreceptor assay for somatomedin A

    Energy Technology Data Exchange (ETDEWEB)

    Takano, K [Tokyo Women' s Medical Coll. (Japan)

    1975-04-01

    Measurement method of somatomedian A by radioreceptor assay using the human placenta membrane was described and discussed. Binding rate of /sup 125/I-somatomedin A to its receptors was studied under various conditions of time and temperature of the incubation, and pH of the system. The influence of somatomedin A, porcine insulin, and porcine calcitonin, on /sup 125/I-somatomedin A bound receptors was studied, and these hormones showed the competitive binding to somatomedin A receptors in some level. The specificity, recovery rate, and clinical applications of somatomedin A were also discussed. Radioreceptor assay for somatomedine A provided easier, faster, and more accurate measurements than conventional bioassay. This technique would be very useful to study somatomedin A receptor and functions of insulin.

  16. 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.

  17. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  18. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  19. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  20. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  1. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  2. 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)

    Garaj-Vrhovac, V.; Kopjar, N.

    2003-01-01

    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

  3. Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria

    Directory of Open Access Journals (Sweden)

    E. Zerbini

    2006-12-01

    Full Text Available El aumento de aislamientos clínicos de Mycobacterium tuberculosis resistentes a las drogas esenciales y de casos de micobacteriosis diseminadas debidas al complejo Mycobacterium avium hacen necesario investigar nuevos agentes antimicobacterianos. Los péptidos antimicrobianos son un nuevo grupo de antibióticos que poseen un mecanismo de acción particular. Algunos de ellos, como la cecropina y la melitina, han sido aislados de insectos y han demostrado buena actividad in vitro contra bacterias gram positivas y gram negativas. Híbridos sintéticos de esos péptidos han presentado mayor actividad que los péptidos individuales. En este trabajo se evaluó la actividad in vitro de dos péptidos híbridos sintéticos de melitina y cecropina contra M. tuberculosis, complejo M. avium, Mycobacterium fortuitum y Mycobacterium smegmatis. Se determinó la concentración inhibitoria mínima empleando la técnica de macrodilución en caldo. Luego se estableció la concentración bactericida mínima en medio Lowenstein Jensen. Los péptidos evaluados mostraron ser activos in vitro contra M. smegmatis, mientras que no presentaron ninguna actividad contra las otras micobacterias estudiadas.The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis

  4. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  5. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  6. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  7. 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...

  8. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  9. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  10. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  11. A multiwell format assay for heparanase.

    Science.gov (United States)

    Behzad, Farhad; Brenchley, Paul E C

    2003-09-15

    This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

  12. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  13. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

    2016-12-15

    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  14. 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+.

  15. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  16. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  17. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

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

    International Nuclear Information System (INIS)

    Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

    1981-01-01

    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 ( 51 Cr-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)

  19. Antioxidants and the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

    It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

  20. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

    Full Text Available An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease. Keywords: Prions, Thickness shear mode quartz sensor

  1. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  2. The narrow therapeutic window of glycated hemoglobin and assay variability.

    Science.gov (United States)

    Hosseini, S S; Bibler, I; Charles, M A

    1999-12-01

    Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

  3. Development of an integrated assay facility

    International Nuclear Information System (INIS)

    Molesworth, T.V.; Bailey, M.; Findlay, D.J.S.; Parsons, T.V.; Sene, M.R.; Swinhoe, M.T.

    1990-01-01

    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

  4. Risk Aversion in Game Shows

    DEFF Research Database (Denmark)

    Andersen, Steffen; Harrison, Glenn W.; Lau, Morten I.

    2008-01-01

    We review the use of behavior from television game shows to infer risk attitudes. These shows provide evidence when contestants are making decisions over very large stakes, and in a replicated, structured way. Inferences are generally confounded by the subjective assessment of skill in some games......, and the dynamic nature of the task in most games. We consider the game shows Card Sharks, Jeopardy!, Lingo, and finally Deal Or No Deal. We provide a detailed case study of the analyses of Deal Or No Deal, since it is suitable for inference about risk attitudes and has attracted considerable attention....

  5. Measuring performance at trade shows

    DEFF Research Database (Denmark)

    Hansen, Kåre

    2004-01-01

    Trade shows is an increasingly important marketing activity to many companies, but current measures of trade show performance do not adequately capture dimensions important to exhibitors. Based on the marketing literature's outcome and behavior-based control system taxonomy, a model is built...... that captures a outcome-based sales dimension and four behavior-based dimensions (i.e. information-gathering, relationship building, image building, and motivation activities). A 16-item instrument is developed for assessing exhibitors perceptions of their trade show performance. The paper presents evidence...

  6. Global optimization in the adaptive assay of subterranean uranium nodules

    International Nuclear Information System (INIS)

    Vulkan, U.; Ben-Haim, Y.

    1989-01-01

    An adaptive assay is one in which the design of the assay system is modified during operation in response to measurements obtained on-line. The present work has two aims: to design an adaptive system for borehole assay of isolated subterranean uranium nodules, and to investigate globality of optimal design in adaptive assay. It is shown experimentally that reasonably accurate estimates of uranium mass are obtained for a wide range of nodule shapes, on the basis of an adaptive assay system based on a simple geomorphological model. Furthermore, two concepts are identified which underlie the optimal design of the assay system. The adaptive assay approach shows promise for successful measurement of spatially random material in many geophysical applications. (author)

  7. Tokyo Motor Show 2003; Tokyo Motor Show 2003

    Energy Technology Data Exchange (ETDEWEB)

    Joly, E.

    2004-01-01

    The text which follows present the different techniques exposed during the 37. Tokyo Motor Show. The report points out the great tendencies of developments of the Japanese automobile industry. The hybrid electric-powered vehicles or those equipped with fuel cells have been highlighted by the Japanese manufacturers which allow considerable budgets in the research of less polluting vehicles. The exposed models, although being all different according to the manufacturer, use always a hybrid system: fuel cell/battery. The manufacturers have stressed too on the intelligent systems for navigation and safety as well as on the design and comfort. (O.M.)

  8. Evaluation of total PSA assay on vitros ECi and correlation with Kryptor-PSA assay.

    Science.gov (United States)

    Cassinat, B; Wacquet, M; Toubert, M E; Rain, J D; Schlageter, M H

    2001-01-01

    An increasing number of multiparametric immuno-analysers for PSA assays are available. As different immuno-assays may vary in their analytical quality and their accuracy for the follow-up of patients, expertise is necessary for each new assay. The PSA assay on the Vitros-ECi analyser has been evaluated and compared with the PSA assay from the Kryptor analyser. Variation coefficients were 0.91 to 1.98% for within-run assays, and 4.2% to 5.4% for interassay (PSA levels = 0.8 microgram/L to 33.6 micrograms/L). Dilution tests showed 93 to 136% recovery until 70 micrograms/L PSA. Functional sensitivity was estimated at 0.03 microgram/L. Equimolarity of the test was confirmed. Correlation of PSA levels measured with Vitros-ECi and Kryptor analysers displayed a correlation coefficient r2 of 0.9716. The half-lives and doubling times of PSA were similar using both methods. Vitros-ECi PSA assay meets the major criteria for the management of prostate cancer patients.

  9. Reality show: um paradoxo nietzschiano

    Directory of Open Access Journals (Sweden)

    Ilana Feldman

    2011-01-01

    Full Text Available

    O fenômeno dos reality shows - e a subseqüente relação entre imagem e verdade - assenta-se sobre uma série de paradoxos. Tais paradoxos podem ser compreendidos à luz do pensamento do filósofo alemão Friedrich Nietzsche, que, através dos usos de formulações paradoxais, concebia a realidade como um mundo de pura aparência e a verdade como um acréscimo ficcional, como um efeito. A ficção é então tomada, na filosofia de Nietzsche, não em seu aspecto falsificante e desrealizador - como sempre pleiteou nossa tradição metafísica -, mas como condição necessária para que certa espécie de invenção possa operar como verdade. Sendo assim, a própria expressão reality show, através de sua formulação paradoxal, engendra explicitamente um mundo de pura aparência, em que a verdade, a parte reality da proposição, é da ordem do suplemento, daquilo que se acrescenta ficcionalmente - como um adjetivo - a show. O ornamento, nesse caso, passa a ocupar o lugar central, apontando para o efeito produzido: o efeito-de-verdade. Seguindo, então, o pensamento nietzschiano e sua atualização na contemporaneidade, investigaremos de que forma os televisivos “shows de realidade” operam paradoxalmente, em consonância com nossas paradoxais práticas culturais.

  10. Determination of antipsychotic drug in human serum by radioreceptor assay

    International Nuclear Information System (INIS)

    Wu Jinchang; Jiang Yimin

    1989-01-01

    Serum antipsychotic drug in 50 psychosis cases were measured by radioreceptor assay (RRA) and the values were compared in parallel with that by radioimmunoassay (RIA). The results showed that the RRA values were lower than the RIA values, but both assays gave significant correlation between the serum drug level and antipsychotic dose

  11. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  12. Micronucleus assay for radiation workers

    International Nuclear Information System (INIS)

    Balasem, A.N.; Ali, A.S.K.

    1997-01-01

    Micronucleus assay was performed on 49 radiation workers and 22 healthy volunteers. Radiation workers were subdivided into two groups according to their employments durations in the radiation field. Group a consisted of 18 radiation workers who have been in this work between 5 and 22 years. Group b included 31 employees who have been classified as radiation workers for 1 to 4.5 years. Statistical analysis showed significant variations between the yields of micronuclei in groups A and B as well as between group A and a group of healthy controls. Meanwhile no significant difference was noticed between the yields of micronuclei in group B and the corresponding values in the healthy controls. The possible effect of age in the induction of micronuclei was discussed and a comparison with the yield of chromosomal aberrations was described. It seems that cytokinesis- blocking method may be used to detect the radiation-induced micronuclei in workers exposed occupationally to ionizing radiation in levels below the maximum permissible limit of 0.05 Sv per year

  13. PAME: plasmonic assay modeling environment

    Directory of Open Access Journals (Sweden)

    Adam Hughes

    2015-08-01

    Full Text Available Plasmonic assays are an important class of optical sensors that measure biomolecular interactions in real-time without the need for labeling agents, making them especially well-suited for clinical applications. Through the incorporation of nanoparticles and fiberoptics, these sensing systems have been successfully miniaturized and show great promise for in-situ probing and implantable devices, yet it remains challenging to derive meaningful, quantitative information from plasmonic responses. This is in part due to a lack of dedicated modeling tools, and therefore we introduce PAME, an open-source Python application for modeling plasmonic systems of bulk and nanoparticle-embedded metallic films. PAME combines aspects of thin-film solvers, nanomaterials and fiber-optics into an intuitive graphical interface. Some of PAME’s features include a simulation mode, a database of hundreds of materials, and an object-oriented framework for designing complex nanomaterials, such as a gold nanoparticles encased in a protein shell. An overview of PAME’s theory and design is presented, followed by example simulations of a fiberoptic refractometer, as well as protein binding to a multiplexed sensor composed of a mixed layer of gold and silver colloids. These results provide new insights into observed responses in reflectance biosensors.

  14. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L

    2010-01-01

    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

  15. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  16. Solution assay instrument operations manual

    International Nuclear Information System (INIS)

    Li, T.K.; Marks, T.; Parker, J.L.

    1983-09-01

    An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

  17. Nondestructive assay of HTGR fuel rods

    International Nuclear Information System (INIS)

    Menlove, H.O.

    1974-01-01

    Performance characteristics of three different radioactive source NDA systems are compared for the assay of HTGR fuel rods and stacks of rods. These systems include the fast neutron Sb-Be assay system, the 252 Cf ''Shuffler,'' and the thermal neutron PAPAS assay system. Studies have been made to determinethe perturbation on the measurements from particle size, kernel Th/U ratio, thorium content, and hydrogen content. In addition to the total 235 U determination, the pellet-to-pellet or rod-to-rod uniformity of HTGR fuel rod stacks has been measured by counting the delayed gamma rays with a NaI through-hole in the PAPAS system. These measurements showed that rod substitutions can be detected easily in a fuel stack, and that detailed information is available on the loading variations in a uniform stack. Using a 1.0 mg 252 Cf source, assay rates of 2 to 4 rods/s are possible, thus facilitating measurement of 100 percent of a plant's throughput. (U.S.)

  18. Radioligand assay in reproductive biology

    International Nuclear Information System (INIS)

    Korenman, S.G.; Sherman, B.M.

    1975-01-01

    Radioligand assays have been developed for the principal reproductive steroids and peptide hormones. Specific binding reagents have included antibodies, plasma binders, and intracellular receptors. In each assay, problems of specificity, sensitivity, and nonspecific inhibitors were encountered. Many features of the endocrine physiology in childhood, during puberty, and in adulthood have been characterized. Hormonal evaluations of endocrine disorders of reproduction are characterized on the basis of their characteristic pathophysiologic alterations. (U.S.)

  19. 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.

  20. Assays for mammalian tyrosinase: a comparative study

    International Nuclear Information System (INIS)

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    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

  1. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. First results with a radioreceptor-assay (TRAK-Assay) for TSH-receptor-autoantibodies

    International Nuclear Information System (INIS)

    Becker, W.; Reiners, C.; Boerner, W.

    1983-01-01

    A new radioreceptor-assay (TRAK-assay) for autoantibodies against TSH-receptors was tested in 48 untreated thyrotoxic patients (26 regional autonomies, 22 toxic diffuse goiters). None of the 26 patients with regional autonomy showed positive autoantibody-titers. 4 patients with toxic diffuse goiter and thyrotoxic exophthalmos were TRAK-positive. Positive titers of microsomal and thyreoglobulin autoantibodies could be seen in 8 of 9 patients with positive TRAK-titers. In accordance with the conventional methods for detecting thyroid-stimulating immunoglobulins the new TRAK-assay seems to be suited for differentiating between immunogenic toxic diffuse goiter (Graves' disease) and goiter with disseminated autonomy as well as for prediction of relapse. (orig.) [de

  3. Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs.

    Science.gov (United States)

    de Waaij, Dewi J; Ouburg, Sander; Dubbink, Jan Henk; Peters, Remco P H; Morré, Servaas A

    2016-08-01

    This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05). Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Identification of irradiated pepper with comet assay

    International Nuclear Information System (INIS)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra; Iglesia Enriquez, Isora

    2007-01-01

    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)

  5. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  6. Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

    Science.gov (United States)

    Maier, Richard H; Maier, Christina J; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2012-12-01

    Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. 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.

  8. Design of radiation dose tumor response assays

    International Nuclear Information System (INIS)

    Suit, H.D.; Hwang, T.; Hsieh, C.; Thames, H.

    1985-01-01

    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

  9. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  10. Automation of the dicentric chromosome assay and related assays

    International Nuclear Information System (INIS)

    Balajee, Adayabalam S.; Dainiak, Nicholas

    2016-01-01

    Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

  11. Assay strategies and methods for phospholipases

    International Nuclear Information System (INIS)

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

  12. Independency of Fe ions in hemoglobin on immunomagnetic reduction assay

    Energy Technology Data Exchange (ETDEWEB)

    Yang, S.Y. [MagQu Co. Ltd., Sindian City, Taipei County 231, Taiwan (China); Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Lan, C.B.; Chen, C.H. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Horng, H.E. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China)], E-mail: phyfv001@scc.ntnu.edu.tw; Hong, Chin-Yih [Department of Mechanical Engineering, Nan-Kai University of Technology, Nantau County, Taiwan (China)], E-mail: cyhong@nkut.edu.tw; Yang, H.C. [Department of Physics, National Taiwan University, Taipei 106, Taiwan (China)], E-mail: hcyang@phys.ntu.edu.tw; Lai, Y.K. [College of Life Sciences, National Tsing Hua University, Hsinchu City 300, Taiwan (China); Department of Bioresources, Da-Yeh University, Changhua 515, Taiwan (China); Lin, Y.H.; Teng, K.S. [Apex Biotechnology Co. Ltd., Hsinchu City 300, Taiwan (China)

    2009-10-15

    Immunomagnetic reduction (IMR), which involves measuring the reduction in the ac magnetic susceptibility of magnetic reagents, is due to the association between bio-functionalized magnetic nanoparticles and target bio-molecules. This has been demonstrated for assaying proteins in solutions free of Fe ions, such as serum. In this work, the validity of IMR assay for samples rich in Fe ions like hemoglobin (Hb) is investigated. According to the results, there is no magnetic signal contributed by Fe-ion-rich Hb. Furthermore, the results show a high sensitivity in assaying hemoglobin A1c (HbA1c) by using IMR.

  13. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    2013-01-01

    Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

  14. Independency of Fe ions in hemoglobin on immunomagnetic reduction assay

    International Nuclear Information System (INIS)

    Yang, S.Y.; Lan, C.B.; Chen, C.H.; Horng, H.E.; Hong, Chin-Yih; Yang, H.C.; Lai, Y.K.; Lin, Y.H.; Teng, K.S.

    2009-01-01

    Immunomagnetic reduction (IMR), which involves measuring the reduction in the ac magnetic susceptibility of magnetic reagents, is due to the association between bio-functionalized magnetic nanoparticles and target bio-molecules. This has been demonstrated for assaying proteins in solutions free of Fe ions, such as serum. In this work, the validity of IMR assay for samples rich in Fe ions like hemoglobin (Hb) is investigated. According to the results, there is no magnetic signal contributed by Fe-ion-rich Hb. Furthermore, the results show a high sensitivity in assaying hemoglobin A1c (HbA1c) by using IMR.

  15. Radioimmunoassay and radioenzymatic assay of a new aminoglycoside antibiotic, netilmicin

    International Nuclear Information System (INIS)

    Broughton, A.; Strong, J.E.; Pickering, L.K.; Knight, J.; Bodey, G.P.

    1978-01-01

    A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min, which is comparable to that for other aminoglycosides

  16. Assessing sediment contamination using six toxicity assays

    OpenAIRE

    Allen G. BURTON Jr.; Carolyn ROWLAND; Renato BAUDO; Monica BELTRAMI

    2001-01-01

    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 ...

  17. Nondestructive assay instrument for measurement of plutonium in solutions

    International Nuclear Information System (INIS)

    Shirk, D.G.; Hsue, F.; Li, T.K.; Canada, T.R.

    1979-01-01

    A nondestructive assay (NDA) instrument that measures the 239 Pu content in solutions, using a passive gamma-ray spectroscopy technique, has been developed and installed in the LASL Plutonium Processing Facility. A detailed evaluation of this instrument has been performed. The results show that the instrument can routinely determine 239 Pu concentrations of 1 to 500 g/l with accuracies of 1 to 5% and assay times of 1 to 2 x 10 3 s

  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.

  19. Assay of vitamin B12

    International Nuclear Information System (INIS)

    Tovey, K.C.; Carrick, D.T.

    1982-01-01

    A radioassay is described for vitamin B12 which involves denaturing serum protein binding proteins with alkali. In the denaturation step a dithiopolyol and cyanide are used and in the intrinsic factor assay step a vitamin B12 analogue such as cobinamide is used to bind with any remaining serum proteins. The invention also includes a kit in which the dithiopolyol is provided in admixture with the alkali. The dithiopolyol may be dithiothreitol or dithioerythritol. (author)

  20. Assay of ribulose bisphosphate carboxylase

    International Nuclear Information System (INIS)

    Pike, C.; Berry, J.

    1987-01-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, [ 14 C]-NaHCO 3 , and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30 0 . The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number

  1. A passive-active neutron device for assaying remote-handled transuranic waste

    International Nuclear Information System (INIS)

    Estep, R.J.; Coop, K.L.; Deane, T.M.; Lujan, J.E.

    1990-01-01

    A combined passive-active neutron assay device was constructed for assaying remote-handled transuranic waste. A study of matrix and source position effects in active assays showed that a knowledge of the source position alone is not sufficient to correct for position-related errors in highly moderating or absorbing matrices. An alternate function for the active assay of solid fuel pellets was derived, although the efficacy of this approach remains to be established

  2. Chromatographic assessment of two hybrid monoliths prepared via epoxy-amine ring-opening polymerization and methacrylate-based free radical polymerization using methacrylate epoxy cyclosiloxane as functional monomer.

    Science.gov (United States)

    Wang, Hongwei; Ou, Junjie; Lin, Hui; Liu, Zhongshan; Huang, Guang; Dong, Jing; Zou, Hanfa

    2014-11-07

    Two kinds of hybrid monolithic columns were prepared by using methacrylate epoxy cyclosiloxane (epoxy-MA) as functional monomer, containing three epoxy moieties and one methacrylate group. One column was in situ fabricated by ring-opening polymerization of epoxy-MA and 1,10-diaminodecane (DAD) using a porogenic system consisting of isopropanol (IPA), H2O and ethanol at 65°C for 12h. The other was prepared by free radical polymerization of epoxy-MA and ethylene dimethacrylate (EDMA) using 1-propanol and 1,4-butanediol as the porogenic solvents at 60°C for 12h. Two hybrid monoliths were investigated on the morphology and chromatographic assessment. Although two kinds of monolithic columns were prepared with epoxy-MA, their morphologies looked rather different. It could be found that the epoxy-MA-DAD monolith possessed higher column efficiencies (25,000-34,000plates/m) for the separation of alkylbenzenes than the epoxy-MA-EDMA monolith (12,000-13,000plates/m) in reversed-phase nano-liquid chromatography (nano-LC). Depending on the remaining epoxy or methacrylate groups on the surface of two pristine monoliths, the epoxy-MA-EDMA monolith could be easily modified with 1-octadecylamine (ODA) via ring-opening reaction, while the epoxy-MA-DAD monolith could be modified with stearyl methacrylate (SMA) via free radical reaction. The chromatographic performance for the separation of alkylbenzenes on SMA-modified epoxy-MA-DAD monolith was remarkably improved (42,000-54,000 plates/m) when compared with that on pristine epoxy-MA-DAD monolith, while it was not obviously enhanced on ODA-modified epoxy-MA-EDMA monolith when compared with that on pristine epoxy-MA-EDMA monolith. The enhancement of the column efficiency of epoxy-MA-DAD monolith after modification might be ascribed to the decreased mass-transfer resistence. The two kinds of hybrid monoliths were also applied for separations of six phenols and seven basic compounds in nano-LC. Copyright © 2014 Elsevier B.V. All

  3. Integrated bioassays in microfluidic devices: botulinum toxin assays.

    Science.gov (United States)

    Mangru, Shakuntala; Bentz, Bryan L; Davis, Timothy J; Desai, Nitin; Stabile, Paul J; Schmidt, James J; Millard, Charles B; Bavari, Sina; Kodukula, Krishna

    2005-12-01

    A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

  4. Performance characteristics of the ARCHITECT anti-HCV assay.

    Science.gov (United States)

    Jonas, Gesa; Pelzer, Claudia; Beckert, Christian; Hausmann, Michael; Kapprell, Hans-Peter

    2005-10-01

    The ARCHITECT Anti-HCV assay is a fully automated high throughput chemiluminescent microparticle immunoassay (CMIA) for the detection of antibodies to structural and nonstructural proteins of the hepatitis C virus (HCV). To further enhance the performance of this test, the assay was modified to improve the specificity for blood donor specimens. The specificity of the enhanced ARCHITECT Anti-HCV assay was evaluated by screening blood donor samples randomly collected from various German blood banks, as well as hospitalized patient samples derived from Germany and the US. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels and on a commercially available worldwide anti-HCV genotype performance panel. Apparent specificity of the modified ARCHITECT Anti-HCV assay in a blood donor population consisting of 3811 specimens was 99.92%, compared to 99.76% for the current on-market assay. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels. Seroconversion sensitivity equivalent to or better than the current on-market product was observed by testing 33 seroconversion panels. This study demonstrates that the modified version of the ARCHITECT Anti-HCV assay shows improved specificity for blood donor specimens compared to the current assay on market without compromising sensitivity. With the availability of the improved ARCHITECT Anti-HCV assay and the recent launch of the ARCHITECT HIV Ag/Ab Combo assay, the ARCHITECT system now offers a full hepatitis/retrovirus menu with excellent performance on a high throughput, random access, automated analyzer, ideally suited for blood screening and diagnostic applications.

  5. Study on quantification of HBs-antibody by immunoradiometric assay

    International Nuclear Information System (INIS)

    Kondo, Yuichi; Itoi, Yoshihiro; Kajiyama, Shizuo

    1989-01-01

    Quantification of HBs-antibody assay was carried out using a commercialized assay kit and standard solutions of HBs-antibody recognised as 1 st reference preparation of hepatitis B immunogloblin by WHO. Standard curve of HBs-antibody was drawn with the function of 3D-spline and the correlation factor was obtained as r = 0.999. Coefficient of intra-assay variance was 3.8 % and that of inter-assay variance was 7.8 %. Dilution tests showed satisfactory results in the range of 2-16 times. Correlation between value of cut-off indices and concentration of HBs-antibody was obtained as the formula of y = 2.599 x-3.894 (r = 0.992) and 2.1 of cut-off index corresponded to about 5 mIU/ml of HBs-antibody concentration. (author)

  6. Radiosotopic assay and binder therefor

    International Nuclear Information System (INIS)

    Caston, J.D.; Kamen, B.A.

    1976-01-01

    A rapid and less costly radioisotopic assay for measuring the concentration of folate in blood serum is described. This procedure utilizes 3 H-pteroylmonoglutamate, unlabeled 5-methyltetrahydrofolic acid, and a partially purified folate binder, such as for example a folate binder extracted from hog kidney. The procedure involves radioisotopically relating the bound amounts of a labeled folate and a known folate at various concentrations of the known folate in a system containing a predetermined amount of the labeled folate, a predetermined amount of the binder factor for the folates, and a predetermined amount of defolated test serum. 16 claims, 8 drawing figures

  7. Biobarcode assay for the oral anticoagulant acenocoumarol.

    Science.gov (United States)

    Broto, Marta; Salvador, J Pablo; Galve, Roger; Marco, M Pilar

    2018-02-01

    A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R 2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Rotor assembly and assay method

    Science.gov (United States)

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  9. Data transformation methods for multiplexed assays

    Science.gov (United States)

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  10. 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...

  11. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

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

    2014-01-01

    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  12. Assaying environmental nickel toxicity using model nematodes.

    Directory of Open Access Journals (Sweden)

    David Rudel

    Full Text Available Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water, we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  13. Assaying environmental nickel toxicity using model nematodes

    Science.gov (United States)

    Rudel, David; Douglas, Chandler; Huffnagle, Ian; Besser, John M.; Ingersoll, Christopher G.

    2013-01-01

    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegansand P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  14. Determination of Nitrate Reductase Assay Depending on the Microbial Growth

    International Nuclear Information System (INIS)

    El-Kabbany, H.M.

    2012-01-01

    A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

  15. Making transuranic assay measurements using modern controllers

    International Nuclear Information System (INIS)

    Kuckertz, T.H.; Caldwell, J.T.; Medvick, P.A.; Kunz, W.E.; Hastings, R.D.

    1987-01-01

    This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

  16. Non destructive assay (NDA) techniques

    International Nuclear Information System (INIS)

    Mafra Guidicini, Olga; Llacer, Carlos D.; Rojo, Marcelo

    2001-01-01

    In the IAEA Safeguards System the basic verification method used is nuclear material accountancy, with containment and surveillance as important complementary measures. If nuclear material accountancy is to be effective, IAEA inspectors have to make independent measurements to verify declared material quantities. Most of the equipment available to the inspectors is designed to measure gamma rays and/or neutrons emitted by various nuclear materials. Equipment is also available to measure the gross weight of an item containing nuclear material. These types of measurement techniques are generally grouped under the title of nondestructive assay (NDA). The paper describes the NDA techniques and instruments used to verify the total amount of nuclear material held at a nuclear facility. (author)

  17. Assay of cysteine dioxygenase activity

    International Nuclear Information System (INIS)

    Bagley, P.J.; Stipanuk, M.H.

    1990-01-01

    It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD + and has a pH optimum of 6.8 and one which is not stimulated by NAD + and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac trademark PA1 (4x250 mm)) was used to separate the [ 35 S]cysteinesulfinic acid produced from [ 35 S]cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine

  18. Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

    Science.gov (United States)

    Kidwell, David A.

    1993-05-01

    A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

  19. Systematic random sampling of the comet assay.

    Science.gov (United States)

    McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

    2009-07-01

    The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

  20. Expert system for transuranic waste assay

    Energy Technology Data Exchange (ETDEWEB)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  1. Expert system for transuranic waste assay

    International Nuclear Information System (INIS)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

  2. Radiometric assays for glycerol, glucose, and glycogen

    International Nuclear Information System (INIS)

    Bradley, D.C.; Kaslow, H.R.

    1989-01-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

  3. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    Prasanna, Pataje G.

    2014-01-01

    The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

  4. 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

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

    Science.gov (United States)

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

    2012-05-01

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

  6. Automated amperometric plutonium assay system

    International Nuclear Information System (INIS)

    Burt, M.C.

    1985-01-01

    The amperometric titration for plutonium assay has been used in the nuclear industry for over twenty years and has been in routine use at the Hanford Engineering Development Laboratory since 1976 for the analysis of plutonium oxide and mixed oxide fuel material for the Fast Flux Test Facility. It has proven itself to be an accurate and reliable method. The method may be used as a direct end point titration or an excess of titrant may be added and a back titration performed to aid in determination of the end point. Due to the slowness of the PuVI-FeII reaction it is difficult to recognize when the end point is being approached and is very time consuming if the current is allowed to decay to the residual value after each titrant addition. For this reason the back titration in which the rapid FeII-CrVI reaction occurs is used by most laboratories. The back titration is performed by the addition of excess ferrous solution followed by two measured aliquots of standard dichromate with measurement of cell current after each addition

  7. TRU assay system and measurements

    International Nuclear Information System (INIS)

    Brodzinski, R.L.

    1984-02-01

    The measurement of the transuranic content of nuclear products or process residues has become increasingly important for the recovery of fissionable material from spent fuel elements, the identification of commercial fuel elements which have not yet reached full burnup, the measurement and recovery of transuranics from discarded or stored waste materials, the determination of the transuranic content in high gamma activity waste material scheduled for disposal, compliance with 10CFR61 by land burial operators/shippers, and the satisfaction of accountability requirements. Active neutron interrogation techniques measure either the prompt neutrons or the beta delayed neutrons from fission products following induced fission. These techniques normally only measure fissile transuranics ( 235 U, 239 Pu, and 241 Pu) and are commonly applied only to contact handleable waste. Passive neutron interrogation techniques, on the other hand, are capable of measuring all transuranics except 235 U with adequate sensitivity and will work on both contact handleable and high gamma activity wastes. Since the passive techniques are senstitive to a wider spectrum of transuranic isotopes than the active techniques, substantially less complex and less expensive than the active systems, and they have proven techniques for measuring small quantities of TRU in high gamma activity packages, the passive neutron TRU assay technology was chosen for development into the instruments discussed in this paper

  8. Assay development status report for total cyanide

    International Nuclear Information System (INIS)

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

  9. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  10. New automated pellet/powder assay system

    International Nuclear Information System (INIS)

    Olsen, R.N.

    1975-01-01

    This paper discusses an automated, high precision, pellet/ powder assay system. The system is an active assay system using a small isotopic neutron source and a coincidence detection system. The handling of the pellet powder samples has been automated and a programmable calculator has been integrated into the system to provide control and data analysis. The versatile system can assay uranium or plutonium in either active or passive modes

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

    International Nuclear Information System (INIS)

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

  12. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. 233U Assay A Neutron NDA System

    Energy Technology Data Exchange (ETDEWEB)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  14. 233U Assay A Neutron NDA System

    International Nuclear Information System (INIS)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-01-01

    The assay of highly enriched 233 U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched 235 U do not convert easily over to the assay of 233 U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with γ ray isotopics information should give a good overall determination of 233 U material now stored in bldg. 3019 at the Oak Ridge National Laboratory

  15. Safeguards and Non-destructive Assay

    International Nuclear Information System (INIS)

    Carchon, R.; Bruggeman, M.

    2001-01-01

    SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

  16. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

    Science.gov (United States)

    Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

    2005-02-01

    The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

  17. Application of radioreceptor assay of benzodiazepines for toxicology

    International Nuclear Information System (INIS)

    Aaltonen, L.; Scheinin, M.

    1982-01-01

    A radioreceptor assay (RRA) for determining benzodiazepines (BZ) has been developed and applied to toxicological analysis of serum samples from 21 patients with acute BZ overdosage. The method was sensitive (e.g., lorazepam 17 nM, diazepam 41 nM), and specific for pharmacologically active BZ derivatives. The reproducibility of the results was good (intra-assay variation < 8%, inter-assay variation < 10%). Concentrations measured by the RRA showed a good correlation with those obtained by gas-liquid chromatographic analysis of the same samples. The quantitative results represent the sum of one or several parent substances and all biologically active metabolites, in proportion to their receptor binding affinities. (author)

  18. Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays.

    Science.gov (United States)

    Fischer, Janine; Prosenc, Marc H; Wolff, Martin; Hort, Norbert; Willumeit, Regine; Feyerabend, Frank

    2010-05-01

    Magnesium (Mg) alloys are promising materials for the development of biodegradable implants. However, the current in vitro test procedures for cytotoxicity, cell viability and proliferation are not always suitable for this class of materials. In this paper we show that tetrazolium-salt-based assays, which are widely used in practice, are influenced by the corrosion products of Mg-based alloys. Corroded Mg converts tetrazolium salts to formazan, leading to a higher background and falsifying the results of cell viability. Tetrazolium-based assays are therefore not a useful tool for testing the cytotoxicity of Mg in static in vitro assays. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  19. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  20. Competitive binding assay for fructose 2,6-bisphosphate

    International Nuclear Information System (INIS)

    Thomas, H.; Uyeda, K.

    1986-01-01

    A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P 2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol promoter) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6[2- 32 P]P 2 and samples containing varying concentrations of fructose 2,6-P 2 . The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P 2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P 2 , ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. The new assay is simple, direct, rapid, and does not require pretreatment

  1. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    Hood, R.L.

    1975-01-01

    A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

  2. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    Science.gov (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  3. UV Photography Shows Hidden Sun Damage

    Science.gov (United States)

    ... mcat1=de12", ]; for (var c = 0; c UV photography shows hidden sun damage A UV photograph gives ... developing skin cancer and prematurely aged skin. Normal photography UV photography 18 months of age: This boy's ...

  4. Radioreceptor assay: theory and applications to pharmacology

    International Nuclear Information System (INIS)

    Perret, G.; Simon, P.

    1984-01-01

    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 [fr

  5. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    DEFF Research Database (Denmark)

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred

    2016-01-01

    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  6. 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 Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  7. The optimal condition of performing MTT assay for the determination of radiation sensitivity

    International Nuclear Information System (INIS)

    Hong, Semie; Kim, Il Han

    2001-01-01

    The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. Four human cancer cell lines - PCI-1, SNU-1066, NCI-H63O and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy, For clonogenic assay, cells in 25 cm 2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10-14 days, For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R 2 value of 0.975-0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was alter 6

  8. Educational Outreach: The Space Science Road Show

    Science.gov (United States)

    Cox, N. L. J.

    2002-01-01

    The poster presented will give an overview of a study towards a "Space Road Show". The topic of this show is space science. The target group is adolescents, aged 12 to 15, at Dutch high schools. The show and its accompanying experiments would be supported with suitable educational material. Science teachers at schools can decide for themselves if they want to use this material in advance, afterwards or not at all. The aims of this outreach effort are: to motivate students for space science and engineering, to help them understand the importance of (space) research, to give them a positive feeling about the possibilities offered by space and in the process give them useful knowledge on space basics. The show revolves around three main themes: applications, science and society. First the students will get some historical background on the importance of space/astronomy to civilization. Secondly they will learn more about novel uses of space. On the one hand they will learn of "Views on Earth" involving technologies like Remote Sensing (or Spying), Communication, Broadcasting, GPS and Telemedicine. On the other hand they will experience "Views on Space" illustrated by past, present and future space research missions, like the space exploration missions (Cassini/Huygens, Mars Express and Rosetta) and the astronomy missions (Soho and XMM). Meanwhile, the students will learn more about the technology of launchers and satellites needed to accomplish these space missions. Throughout the show and especially towards the end attention will be paid to the third theme "Why go to space"? Other reasons for people to get into space will be explored. An important question in this is the commercial (manned) exploration of space. Thus, the questions of benefit of space to society are integrated in the entire show. It raises some fundamental questions about the effects of space travel on our environment, poverty and other moral issues. The show attempts to connect scientific with

  9. 2008 LHC Open Days Physics: the show

    CERN Multimedia

    2008-01-01

    A host of events and activities await visitors to the LHC Open Days on 5 and 6 April. A highlight will be the physics shows funded by the European Physical Society (EPS), which are set to surprise and challenge children and adults alike! School children use their experience of riding a bicycle to understand how planets move around the sun (Copyright : Circus Naturally) Participating in the Circus Naturally show could leave a strange taste in your mouth! (Copyright : Circus Naturally) The Rino Foundation’s experiments with liquid nitrogen can be pretty exciting! (Copyright: The Rino Foundation)What does a bicycle have in common with the solar system? Have you ever tried to weigh air or visualise sound? Ever heard of a vacuum bazooka? If you want to discover the answers to these questions and more then come to the Physics Shows taking place at the CERN O...

  10. Online Italian fandoms of American TV shows

    Directory of Open Access Journals (Sweden)

    Eleonora Benecchi

    2015-06-01

    Full Text Available The Internet has changed media fandom in two main ways: it helps fans connect with each other despite physical distance, leading to the formation of international fan communities; and it helps fans connect with the creators of the TV show, deepening the relationship between TV producers and international fandoms. To assess whether Italian fan communities active online are indeed part of transnational online communities and whether the Internet has actually altered their relationship with the creators of the original text they are devoted to, qualitative analysis and narrative interviews of 26 Italian fans of American TV shows were conducted to explore the fan-producer relationship. Results indicated that the online Italian fans surveyed preferred to stay local, rather than using geography-leveling online tools. Further, the sampled Italian fans' relationships with the show runners were mediated or even absent.

  11. Evaluation of multiplex assay platforms for detection of influenza hemagglutinin subtype specific antibody responses.

    Science.gov (United States)

    Li, Zhu-Nan; Weber, Kimberly M; Limmer, Rebecca A; Horne, Bobbi J; Stevens, James; Schwerzmann, Joy; Wrammert, Jens; McCausland, Megan; Phipps, Andrew J; Hancock, Kathy; Jernigan, Daniel B; Levine, Min; Katz, Jacqueline M; Miller, Joseph D

    2017-05-01

    Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex ® , and ForteBio ® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections. Published by Elsevier B.V.

  12. Duchenne muscular dystrophy models show their age

    OpenAIRE

    Chamberlain, Jeffrey S.

    2010-01-01

    The lack of appropriate animal models has hampered efforts to develop therapies for Duchenne muscular dystrophy (DMD). A new mouse model lacking both dystrophin and telomerase (Sacco et al., 2010) closely mimics the pathological progression of human DMD and shows that muscle stem cell activity is a key determinant of disease severity.

  13. Show Them You Really Want the Job

    Science.gov (United States)

    Perlmutter, David D.

    2012-01-01

    Showing that one really "wants" the job entails more than just really wanting the job. An interview is part Broadway casting call, part intellectual dating game, part personality test, and part, well, job interview. When there are 300 applicants for a position, many of them will "fit" the required (and even the preferred) skills listed in the job…

  14. A Talk Show from the Past.

    Science.gov (United States)

    Gallagher, Arlene F.

    1991-01-01

    Describes a two-day activity in which elementary students examine voting rights, the right to assemble, and women's suffrage. Explains the game, "Assemble, Reassemble," and a student-produced talk show with five students playing the roles of leaders of the women's suffrage movement. Profiles Elizabeth Cady Stanton, Lucretia Mott, Susan…

  15. Laser entertainment and light shows in education

    Science.gov (United States)

    Sabaratnam, Andrew T.; Symons, Charles

    2002-05-01

    Laser shows and beam effects have been a source of entertainment since its first public performance May 9, 1969, at Mills College in Oakland, California. Since 1997, the Photonics Center, NgeeAnn Polytechnic, Singapore, has been using laser shows as a teaching tool. Students are able to exhibit their creative skills and learn at the same time how lasers are used in the entertainment industry. Students will acquire a number of skills including handling three- phase power supply, operation of cooling system, and laser alignment. Students also acquire an appreciation of the arts, learning about shapes and contours as they develop graphics for the shows. After holography, laser show animation provides a combination of the arts and technology. This paper aims to briefly describe how a krypton-argon laser, galvanometer scanners, a polychromatic acousto-optic modulator and related electronics are put together to develop a laser projector. The paper also describes how students are trained to make their own laser animation and beam effects with music, and at the same time have an appreciation of the operation of a Class IV laser and the handling of optical components.

  16. The Last Great American Picture Show

    NARCIS (Netherlands)

    Elsaesser, Thomas; King, Noel; Horwath, Alexander

    2004-01-01

    The Last Great American Picture Show brings together essays by scholars and writers who chart the changing evaluations of the American cinema of the 1970s, sometimes referred to as the decade of the lost generation, but now more and more recognized as the first New Hollywood, without which the

  17. Individual response to ionising radiation: What predictive assay(s) to choose?

    International Nuclear Information System (INIS)

    Granzotto, A.; Viau, M.; Devic, C.; Maalouf, M.; Thomas, Ch.; Vogin, G.; Foray, N.; Granzotto, A.; Vogin, G.; Balosso, J.; Joubert, A.; Maalouf, M.; Vogin, G.; Colin, C.; Malek, K.; Balosso, J.; Colin, C.

    2011-01-01

    Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomics technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensing and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavored to analyse the contributions of these major approaches to predict human radiosensitivity. (authors)

  18. Optimized candidal biofilm microtiter assay

    NARCIS (Netherlands)

    Krom, Bastiaan P.; Cohen, Jesse B.; Feser, Gail E. McElhaney; Cihlar, Ronald L.

    Microtiter based candidal biofilm formation is commonly being used. Here we describe the analysis of factors influencing the development of candidal biofilms such as the coating with serum, growth medium and pH. The data reported here show that optimal candidal biofilm formation is obtained when

  19. Eccentric muscle challenge shows osteopontin polymorphism modulation of muscle damage.

    Science.gov (United States)

    Barfield, Whitney L; Uaesoontrachoon, Kitipong; Wu, Chung-Sheih; Lin, Stephen; Chen, Yue; Wang, Paul C; Kanaan, Yasmine; Bond, Vernon; Hoffman, Eric P

    2014-08-01

    A promoter polymorphism of the osteopontin (OPN) gene (rs28357094) has been associated with multiple inflammatory states, severity of Duchenne muscular dystrophy (DMD) and muscle size in healthy young adults. We sought to define the mechanism of action of the polymorphism, using allele-specific in vitro reporter assays in muscle cells, and a genotype-stratified intervention in healthy controls. In vitro reporter constructs showed the G allele to respond to estrogen treatment, whereas the T allele showed no transcriptional response. Young adult volunteers (n = 187) were enrolled into a baseline study, and subjects with specific rs28357094 genotypes enrolled into an eccentric muscle challenge intervention [n = 3 TT; n = 3 GG/GT (dominant inheritance model)]. Female volunteers carrying the G allele showed significantly greater inflammation and increased muscle volume change as determined by magnetic resonance imaging T1- and T2-weighted images after eccentric challenge, as well as greater decrement in biceps muscle force. Our data suggest a model where the G allele enables enhanced activities of upstream enhancer elements due to loss of Sp1 binding at the polymorphic site. This results in significantly greater expression of the pro-inflammatory OPN cytokine during tissue remodeling in response to challenge in G allele carriers, promoting muscle hypertrophy in normal females, but increased damage in DMD patients. © The Author 2014. Published by Oxford University Press.

  20. Detection of hepatitis B surface antigen by solid phase radioimmunoassay and immunometric assay (using enhanced luminescence)

    International Nuclear Information System (INIS)

    Nicholson, S.; Efandis, T.; Gust, I.

    1991-01-01

    A study was performed to assess the sensitivity and specificity of the amerlite monoclonal immunoassay for detection of hepatitis B surface antigen (HBsAG) by comparison with the Abbott Ausria II radioimmunoassay (RI). Serial bleeds from 34 patients with acute or chronic hepatitis B were tested by both assays. The Abbott Ausria II assay detected HBsAG longer than the Amersham Amerlite assay on two occasions and earlier on one occasion. Twelve patients with low HBsAg positive results (confirmed by Ausria II) were tested by the Amerlite assay, four were repeatably positive, five repeatably negative and three gave borderline results (which on repeat testing were negative). A similar trend was seen when a panel of sera containing known concentrations of HBsAG was tested. Replicate testing of 10 specimens eight times showed very good reproducibility by the Amerlite assay. Overall, the specificity of both assays was comparable, however differences in sensitivity were observed. 3 tabs

  1. Nano-immunosafety: issues in assay validation

    International Nuclear Information System (INIS)

    Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

    2011-01-01

    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  2. Radioactive wastes assay technique and equipment

    International Nuclear Information System (INIS)

    Lee, K. M.; Hong, D. S; Kim, T. K.; Bae, S. M.; Shon, J. S.; Hong, K. P.

    2004-12-01

    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

  3. A study on the toxicity of three radiosensitizers on retinoblastoma cells by MTT assay

    International Nuclear Information System (INIS)

    Yi Xianjin; Jin Yizun; Ding Li; Ni Zhou; Wang Wenji

    1994-01-01

    The toxicity of three radiosensitizers BSO, CM and RSU-1069 on retinoblastoma cells was determined and the efficiency of in vitro MTT assay on drug-screening for retinoblastoma was also evaluated. The results showed that the MTT assay is very useful. The toxicity of radiosensitizers on retinoblastoma cells is dependent on cell line characteristics, drug concentration and time of exposure to it

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

    Directory of Open Access Journals (Sweden)

    Ta-Wei Liu

    2015-01-01

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

  5. Reality, ficción o show

    Directory of Open Access Journals (Sweden)

    Sandra Ruíz Moreno

    2002-01-01

    Full Text Available Para tener un punto de vista claro y objetivo frente a la polémica establecida en torno al programa “Protagonistas de novela” y la tendiente proliferación de los reality show en las parrillas de programación de la televisión colombiana, se realizó un análisis de texto y contenido de dicho programa, intentando definirlo desde sus posibilidades de realidad, ficción y show. Las unidades de análisis y el estudio de su tratamiento arrojaron un alto contenido que gira en torno a las emociones del ser humano relacionadas con la convivencia, tratadas a manera de show y con algunos aportes textuales de ficción, pero sin su elemento mediador básico, el actor, quitándole toda la posibilidad de tener un tratamiento con la profundidad, distancia y ética que requieren los temas de esta índole. El resultado es un formato que sólo busca altos índices de sintonía y que pertenece más a la denominada televisión “trash”, que a una búsqueda de realidad del hombre y mucho menos de sociedad.

  6. Comparative evaluation of two radioenzymatic procedures designed to determine noradrenaline in the plasma (COMT assay and PNMT assay)

    International Nuclear Information System (INIS)

    Barth, A.

    1984-01-01

    A comparative evaluation of two radioenzymatic procedures to determine the concentration of noradrenaline in the plasma - with linearity, sensitivity, specifity and accuracy serving as test criteria - led to the following results: In view of a probability of error in the order of 2% both methods were judged to show a satisfactory sensitivity. The specific of the COMT assay, by contrast with that of the PNMT assay, was found to be wanting, as the noradrenaline measurements in the presence of other biogenic amines were biassed in such a way that the values determined were higher than the actual concentrations. During antihypertensive treatment even minimal changes in the noradrenaline concentration can be ascertained on a quantitative basis. If suitable hardware is available, the COMT assay permits up to 25 single determinations to be carried out per day, while the number of double determinations is restricted to 7 per day. One advantage, however, lies in the fact that several catecholamines in the plasma can be detected simultaneously, if required. In cases where the noradrenaline concentration alone is to be determined for clinical purposes, preference should be given to the PNMT assay, as both tests showed equal linearity and sensitivity. (TRV) [de

  7. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

    Directory of Open Access Journals (Sweden)

    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

  8. Nondestructive assay measurements applied to reprocessing plants

    International Nuclear Information System (INIS)

    Ruhter, Wayne D.; Lee, R. Stephen; Ottmar, Herbert; Guardini, Sergio

    1999-01-01

    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 [ru

  9. Thermometric enzyme linked immunosorbent assay: TELISA.

    Science.gov (United States)

    Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

    1977-08-11

    A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

  10. Multisite analytical evaluation of the Abbott ARCHITECT cyclosporine assay.

    Science.gov (United States)

    Wallemacq, Pierre; Maine, Gregory T; Berg, Keith; Rosiere, Thomas; Marquet, Pierre; Aimo, Giuseppe; Mengozzi, Giulio; Young, Julianna; Wonigeit, Kurt; Kretschmer, Robert; Wermuth, Bendicht; Schmid, Rainer W

    2010-04-01

    The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Cyclosporine (CsA) immunoassay in 7 clinical laboratories in comparison to liquid chromatography/tandem mass spectrometry (LC/MS/MS), Abbott TDx, Cobas Integra 800, and the Dade Dimension Xpand immunoassay. The ARCHITECT assay uses a whole blood specimen, a pretreatment step with organic reagents to precipitate proteins and extract the drug, followed by a 2-step automated immunoassay with magnetic microparticles coated with anti-CsA antibody and an acridinium-CsA tracer. Imprecision testing at the 7 evaluation sites gave a range of total % coefficient of variations of 7.5%-12.2% at 87.5 ng/mL, 6.6%-14.3% at 411 ng/mL, and 5.2%-10.7% at 916 ng/mL. The lower limit of quantification ranged from 12 to 20 ng/mL. Purified CsA metabolites AM1, AM1c, AM4N, AM9, and AM19 were tested in whole blood by the ARCHITECT assay and showed minimal cross-reactivity at all 7 sites. In particular, AM1 and AM9 cross-reactivity in the ARCHITECT assay, ranged from -2.5% to 0.2% and -0.8% to 2.2%, respectively, and was significantly lower than for the TDx assay, in which the values were 3.2% and 16.1%, respectively. Comparable testing of metabolites in the Dade Dimension Xpand assay at 2 evaluation sites showed cross-reactivity to AM4N (6.4% and 6.8%) and AM9 (2.6% and 3.6%) and testing on the Roche Integra 800 showed cross-reactivity to AM1c (2.4%), AM9 (10.7%), and AM19 (2.8%). Cyclosporine International Proficiency Testing Scheme samples, consisting of both pooled specimens from patients receiving CsA therapy as well as whole-blood specimens supplemented with CsA, were tested by the ARCHITECT assay at 6 sites and showed an average bias of -24 to -58 ng/mL versus LC/MSMS CsA and -2 to -37 ng/mL versus AxSYM CsA. Studies were performed with the ARCHITECT CsA assay on patient specimens with the following results: ARCHITECT CsA assay versus LC/MSMS, average bias of 31 ng/mL; ARCHITECT versus the

  11. Neutron Assay System for Con?nement Vessel Disposition

    International Nuclear Information System (INIS)

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Valdez, Jose I.; Vigil, Georgiana M.

    2012-01-01

    Waste will be removed from confinement vessels remaining from 1970s-era experiments. Los Alamos has 9+ spherical confinement vessels remaining from experiments. Each vessel contains ∼ 500 lbs of radioactive debris such as actinide metals and oxides, metals, powdered silica, graphite, and wires and hardware. In order to dispose of the vessels, debris and contamination must be removed. Neutron assay system was designed to assay vessels before and after cleanout. System requirements are: (1) Modular and moveable; (2) Capable of detecting ∼100g 239 Pu equivalent in a 2-inch thick steel sphere with 6 foot diameter; and (3) Capable of safeguards-quality assays. Initial design parameters arethe use of 4-atm 3 He tubes with length of 6 feet, and 3 He tubes embedded in polyethelene for moderation. This paper describes the calibration of the Confinement Vessel Assay System (CVAS) and quantification of its uncertainties. Assay uncertainty depends on five factors: (1) Statistical uncertainty in the assay measurement; (2) Statistical uncertainty in the background measurement; (3) Statistical uncertainty in the isotopics determination - This should be much smaller than the other uncertainties; (4) Systematic uncertainty due to position bias; and (5) Systematic uncertainty due to fluctuations in cosmic ray spallation. This one can be virtually eliminated by performing the background measurement with an empty vessel - but that may not be possible. We used modeling and experiments to quantify the systematic uncertainties. The calibration assumes a uniform distribution of material, but reality will be different. MCNPX modeling was used to quantify the positional bias. The model was benchmarked to build confidence in its results. Material at top of vessel is 44% greater than amount assayed, according to singles. Material near 19-tube detector is 38% less than amount assayed, according to singles. Cosmic ray spallation contributes significantly to the background. Comparing rates

  12. Prandiology of Drosophila and the CAFE assay

    Science.gov (United States)

    Ja, William W.; Carvalho, Gil B.; Mak, Elizabeth M.; de la Rosa, Noelle N.; Fang, Annie Y.; Liong, Jonathan C.; Brummel, Ted; Benzer, Seymour

    2007-01-01

    Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7× their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant. PMID:17494737

  13. Performance evaluation of a particle-enhanced turbidimetric cystatin C assay on the Abbott ci8200 analyzer.

    Science.gov (United States)

    Flodin, Mats; Larsson, Anders

    2009-06-01

    Glomerular filtration rate (GFR) is widely accepted as the best overall measure of kidney function. Cystatin C is a novel endogenous GFR marker that has been shown to be superior to creatinine for estimation of GFR in several studies. There is a need for cystatin C assays adapted to routine chemistry instrument to minimize turnaround times and allowing 24 h/day availability. We have evaluated a new cystatin C assay developed for Architect cSystem (Abbott Laboratories, Abbott Park, IL, USA). The cystatin C assay showed good agreement with the corresponding assay from Dade Behring (Deerfield, IL, USA). The assay has a very low total imprecision and a good linearity. The new cystatin C assay is an interesting alternative to current cystatin C assays. On an Architect cSystem the assay can be performed with the same turnaround times and availability as creatinine.

  14. Myopes show increased susceptibility to nearwork aftereffects.

    Science.gov (United States)

    Ciuffreda, K J; Wallis, D M

    1998-09-01

    Some aspects of accommodation may be slightly abnormal (or different) in myopes, compared with accommodation in emmetropes and hyperopes. For example, the initial magnitude of accommodative adaptation in the dark after nearwork is greatest in myopes. However, the critical test is to assess this initial accommodative aftereffect and its subsequent decay in the light under more natural viewing conditions with blur-related visual feedback present, if a possible link between this phenomenon and clinical myopia is to be considered. Subjects consisted of adult late- (n = 11) and early-onset (n = 13) myopes, emmetropes (n = 11), and hyperopes (n = 9). The distance-refractive state was assessed objectively using an autorefractor immediately before and after a 10-minute binocular near task at 20 cm (5 diopters [D]). Group results showed that myopes were most susceptible to the nearwork aftereffect. It averaged 0.35 D in initial magnitude, with considerably faster posttask decay to baseline in the early-onset (35 seconds) versus late-onset (63 seconds) myopes. There was no myopic aftereffect in the remaining two refractive groups. The myopes showed particularly striking accommodatively related nearwork aftereffect susceptibility. As has been speculated and found by many others, transient pseudomyopia may cause or be a precursor to permanent myopia or myopic progression. Time-integrated increased retinal defocus causing axial elongation is proposed as a possible mechanism.

  15. Split Beta-Lactamase Complementation Assay

    Indian Academy of Sciences (India)

    IAS Admin

    A Search for the Molecular Better Half! Vaishali Verma ... These assays comprise of a protein molecule, ... ciferase, beta-galactosidase, GFP, g3p of M13 filamentous ph- .... sensors of protein–protein interactions, Nature Biotechnology, Vol.20,.

  16. Linearization of the bradford protein assay.

    Science.gov (United States)

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  17. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a... intended for use in conjunction with other laboratory findings and clinical assessment of the patient to...

  18. Evaluation of environmental genotoxicity by comet assay in Columba livia.

    Science.gov (United States)

    González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I

    2016-01-01

    The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

  19. Detection and removal of spatial bias in multiwell assays.

    Science.gov (United States)

    Lachmann, Alexander; Giorgi, Federico M; Alvarez, Mariano J; Califano, Andrea

    2016-07-01

    Multiplex readout assays are now increasingly being performed using microfluidic automation in multiwell format. For instance, the Library of Integrated Network-based Cellular Signatures (LINCS) has produced gene expression measurements for tens of thousands of distinct cell perturbations using a 384-well plate format. This dataset is by far the largest 384-well gene expression measurement assay ever performed. We investigated the gene expression profiles of a million samples from the LINCS dataset and found that the vast majority (96%) of the tested plates were affected by a significant 2D spatial bias. Using a novel algorithm combining spatial autocorrelation detection and principal component analysis, we could remove most of the spatial bias from the LINCS dataset and show in parallel a dramatic improvement of similarity between biological replicates assayed in different plates. The proposed methodology is fully general and can be applied to any highly multiplexed assay performed in multiwell format. ac2248@columbia.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

    Johnson, Sarah Stewart; Hebsgaard, Martin B; Christensen, Torben R

    2007-01-01

    -term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  1. Microbiological and environmental issues in show caves.

    Science.gov (United States)

    Saiz-Jimenez, Cesareo

    2012-07-01

    Cultural tourism expanded in the last half of the twentieth century, and the interest of visitors has come to include caves containing archaeological remains. Some show caves attracted mass tourism, and economical interests prevailed over conservation, which led to a deterioration of the subterranean environment and the rock art. The presence and the role of microorganisms in caves is a topic that is often ignored in cave management. Knowledge of the colonisation patterns, the dispersion mechanisms, and the effect on human health and, when present, over rock art paintings of these microorganisms is of the utmost importance. In this review the most recent advances in the study of microorganisms in caves are presented, together with the environmental implications of the findings.

  2. Passive nondestructive assay of nuclear materials

    International Nuclear Information System (INIS)

    Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

    1991-03-01

    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

  3. Optical assay for biotechnology and clinical diagnosis.

    Science.gov (United States)

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

    2011-08-01

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

  4. Calibration method for a radwaste assay system

    International Nuclear Information System (INIS)

    Dulama, C.; Dobrin, R.; Toma, Al.; Paunoiu, C.

    2004-01-01

    A waste assay system entirely designed and manufactured in the Institute for Nuclear Research is used in radwaste treatment and conditioning stream to ensure compliance with national repository radiological requirements. Usually, waste assay systems are calibrated by using various experimental arrangements including calibration phantoms. The paper presents a comparative study concerning the efficiency calibration performed by shell source method and a semiempirical, computational method based on a Monte Carlo algorithm. (authors)

  5. Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

    Science.gov (United States)

    Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

    2017-06-28

    The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

  6. Evaluation of a Noncontact, Alternative Mosquito Repellent Assay System.

    Science.gov (United States)

    Tisgratog, Rungarun; Kongmee, Monthathip; Sanguanpong, Unchalee; Prabaripai, Atchariya; Bangs, Michael J; Chareonviriyaphap, Theeraphap

    2016-09-01

    A novel noncontact repellency assay system (NCRAS) was designed and evaluated as a possible alternative method for testing compounds that repel or inhibit mosquitoes from blood feeding. Deet and Aedes aegypti were used in a controlled laboratory setting. Using 2 study designs, a highly significant difference were seen between deet-treated and untreated skin placed behind the protective screens, indicating that deet was detected and was acting as a deterrence to mosquito landing and probing behavior. However, a 2nd study showed significant differences between protected (behind a metal screen barrier) and unprotected (exposed) deet-treated forearms, indicating the screen mesh might restrict the detection of deet and thus influences landing/biting response. These findings indicate the prototype NCRAS shows good promise but requires further evaluation and possible modification in design and testing protocol to achieve more desirable operational attributes in comparison with direct skin-contact repellency mosquito assays.

  7. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Roth, G.J.; Machuga, E.T.

    1982-01-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 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 [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]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 10 9 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

  8. Nondestructive assay methods for irradiated nuclear fuels

    International Nuclear Information System (INIS)

    Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

    1978-01-01

    This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed

  9. Thermoluminescence authenticity assay of Chinese pottery in Hong Kong

    International Nuclear Information System (INIS)

    Tso, W.M.

    1989-01-01

    The method used is similar to the dating of sherd samples with some modification to minimize the damage to the delicate pottery but still produce a reasonable accurate result. During the past year, the author has done 31 samples successfully. Most of them claimed to be 1100-4500 years of age. After testing, nineteen samples showed out to be compatible with the claimed age. The general procedures and the encountered problems for the assay are described

  10. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    Science.gov (United States)

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. NASA GIBS Use in Live Planetarium Shows

    Science.gov (United States)

    Emmart, C. B.

    2015-12-01

    The American Museum of Natural History's Hayden Planetarium was rebuilt in year 2000 as an immersive theater for scientific data visualization to show the universe in context to our planet. Specific astrophysical movie productions provide the main daily programming, but interactive control software, developed at AMNH allows immersive presentation within a data aggregation of astronomical catalogs called the Digital Universe 3D Atlas. Since 2006, WMS globe browsing capabilities have been built into a software development collaboration with Sweden's Linkoping University (LiU). The resulting Uniview software, now a product of the company SCISS, is operated by about fifty planetariums around that world with ability to network amongst the sites for global presentations. Public presentation of NASA GIBS has allowed authoritative narratives to be presented within the range of data available in context to other sources such as Science on a Sphere, NASA Earth Observatory and Google Earth KML resources. Specifically, the NOAA supported World Views Network conducted a series of presentations across the US that focused on local ecological issues that could then be expanded in the course of presentation to national and global scales of examination. NASA support of for GIBS resources in an easy access multi scale streaming format like WMS has tremendously enabled particularly facile presentations of global monitoring like never before. Global networking of theaters for distributed presentations broadens out the potential for impact of this medium. Archiving and refinement of these presentations has already begun to inform new types of documentary productions that examine pertinent, global interdependency topics.

  12. SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

    Science.gov (United States)

    Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.

    2016-01-01

    The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

  13. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

    Directory of Open Access Journals (Sweden)

    Chao Shan

    2017-03-01

    Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  14. Tungsten carbide-cobalt as a nanoparticulate reference positive control in in vitro genotoxicity assays.

    Science.gov (United States)

    Moche, Hélène; Chevalier, Dany; Barois, Nicolas; Lorge, Elisabeth; Claude, Nancy; Nesslany, Fabrice

    2014-01-01

    With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

  15. Geoscience is Important? Show Me Why

    Science.gov (United States)

    Boland, M. A.

    2017-12-01

    "The public" is not homogenous and no single message or form of messaging will connect the entire public with the geosciences. One approach to promoting trust in, and engagement with, the geosciences is to identify specific sectors of the public and then develop interactions and communication products that are immediately relevant to that sector's interests. If the content and delivery are appropriate, this approach empowers people to connect with the geosciences on their own terms and to understand the relevance of the geosciences to their own situation. Federal policy makers are a distinct and influential subgroup of the general public. In preparation for the 2016 presidential election, the American Geosciences Institute (AGI) in collaboration with its 51 member societies prepared Geoscience for America's Critical Needs: Invitation to a National Dialogue, a document that identified major geoscience policy issues that should be addressed in a national policy platform. Following the election, AGI worked with eight other geoscience societies to develop Geoscience Policy Recommendations for the New Administration and the 115th Congress, which outlines specific policy actions to address national issues. State and local decision makers are another important subgroup of the public. AGI has developed online content, factsheets, and case studies with different levels of technical complexity so people can explore societally-relevant geoscience topics at their level of technical proficiency. A related webinar series is attracting a growing worldwide audience from many employment sectors. Partnering with government agencies and other scientific and professional societies has increased the visibility and credibility of these information products with our target audience. Surveys and other feedback show that these products are raising awareness of the geosciences and helping to build reciprocal relationships between geoscientists and decision makers. The core message of all

  16. Bacteriophages show promise as antimicrobial agents.

    Science.gov (United States)

    Alisky, J; Iczkowski, K; Rapoport, A; Troitsky, N

    1998-01-01

    The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed

  17. Selection of non-destructive assay methods: Neutron counting or calorimetric assay?

    International Nuclear Information System (INIS)

    Cremers, T.L.; Wachter, J.R.

    1994-01-01

    The transition of DOE facilities from production to D ampersand D has lead to more measurements of product, waste, scrap, and other less attractive materials. Some of these materials are difficult to analyze by either neutron counting or calorimetric assay. To determine the most efficacious analysis method, variety of materials, impure salts and hydrofluorination residues have been assayed by both calorimetric assay and neutron counting. New data will be presented together with a review of published data. The precision and accuracy of these measurements are compared to chemistry values and are reported. The contribution of the gamma ray isotopic determination measurement to the overall error of the calorimetric assay or neutron assay is examined and discussed. Other factors affecting selection of the most appropriate non-destructive assay method are listed and considered

  18. Comparison of antibacterial activities of root-end filling materials by an agar diffusion assay and Alamar blue assay

    Directory of Open Access Journals (Sweden)

    Tsui-Hsien Huang

    2012-12-01

    Conclusion: We concluded that both the agar diffusion test and Alamar blue assay gave comparable findings of assessing the antimicrobial activity present in root-end filling materials. No antimicrobial activity was detected for mineral trioxide aggregate, calcium silicate cement, or amalgam after coming into contact with S. mutans, S. sanguinis and E. coli. IRM showed high antimicrobial activity against both S. sanguinis and E. coli.

  19. 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.

  20. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-07-01

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

  1. 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.

  2. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

    Directory of Open Access Journals (Sweden)

    Lyerly Herbert K

    2008-03-01

    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  3. Plasma catecholamine content using radioenzymatic assay

    International Nuclear Information System (INIS)

    Minami, Masaru; Togashi, Hiroko; Koike, Yuichi; Shimamura, Keiichi; Yamazaki, Noriko

    1980-01-01

    Catecholamine (CA) contents in blood plasma of spontaneously hypertensive rats (SHR) and human blood plasma were measured by radioenzymatic assay (REA) and trihydroxyindol (THI) fluorescent method using high performance liquid chromatography (HPLC), and both measurement methods were compared. The standard curve of REA showed a good linear relationship between total CA contents and separated CA contents. Though there was a danger of exposure to β-ray when REA was performed, this method was useful for measurement of CA contents in blood of small animals and small quantity of blood because CA content of only 50 μg of blood plasma could be measured by this method. Norepinephrine (NE) and epinephrine (E) contents in men with normal blood pressure measured by REA was 250 +- 61 pg/ml and 37 +- 22 pg/ml, respectively. NE and E contents in patients with mild hypertension were 460 +- 128 pg/ml and 50 +- 20 pg/ml, respectively. There was not a significant difference between NE and E contents in men with normal blood pressure and those in patients with mild hypertension. Total CA content in blood plasma of SHR killed by decapitation was 5,000 +- 1,131 pg/ml, which was 5 times NE and E contents in blood plasma obtained from femoral vein of anesthetized SHR (816 +- 215 pg/ml and 209 +- 44 pg/ml). Total CA content in the same sample was measured by REA and HPLC. As a result, total CA content measured by REA was higher than that measured by HPLC, but there was a good relationship between total CA content measured by REA and that measured by HPLC. NE content in men with normal blood pressure measured by HPLC was elevated significantly according to an increase in their age, but this tendency was not observed in patients with hypertension. (Tsunoda, M.)

  4. Limonene hydroperoxide analogues show specific patch test reactions.

    Science.gov (United States)

    Christensson, Johanna Bråred; Hellsén, Staffan; Börje, Anna; Karlberg, Ann-Therese

    2014-05-01

    The fragrance terpene R-limonene is a very weak sensitizer, but forms allergenic oxidation products upon contact with air. The primary oxidation products of oxidized limonene, the hydroperoxides, have an important impact on the sensitizing potency of the oxidation mixture. One analogue, limonene-1-hydroperoxide, was experimentally shown to be a significantly more potent sensitizer than limonene-2-hydroperoxide in the local lymph node assay with non-pooled lymph nodes. To investigate the pattern of reactivity among consecutive dermatitis patients to two structurally closely related limonene hydroperoxides, limonene-1-hydroperoxide and limonene-2-hydroperoxide. Limonene-1-hydroperoxide, limonene-2-hydroperoxide, at 0.5% in petrolatum, and oxidized limonene 3.0% pet. were tested in 763 consecutive dermatitis patients. Of the tested materials, limonene-1-hydroperoxide gave most reactions, with 2.4% of the patients showing positive patch test reactions. Limonene-2-hydroperoxide and oxidized R-limonene gave 1.7% and 1.2% positive patch test reactions, respectively. Concomitant positive patch test reactions to other fragrance markers in the baseline series were frequently noted. The results are in accordance with the experimental studies, as limonene-1-hydroperoxide gave more positive patch test reactions in the tested patients than limonene-2-hydroperoxide. Furthermore, the results support the specificity of the allergenic activity of the limonene hydroperoxide analogues and the importance of oxidized limonene as a cause of contact allergy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. 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.

  6. Immune chromatography: a quantitative radioimmunological assay

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  7. Evaluation of three gentamicin serum assay techniques

    International Nuclear Information System (INIS)

    Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

    1982-01-01

    This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  9. A new semiquantitative radiometric opsonin assay

    International Nuclear Information System (INIS)

    Yamamura, M.; Valdimarsson, H.

    1978-01-01

    A new semiquantitative radiometric opsonin assay is described. It was found that the opsonin activity generated by incubating brewer's yeast, Saccharomyces cerevisiae, in medium containing less than 5% human serum was exclusively complement dependent. In contrast, C.albicans was effectively opsonized in the absence of complement. Antibodies and the early classical complement pathway did not contribute to the opsonization of S.cerevisiae and neither did C5-9. The brewer's yeast assay can therefore be used for measuring selectively the opsonizing capacity of the alternative pathway. Sera from approximately 7% of apparently healthy adult controls consistently failed to generate significant opsonin activity while 8 out of 26 patients with suspected immune deficiency of unknown cause were defective in this assay. All opsonin deficient sera so far tested had haemolytically normal alternative pathway and Factor B activity. (author)

  10. Evaluation of a molybdenum assay canister

    International Nuclear Information System (INIS)

    Yoshizumi, T.T.; Keener, S.J.

    1988-01-01

    The performance characteristics of a commercial molybdenum assay canister were evaluated. The geometrical variation of the technetium-99m (/sup 99m/Tc) activity reading was studied as a function of the elution volume for the standard vials. It was found that the /sup 99m/Tc canister activity reading was ∼ 5% lower than that of the standard method. This is due to attenuation by the canister wall. However, the effect of the geometric variation on the clinical dose preparation was found to be insignificant. The molybdenum-99 ( 99 Mo) contamination level was compared by two methods: (1) the commercial canister and (2) the standard assay kit. The 99 Mo contamination measurements with the canister indicated consistently lower readings than those with the standard 99 Mo assay kit. The authors conclude that the canister may be used in the clinical settings. However, the user must be aware of the problems and the limitations associated with this canister

  11. Elements of nondestructive assay (NDA) technology

    International Nuclear Information System (INIS)

    Anon.

    1981-01-01

    This session provides an introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards. The purpose of the sessions is to enable participants to: (1) discuss the general principles and major applications of NDA; (2) describe situations in which NDA is particularly useful for nuclear safeguards purposes; (3) distinguish between various passive and active gamma-ray and neutron NDA methods; (4) describe several NDA instruments that measure gamma rays, and identify assay situations particularly suited to gamma-ray techniques; (5) describe several NDA instruments that measure neutrons, and identify assay situations particularly suited to neutron techniques; (6) discuss the role of calorimetry in the NDA of plutonium-bearing materials; and (7) compare the advantages and disadvantages of various NDA methods for different types of nuclear materials

  12. 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....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  13. Monitoring environmental exposures with semen assays

    International Nuclear Information System (INIS)

    Anon.

    1979-01-01

    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

  14. Have you stress tested your assay?

    Directory of Open Access Journals (Sweden)

    Zheng Cao

    2016-08-01

    Full Text Available Objectives: When a clinical assay is stressed with extraordinarily high volume of specimens over a short period of time, extra caution may be needed to avoid systematic errors and biases. Here we report our experience with a HgbA1c assay used for high volume wellness screening purpose, to illustrate the importance of stress testing during assay validation. Design and Methods: Over 15,000 whole blood specimens were tested for HgbA1c in a period of 2 months. HgbA1c was tested by an immunoturbidimetric method on a high through-put automation line. The HgbA1c population distribution in our study was compared to that from the NHANES database. Daily distributions of HgbA1c values ≥6%, means and medians were plotted. Correlation studies were performed between the high through-put immunoturbidimetric assay and a medium through-put HPLC method. Results: We observed a shift of HgbA1c distribution to the higher values compared to the NHANES. A bias of 15–20% was noted from further stress testing where large number of samples were batched and tested using the immunoturbidimetric assay. A 5–7% higher bias remained after implementing a cuvette washing program after each HgbA1c sample. We hypothesized this bias was caused by build-up of blood cell fragments in the cuvettes when continuous whole blood samples are run through the system. Our experience suggests stress testing needs to be incorporated early in the test validation process for high volume batched screening applications. This seemingly extra validation step may save significant troubleshooting and retesting efforts down the road. Keywords: Hemoglobin A1c, Immunoturbidimetric assay, HPLC, Quality assurance, Systematic bias, High volume, Automation

  15. Rapid colorimetric assay for gentamicin injection.

    Science.gov (United States)

    Tarbutton, P

    1987-01-01

    A rapid colorimetric method for determining gentamicin concentration in commercial preparations of gentamicin sulfate injection was developed. Methods currently available for measuring gentamicin concentration via its colored complex with cupric ions in alkaline solution were modified to reduce the time required for a single analysis. The alkaline copper tartrate (ACT) reagent solution was prepared such that each milliliter contained 100 mumol cupric sulfate, 210 mumol potassium sodium tartrate, and 1.25 mmol sodium hydroxide. The assay involves mixing 0.3 mL gentamicin sulfate injection 40 mg/mL (of gentamicin), 1.0 mL ACT reagent, and 0.7 mL water; the absorbance of the resulting solution at 560 nm was used to calculate the gentamicin concentration in the sample. For injections containing 10 mg/mL of gentamicin, the amount of the injection was increased to 0.5 mL and water decreased to 0.5 mL. The concentration of gentamicin in samples representing 11 lots of gentamicin sulfate injection 40 mg/mL and 8 lots of gentamicin sulfate injection 10 mg/mL was determined. The specificity, reproducibility, and accuracy of the assay were assessed. The colored complex was stable for at least two hours. Gentamicin concentration ranged from 93.7 to 108% and from 95 to 109% of the stated label value of the 40 mg/mL and the 10 mg/mL injections, respectively. No components of the preservative system present in the injections interfered with the assay. Since other aminoglycosides produced a colored complex, the assay is not specific for gentamicin. The assay was accurate and reproducible over the range of 4-20 mg of gentamicin. This rapid and accurate assay can be easily applied in the hospital pharmacy setting.

  16. Immunological measurements on the disappearance of creatine kinase MM from the circulation. [Immunoradiometric assay

    Energy Technology Data Exchange (ETDEWEB)

    Wevers, R A; van Landeghem, A A.J.; Mul-Steinbusch, M W.F.J.; Bijdendijk, J G; Weerts, P; Kloeg, P; Soons, J B.J. [Rijksuniversiteit Utrecht (Netherlands)

    1983-07-15

    Both a two-site immunoradiometric assay and a two-site enzyme-linked immunosorbent assay for creatine kinase MM are described. Linearity, reproducibility and cross-reactivity of the assays are satisfactory. Creatine kinase MM incubated in a pH-controlled serum matrix loses its activity, but has its antigenic determinants affected as well. Of all the techniques used, only the immunoradiometric assay is capable of measuring part of the inactivated enzyme. Measurements with this assay on the sera of patients after a myocardial infarction show identical results for enzyme activity and creatine kinase protein quantity. The in vitro disappearance rate of creatine kinase activity is slow compared with the in vivo half-life of the enzyme. These two observations lead to the conclusion that creatine kinase is removed from the circulation long before it is inactivated in the blood stream.

  17. Elements of nondestructive assay (NDA) technology

    International Nuclear Information System (INIS)

    Hatcher, C.R.; Smith, H.

    1984-01-01

    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

  18. Assay of low-level plutonium effluents

    International Nuclear Information System (INIS)

    Hsue, S.T.; Hsue, F.; Bowersox, D.F.

    1981-01-01

    In the plutonium recovery section at the Los Alamos National Laboratory, an effluent solution is generated that contains low plutonium concentration and relatively high americium concentration. Nondestructive assay of this solution is demonstrated by measuring the passive L x-rays following alpha decay. Preliminary results indicate that an average deviation of 30% between L x-ray and alpha counting can be achieved for plutonium concentrations above 10 mg/L and Am/Pu ratios of up to 3; for plutonium concentrations less than 10 mg/L, the average deviation is 40%. The sensitivity of the L x-ray assay is approx. 1 mg Pu/L

  19. A sensitive assay for Staphylococcus aureus nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Kohli, J K; Vakil, B V; Patil, M S; Pandey, V N; Pradhan, D S [Bhabha Atomic Reserach Centre, Bombay (India). Biochemistry Div.

    1989-10-01

    A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured ({sup 3}H) 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.

  20. Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

    Science.gov (United States)

    Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

    2016-12-01

    Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Automated two-site immunofluorescent assay for the measurement of serum chromogranin A.

    Science.gov (United States)

    Popovici, Théodora; Moreira, Baptiste; Schlageter, Marie-Hélène; Bories, Phuong-Nhi

    2014-01-01

    Chromogranin A (CgA) is the best-characterized biological marker common to neuroendocrine tumours and is therefore recommended for their diagnosis. The measurement of serum CgA is of great importance for reaching an early diagnosis and thus reducing the delay before treatment is instigated. The Kryptor CgA assay is the first fully automated assay available. The aim of this study was to evaluate its analytical performance. The imprecision and linearity of the Kryptor CgA assay were evaluated. This assay was compared with the Cis Bio CgA RIA assay in 78 serum samples. Its clinical utility was assessed in serum from 229 patients. The study performed on imprecision of Kryptor measurements showed intra- and inter-run CVs ≤ 5%. The study of linearity showed a satisfactory recovery rate for CgA concentrations up to 1200 μg/L. The Kryptor and RIA assays agreed well on the basis of the cut-off values provided by the two manufacturers. The Bland and Altman plot of the values obtained (range: 20-5560 μg/L) provided a mean difference of -10.1 μg/L (SD: 116). The clinical sensitivities of Kryptor CgA for diagnosis of pheochromocytoma and paraganglioma (n 20) and gastroenteropancreatic NETs (n 17) were respectively 100 and 94%. The Kryptor assay for CgA shows reliable analytical and clinical characteristics and allows a fast delivery of results. © 2013.

  2. Fundamental and clinical evaluation of ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen

    International Nuclear Information System (INIS)

    Koizumi, Mitsuru; Endo, Keigo; Nakajima, Kotoko

    1987-01-01

    A commercial ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen (SCC antigen) was fundamentally and clinically evaluated. Laboratory performance was satisfactory for intra-assay and inter-assay reproducibility, recovery, and dilution, with rapid and simple measurement techniques. Seropositivity for SCC antigen was significantly higher for squamous cell carcinoma of the liver and uterine cervix than the other histology types. In the case of cervical squamous cell carcinoma, it increased with progressing disease. Post-treatment serum levels of SCC antigen returned to negative. SCC antigen is considered to be a useful tumor marker for these diseases. There was a good correlation between the measurement values obtained from the present and conventional (SCC RIAKIT) assays. The present assay remarkably decreased false-positive cases of pulmonary benign diseases. The results showed a ''SCC RIABEAD'' to be a favorable kit for immunoradiometric assay of SSC antigen, as compared with conventional assay kit. (Namekawa, K.)

  3. Liquor oligoclonal bands assay: interpretation, correlation with other laboratory assays and importance for diagnostics of neurological disorders

    OpenAIRE

    Bagdonas, Dovydas

    2017-01-01

    Aim: to analyse the possible relationship between liquor IgG oligoclonal bands assay and other laboratory assays in neurological patients. Objectives: to determine the frequency of oligoclonal bands in neurological patients; to compare the results between serum and liquor laboratory assays in dependence of oligoclonal bands assay results; to evaluate the relationships between oligoclonal bands assay and serological-immunological assays for infectious diseases, gender, age and neurological ...

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  5. Evaluation of the reliability of maize reference assays for GMO quantification.

    Science.gov (United States)

    Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel

    2010-03-01

    A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb

  6. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    Science.gov (United States)

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  7. Performance of hepatitis B assays on the Bayer ADVIA Centaur Immunoassay System.

    Science.gov (United States)

    van Helden, Josef; Denoyel, Gérard; Karwowska, Sylwia; Reamer, Randy; Schmalz, John; Wright, Ted; Preisel-Simmons, Barbara

    2004-01-01

    Bayer HealthCare LLC, Diagnostics Division, has developed several new assays on the ADVIA Centaur immunoassay system for the detection of markers of hepatitis B virus infection in human serum and plasma. This panel includes assays for: hepatitis B surface antigen (HBsAg), a confirmatory test method for HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). These assays employ magnetic particle separation technology with direct chemiluminescence for optimal assay performance. All of the assays are fully automated, require sample volumes ranging from 15 microl to 100 microl (with the exception of the ADVIA Centaur HBsAg Confirmatory Assay, which requires 2 x 100 microl), and have throughputs of up to 240 tests per hour. The five ADVIA Centaur HBV assays were tested in extensive performance evaluations conducted at two sites in Europe. The performance evaluations, which included samples from HBV-infected individuals, blood donors, hospitalized/clinical patients, and HBV vaccinees (for Anti-HBs evaluation), generated performance data in support of obtaining the Communautés Européennes (CE) mark for European market distribution. The HBV performance evaluations resulted in an overall diagnostic specificity > 99%, i.e. 99.94% for the ADVIA Centaur HBsAg Assay, 100% for the ADVIA Centaur Anti-HBs Assay, 100% for the ADVIA Centaur HBc IgM Assay and 99.94% for the ADVIA Centaur HBc Total Assay. All of the ADVIA Centaur assays showed a very good diagnostic sensitivity on these populations with 100% for the ADVIA Centaur HBsAg Assay, 99.0% for the ADVIA Centaur Anti-HBs Assay, 98.53% for the ADVIA Centaur HBc IgM Assay and 100% for the ADVIA Centaur HBc Total Assay. The ADVIA Centaur HBsAg Confirmatory Test confirmed 100% of the positive HBsAg samples. Testing of interfering substances and potential cross-reacting samples for all ADVIA

  8. High throughput comet assay to study genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Naouale El Yamani

    2015-06-01

    per slide. Subsequent incubation with FPG revealed damage not seen with the basic assay for strand breaks (without FPG (Harris et al., 2015. Statistical evaluation showed that oleic acid coated Fe3O4 and TiO2 NMs are genotoxic, in the experimental conditions used. No differences were seen between cell lines representing a range of different tissues – demonstrating the general usefulness of in vitro models and the ability of cells to classify NMs as genotoxic and non-genotoxic (Cowie et al., 2015. We are currently studying the effects of 20 NMs in the NANoREG project using A549, BEAS B2 and TK6 cells - again demonstrating the usefulness of the HTP comet assay for nanogenotoxicity testing.

  9. 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...

  10. Comet assay. Pt.1. Theory and practice

    International Nuclear Information System (INIS)

    Kruszewski, M.; Wojewodzka, M.; Iwanenko, T.

    1996-01-01

    Comet assay is a new method for measuring DNA breakage in a single cell. The main applications of the method are estimation of DNA single and double strand breaks, oxidative damage, pyrimidine dimers and (6-4)photoproducts, DNA-DNA and DNA-protein crosslinks. The method is used for studying DNA damage and its repair. (author).19 refs, 9 figs

  11. 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…

  12. The use of calorimetry for plutonium assay

    International Nuclear Information System (INIS)

    Mason, J.A.

    1982-12-01

    Calorimetry is a technique for measuring the thermal power of heat-producing substances. The technique may be applied to the measurement of plutonium-bearing materials which evolve heat as a result of alpha and beta decay. A calorimetric measurement of the thermal power of a plutonium sample, combined with a knowledge or measurement of the plutonium isotopic mass ratios of the sample provides a convenient and accurate, non-destructive measure of the total plutonium mass of the sample. The present report provides a description, and an assessment of the calorimetry technique applied to the assay of plutonium-bearing materials. Types and characteristics of plutonium calorimeters are considered, as well as calibration and operating procedures. The instrumentation used with plutonium calorimeters is described and the use of computer control for calorimeter automation is discussed. A critical review and assessment of plutonium calorimetry literature since 1970 is presented. Both fuel element and plutonium-bearing material calorimeters are considered. The different types of plutonium calorimeters are evaluated and their relative merits are discussed. A combined calorimeter and gamma-ray measurement assay system is considered. The design principles of plutonium assay calorimeters are considered. An automatic, computer-based calorimeter control system is proposed in conjunction with a general plutonium assay calorimeter design. (author)

  13. 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.

  14. 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) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  15. 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 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250 Erythropoietin...

  16. 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) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  17. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    Rettenmaier, R.

    1979-01-01

    A radioligand assay for biotin in liver tissue is described. 3 H-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.)

  18. Relationships between ytterbium precipitation assay, colorimetric ...

    African Journals Online (AJOL)

    digestion and metabolism of protein (Komolong et al., 2001). ... room temperature (25 °C) pending chemical analyses and in vitro ... assayed without sodium sulphite but with a heat-stable α-amylase due to the high ... of starch in the tree fruits.

  19. Nondestructive assay of boxed radioactive waste

    International Nuclear Information System (INIS)

    Gilles, W.P.; Roberts, R.J.; Jasen, W.G.

    1992-12-01

    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

  20. Production and assay of forskolin antibodies

    International Nuclear Information System (INIS)

    Ho, L.T.; Ho, R.J.

    1986-01-01

    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 3 H-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 3 H-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 IC 50 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

  1. A multiparametric assay for quantitative nerve regeneration evaluation.

    Science.gov (United States)

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

    2005-08-01

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

  2. Development of a lion-specific interferon-gamma assay.

    Science.gov (United States)

    Maas, M; van Kooten, P J S; Schreuder, J; Morar, D; Tijhaar, E; Michel, A L; Rutten, V P M G

    2012-10-15

    The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

    Science.gov (United States)

    Rizzo, Larissa Y; Golombek, Susanne K; Mertens, Marianne E; Pan, Yu; Laaf, Dominic; Broda, Janine; Jayapaul, Jabadurai; Möckel, Diana; Subr, Vladimir; Hennink, Wim E; Storm, Gert; Simon, Ulrich; Jahnen-Dechent, Willi; Kiessling, Fabian; Lammers, Twan

    2013-06-10

    Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.

  4. Comparison of Six Automated Treponema-Specific Antibody Assays.

    Science.gov (United States)

    Park, Borae G; Yoon, Jihoon G; Rim, John Hoon; Lee, Anna; Kim, Hyon-Suk

    2016-01-01

    Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Show Horse Welfare: Horse Show Competitors' Understanding, Awareness, and Perceptions of Equine Welfare.

    Science.gov (United States)

    Voigt, Melissa A; Hiney, Kristina; Richardson, Jennifer C; Waite, Karen; Borron, Abigail; Brady, Colleen M

    2016-01-01

    The purpose of this study was to gain a better understanding of stock-type horse show competitors' understanding of welfare and level of concern for stock-type show horses' welfare. Data were collected through an online questionnaire that included questions relating to (a) interest and general understanding of horse welfare, (b) welfare concerns of the horse show industry and specifically the stock-type horse show industry, (c) decision-making influences, and (d) level of empathic characteristics. The majority of respondents indicated they agree or strongly agree that physical metrics should be a factor when assessing horse welfare, while fewer agreed that behavioral and mental metrics should be a factor. Respondent empathy levels were moderate to high and were positively correlated with the belief that mental and behavioral metrics should be a factor in assessing horse welfare. Respondents indicated the inhumane practices that most often occur at stock-type shows include excessive jerking on reins, excessive spurring, and induced excessive unnatural movement. Additionally, respondents indicated association rules, hired trainers, and hired riding instructors are the most influential regarding the decisions they make related to their horses' care and treatment.

  6. Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    International Nuclear Information System (INIS)

    Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.; Macpherson, J.S.

    1982-01-01

    Two direct radioimmunoassays for progesterone in 50 μL of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11α-hemisuccinyl conjugate and the radioligand 125 I-labeled progesterone 11α-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum

  7. Automation of the anthrone assay for carbohydrate concentration determinations.

    Science.gov (United States)

    Turula, Vincent E; Gore, Thomas; Singh, Suddham; Arumugham, Rasappa G

    2010-03-01

    Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

  8. Multicenter Evaluation of Cystatin C Measurement after Assay Standardization.

    Science.gov (United States)

    Bargnoux, Anne-Sophie; Piéroni, Laurence; Cristol, Jean-Paul; Kuster, Nils; Delanaye, Pierre; Carlier, Marie-Christine; Fellahi, Soraya; Boutten, Anne; Lombard, Christine; González-Antuña, Ana; Delatour, Vincent; Cavalier, Etienne

    2017-04-01

    Since 2010, a certified reference material ERM-DA471/IFCC has been available for cystatin C (CysC). This study aimed to assess the sources of uncertainty in results for clinical samples measured using standardized assays. This evaluation was performed in 2015 and involved 7 clinical laboratories located in France and Belgium. CysC was measured in a panel of 4 serum pools using 8 automated assays and a candidate isotope dilution mass spectrometry reference measurement procedure. Sources of uncertainty (imprecision and bias) were evaluated to calculate the relative expanded combined uncertainty for each CysC assay. Uncertainty was judged against the performance specifications derived from the biological variation model. Only Siemens reagents on the Siemens systems and, to a lesser extent, DiaSys reagents on the Cobas system, provided results that met the minimum performance criterion calculated according to the intraindividual and interindividual biological variations. Although the imprecision was acceptable for almost all assays, an increase in the bias with concentration was observed for Gentian reagents, and unacceptably high biases were observed for Abbott and Roche reagents on their own systems. This comprehensive picture of the market situation since the release of ERM-DA471/IFCC shows that bias remains the major component of the combined uncertainty because of possible problems associated with the implementation of traceability. Although some manufacturers have clearly improved their calibration protocols relative to ERM-DA471, most of them failed to meet the criteria for acceptable CysC measurements. © 2016 American Association for Clinical Chemistry.

  9. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  10. Short communication. Microculture syncytia assay for bovine leukemia virus

    Energy Technology Data Exchange (ETDEWEB)

    Paul, P.S.; Castro, A.E.; Pomeroy, K.A.; Johnson, D.W.; Muscoplat, C.C.

    1978-01-01

    A microculture syncytia assay for the detection of bovine leukemia virus (BLV) has been described and compared with the conventional macroculture assay. The microculture assay required fewer indicator cells, was as sensitive as the macroculture assay and provided a reproducible test for the detection and titration of BLV.

  11. Nondestructive assay of subassemblies of various spent or fresh fuels by active neutron interrogation

    International Nuclear Information System (INIS)

    Ragan, G.L.; Ricker, C.W.; Chiles, M.M.; Ingersoll, D.T.; Slaughter, G.G.

    1979-01-01

    Recent studies show that subassemblies containing various spent fuels could be assayed rapidly and accurately by a nondestructive assay system using active neutron interrogation and prompt-neutron detection. Subassembly penetration is achieved by 24-keV (Sb--Be) interrogation neutrons; the spent-fuel neutron background is overridden by using strong interrogating sources and prompt-neutron signals, and background gammas are absorbed by lead. Experiments have demonstrated the potential for assaying with better than 5% accuracy, three spent plutonium-fueled subassemblies per hour. Calculations, validated by experiments, predict even better performance for fresh or uranium-fueled subassemblies; several performance estimates are given

  12. A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

    Science.gov (United States)

    Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

    2017-01-01

    This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

  13. The influence of the number of cells scored on the sensitivity in the comet assay

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar; Soussaline, Françoise; Sallette, Jerome

    2012-01-01

    The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different che...... of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables “the number of cells scored” and “concentration” on the total variation in the coefficients of variance dataset was statistically significant (p...

  14. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    Directory of Open Access Journals (Sweden)

    Dabisch-Ruthe Mareike

    2012-07-01

    Full Text Available Abstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP, were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml and RSV (103 copies/ml. The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS samples (n = 100 of mechanically ventilated patients in winter (n = 50 and summer (n = 50. In winter, respiratory viruses were detected in 32 TS samples (64% by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32% and PIV-2 (20%. Multiple infections were detected

  15. Best in show but not best shape: a photographic assessment of show dog body condition.

    Science.gov (United States)

    Such, Z R; German, A J

    2015-08-01

    Previous studies suggest that owners often wrongly perceive overweight dogs to be in normal condition. The body shape of dogs attending shows might influence owners' perceptions, with online images of overweight show winners having a negative effect. This was an observational in silico study of canine body condition. 14 obese-prone breeds and 14 matched non-obese-probe breeds were first selected, and one operator then used an online search engine to identify 40 images, per breed, of dogs that had appeared at a major national UK show (Crufts). After images were anonymised and coded, a second observer subjectively assessed body condition, in a single sitting, using a previously validated method. Of 1120 photographs initially identified, 960 were suitable for assessing body condition, with all unsuitable images being from longhaired breeds. None of the dogs (0 per cent) were underweight, 708 (74 per cent) were in ideal condition and 252 (26 per cent) were overweight. Pugs, basset hounds and Labrador retrievers were most likely to be overweight, while standard poodles, Rhodesian ridgebacks, Hungarian vizslas and Dobermanns were least likely to be overweight. Given the proportion of show dogs from some breeds that are overweight, breed standards should be redefined to be consistent with a dog in optimal body condition. British Veterinary Association.

  16. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    Science.gov (United States)

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. Copyright © 2016 Kremastinou et al.

  17. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection

    Science.gov (United States)

    Kremastinou, J.; Polymerou, V.; Lavranos, D.; Aranda Arrufat, A.; Harwood, J.; Martínez Lorenzo, M. J.; Ng, K. P.; Queiros, L.; Vereb, I.

    2016-01-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum. The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  18. Detection of thyroid stimulating hormone receptor antibodies (TRAb) by radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA)

    International Nuclear Information System (INIS)

    Dumrongpisutikul, S.; Tuchinda, S.

    1990-01-01

    Thyroid stimulating hormone receptor antibodies (TRAb) were determined in 100 patients using radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of RRA and ELISA were found to be 70.6% and 88.2% respectively (n=51). The specificity of both assays were 100% (n=16). With RRA as the standard test the sensitivity and specificity of ELISA were 75.8% and 86.8%. In the untreated hyperthyroid the RRA result which expressed as % specific 125 I-TSH inhibition was 33.6% (n=51), decline to 26.9% in the treated hyperthyroid (n=33) and 14.1% in the euthyroid (n=16). The mean 0.D 492nm of TRAb-ELISA were 0.861 in untreated hyperthyroid, 0.437 in treated hyperthyroid and 0.135 in euthyroid Phi coefficient analysis show that the RRA was 60.4% correlated to hyperthyroidism where as TRAb-ELISA was 80.1%

  19. Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH) Assay

    OpenAIRE

    Daniel Lerda; Marta Cabrera; Jorge Flores; Luis Gutierrez; Armando Chierichetti; Martin Revol; Hernan Garcia Onto

    2013-01-01

    Objetive: The overall purpose of the study was to demonstrate applicability of the Dako dual-color chromogenic in situ hybridization (CISH) assay (DAKO Denmark, Glostrup) with respect to fluorescence in situ hybridization (FISH) probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals. The dual –color CISH assay was p...

  20. Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

    Science.gov (United States)

    Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

    2017-10-15

    Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Evaluation of different in vitro assays of inherent sensitivity as predictors of radiotherapy response

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Chicago Univ., IL; Beckett, M.A.; Mustafi, R.; Weichselbaum, R.R.; Vaughan, A.T.M.

    1991-01-01

    The inherent sensitivity of cells within a tumor plays an important role in the response of the tumor to radiotherapy. Clonogenic assays show that cells established from in-field radiotherapy failures are significantly more resistant to radiation than cell lines established from pre-treatment samples. Clonogenic assays fail to predict tumor response to radiotherapy, however. The failure might be due to the small sample size in this study, or the complicating factors of staging, surgery, and chemotherapy, and/or in vivo selection by radiotherapy for resistant tumor cells. In vitro selection for resistant cell lines does not appear to be a complicating factor. Nonclonogenic assays such as those that measure DNA strand break rejoining rates (filter elution, pulse-field gel electrophoresis) or chromosome structure (flow cytometric analysis) show promise as alternative rapid assays of radiation sensitivity and possibly tumor response. 16 refs., 2 figs

  2. C14 Assays and Autoradiographic Studies on the Rooster Comb

    Science.gov (United States)

    Balazs, Endre A.; Szirmai, John A.; Bergendahl, Gudrun

    1959-01-01

    The distribution of C14 was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C14 in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C14 in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C14, glucose-1-C14, and glucuronolactone-U-C14, was administered. The highest values were obtained after the administration of glucose-6-C14; glucuronolactone-U-C14 gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C14 assays and autoradiograms indicate that C14 is also present in other parts of the comb. As seen in autoradiography, the concentration of C14 was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies, suggest that the high specific activity of carbon in the mucoid layer is due mainly to the presence of C14-labelled hyaluronic acid. Autoradiograms and PAS staining suggest that a significant amount of C14 is also incorporated into the glycoproteins associated with the collagen fibers. PMID:13654453

  3. Two hybrids based on Keggin polyoxometalates and dinuclear ...

    Indian Academy of Sciences (India)

    YAN HOU

    2017-10-13

    Oct 13, 2017 ... obtained in identical hydrothermal conditions and further characterized by elemental analyses, ... oxalate-bipyridine cationic complexes and copper(II)- ... ethylenediamine and ox = oxalic acid anion) have been obtained.

  4. Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

    NARCIS (Netherlands)

    Irmak, Hasan; Buzgan, Turan; Evirgen, Omer; Akdeniz, Hayrettin; Demiroz, A. Pekcan; Abdoel, Theresia H.; Smits, Henk L.

    2004-01-01

    The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in

  5. Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

    DEFF Research Database (Denmark)

    Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine

    2015-01-01

    shown to inhibit the aromatase enzyme in both types of aromatase assays. The IC50 values ranged from 3 to 600μM. All five SSRIs, were further investigated in the H295R cell line. All compounds altered the steroid secretion from the cells, the lowest observed effect levels were 0.9μM and 3.1μ....... In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were......M for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine...

  6. The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

    Science.gov (United States)

    Bosserman, Linda; Prendergast, Franklyn; Herbst, Roy; Fleisher, Martin; Salom, Emery; Strickland, Steven; Raptis, Anastasios; Hallquist, Allan; Perree, Mathieu; Rajurkar, Swapnil; Karimi, Misagh; Rogers, Karl; Davidson, Dirk; Willis, Carl; Penalver, Manuel; Homesley, Howard; Burrell, Matthew; Garrett, Audrey; Rutledge, James; Chernick, Michael; Presant, Cary A

    2012-08-15

    A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development. ©2012 AACR.

  7. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    Directory of Open Access Journals (Sweden)

    Bruynseels Peggy

    2009-07-01

    Full Text Available Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

  8. Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator

    Science.gov (United States)

    Chen, Chia-Hung; Sarkar, Aniruddh; Song, Yong-Ak; Miller, Miles A.; Kim, Sung Jae; Griffith, Linda G.; Lauffenburger, Douglas A.; Han, Jongyoon

    2011-01-01

    We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultra low levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (∼10-fold) reduced the time required to complete the assay and the sample volume used. PMID:21671557

  9. Storage and Assay of Tritium in STAR

    International Nuclear Information System (INIS)

    Longhurst, Glen R.; Anderl, Robert A.; Pawelko, Robert J.; Stoots, Carl J.

    2005-01-01

    The Safety and Tritium Applied Research (STAR) facility at the Idaho National Engineering and Environmental Laboratory (INEEL) is currently being commissioned to investigate tritium-related safety questions for fusion and other technologies. The tritium inventory for the STAR facility will be maintained below 1.5 g to avoid the need for STAR to be classified as a Category 3 nuclear facility. A key capability in successful operation of the STAR facility is the ability to receive, inventory, and dispense tritium to the various experiments underway there. The system central to that function is the Tritium Storage and Assay System (SAS).The SAS has four major functions: (1) receiving and holding tritium, (2) assaying, (3) dispensing, and (4) purifying hydrogen isotopes from non-hydrogen species.This paper describes the design and operation of the STAR SAS and the procedures used for tritium accountancy in the STAR facility

  10. Radioligand purification prior to routine receptor assays

    International Nuclear Information System (INIS)

    Le Goff, J.-M.; Berthois, Y.; Martin, P.-M.

    1988-01-01

    The need to repurify the commercially available radioligands [ 3 H]estradiol and [ 3 H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [ 3 H]estradiol compared to [ 3 H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [ 3 H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a concomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. The authors therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [ 3 H]estradiol by solvent differential partition which requires no high-cost investment in apparatus. (author)

  11. Assay of 25-OH vitamin D3

    International Nuclear Information System (INIS)

    Nayer, P. de; Thalasso, M.; Beckers, C.

    1977-01-01

    A simplified version of the competitive protein binding assay for 25-OH vit D3 derived from the method of Belsey et al. is presented. The procedure does not include a chromatography step, and is performed on an alcoolic extract of 0.1 ml plasma or serum. Normal rat serum (1:20,000) 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 50% at 0.15 ng/tube, allowing for the measurement of 25-OH vit D3 concentrations between 2 ng/ml and 200 ng/ml. Mean values in a normal group was 23.1 +- 6.5 ng/ml (range 16-37 ng/ml, n = 11). (orig.) [de

  12. Developments in plutonium waste assay at AWE

    International Nuclear Information System (INIS)

    Miller, T J

    2009-01-01

    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.

  13. Neutron Assay System for Confinement Vessel Disposition

    International Nuclear Information System (INIS)

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Valdez, Jose I.; Vigil, Georgiana M.

    2012-01-01

    Los Alamos National Laboratory has a number of spherical confinement vessels (CVs) remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the CVs. The Confinement Vessel Assay System (CVAS) was developed to measure the amount of special nuclear material (SNM) in CVs before and after cleanout. Prior to cleanout, the system will be used to perform a verification measurement of each vessel. After cleanout, the system will be used to perform safeguards-quality assays of (le)100-g 239 Pu equivalent in a vessel for safeguards termination. The CVAS has been tested and calibrated in preparation for verification and safeguards measurements.

  14. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

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

  15. Expert system technology for nondestructive waste assay

    International Nuclear Information System (INIS)

    Becker, G.K.; Determan, J.C.

    1998-01-01

    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

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

    Science.gov (United States)

    Schneider, K; Akkan, Z

    2004-06-01

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

  17. LDLCHOLESTEROLEXAMINATION (LDL-C USINGHOMOGENEOUS ASSAY

    Directory of Open Access Journals (Sweden)

    Made DwiAmbara Putra

    2013-07-01

    Full Text Available Homogeneous method describe as a method that does not require separation of free and bound label. This method has the ability tofully automate the determination of LDL-C directly small sample volume sand short examination time. In addition this method use automated pipette and control of time and temperature more accurate. There are 5 methods i.e. Solubilization homogeneous LDL-C assay (SOL from KyowaMedex, Surfactant LDL-C assay (SUR from Daiichi Pure Chemicals, Protecting LDL-assay reagent (PRO from Wako Chemicals, LDL-C assaycatalase (CAT Denka Seiken and Calixarene of LDL-C assay (CAL from International Reagents Corporation. All method is to use a variety of detergents and other chemicals that cause blocking or dissolution of specific lipoprotein classes to achieve specificity for LDL. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  18. Assaying the reporter gene chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

    Crabb, D.W.; Minth, C.D.; Dixon, J.E.

    1989-01-01

    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

  19. The electrophoretic mobility shift assay (EMSA)

    OpenAIRE

    sprotocols

    2015-01-01

    The electrophoretic mobility shift assay (EMSA), also known as “gel shift assay”, is used to examine the binding parameters and relative affinities of protein and DNA interactions. We produced recombinant CCA1 protein and tested its binding affinity for the promoter fragments that contain CBS (AAAAATCT) or evening element (EE, AAAATATCT) (1) using a modified procedure adopted from published protocols (2,3).

  20. Nondestructive assay methodologies in nuclear forensics analysis

    International Nuclear Information System (INIS)

    Tomar, B.S.

    2016-01-01

    In the present chapter, the nondestructive assay (NDA) methodologies used for analysis of nuclear materials as a part of nuclear forensic investigation have been described. These NDA methodologies are based on (i) measurement of passive gamma and neutrons emitted by the radioisotopes present in the nuclear materials, (ii) measurement of gamma rays and neutrons emitted after the active interrogation of the nuclear materials with a source of X-rays, gamma rays or neutrons

  1. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    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.

  2. Quality control of estrogen receptor assays.

    Science.gov (United States)

    Godolphin, W; Jacobson, B

    1980-01-01

    Four types of material have been used for the quality control of routine assays of estrogen receptors in human breast tumors. Pieces of hormone-dependent Nb rat mammary tumors gave a precision about 40%. Rat uteri and rat tumors pulverized at liquid nitrogen temperature and stored as powder yielded precision about 30%. Powdered and lyophilised human tumors appear the best with precision as good as 17%.

  3. 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.

  4. Chromosome aberration assays in barley (Hordeum vulgare)

    Energy Technology Data Exchange (ETDEWEB)

    Constantin, M J [Univ. of Tennessee, Knoxville; Nilan, R A

    1982-01-01

    Barley is an exceellent organism for studies of induced chromosome aberrations because of its few (2n = 2x = 14) relatively large chromosomes. Root-tip and shoot-tip cells have been used extensively for the study of ionizing radiation-induced chromosome aberrations. The general procedures are well known, the technology is simple and easy to learn, and the assays are relatively quick and inexpensive. Both root tips and shoot tips can be used for the study of chemical mutagens as well as ionizing radiations. Pollen mother cells are well suited for studying the effects of mutagens on meiotic chromosomes. The literature review for the Gene-Tox Program reported on 61 chemicals tested for their effects on barley chromosomes. Of these, 90% were reported to be either positive or positive dose-related, while 7% were negative and 3% were questionable. Barley assays based on chromosomal aberrations are useful to detect the clastogenic potency of chemicals under laboratory conditions. Indications are that the data from barley can be used to corroborate data obtained from other organisms. Among the classes of chemicals assayed were: alcohols and phenols; alkaloids; epoxides; alkyl sulfates; amides and sulfonamides; aromatic amines; aryl halides; aziridines; alkenes; carbamates; hydroazides; nitroaromatics; nitrosamides; nitrosources; phenothiazines; and polycyclic aromatic hydrocarbons.

  5. Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

    International Nuclear Information System (INIS)

    Schulster, D.

    1977-01-01

    Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

  6. A European multicenter study on the analytical performance of the VERIS HBV assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Maria Angeles; Sauné, Karine; O Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    Hepatitis B viral load monitoring is an essential part of managing patients with chronic Hepatits B infection. Beckman Coulter has developed the VERIS HBV Assay for use on the fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the analytical performance of the VERIS HBV Assay at multiple European virology laboratories. Precision, analytical sensitivity, negative sample performance, linearity and performance with major HBV genotypes/subtypes for the VERIS HBV Assay was evaluated. Precision showed an SD of 0.15 log 10 IU/mL or less for each level tested. Analytical sensitivity determined by probit analysis was between 6.8-8.0 IU/mL. Clinical specificity on 90 unique patient samples was 100.0%. Performance with 754 negative samples demonstrated 100.0% not detected results, and a carryover study showed no cross contamination. Linearity using clinical samples was shown from 1.23-8.23 log 10 IU/mL and the assay detected and showed linearity with major HBV genotypes/subtypes. The VERIS HBV Assay demonstrated comparable analytical performance to other currently marketed assays for HBV DNA monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer.

    Science.gov (United States)

    To, Kenneth K W; Poon, Daniel C; Wei, Yuming; Wang, Fang; Lin, Ge; Fu, Li-Wu

    2016-06-01

    We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

  8. Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer

    Directory of Open Access Journals (Sweden)

    Kenneth K.W. To

    2016-06-01

    Full Text Available We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1], can sensitize multidrug resistant (MDR colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]. This data article describes the possible circumvention of resistance to specifically platinum (Pt-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

  9. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

    Directory of Open Access Journals (Sweden)

    Vijee Mohan

    Full Text Available Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS, carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1 involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

  10. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    Science.gov (United States)

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

    Science.gov (United States)

    Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

    2015-01-01

    Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

  12. Amelioration of oxidative stress by anthraquinones in various in vitro assays

    Directory of Open Access Journals (Sweden)

    Manish Kumar

    2012-10-01

    Full Text Available Objective: The use of natural phytoconstituents for food and as nutritional supplements is an easiest way to be healthier. Anthraquinone pigments have been traditionally used for various purposes viz. food colorants, textile staining, color paints and medicines. Rubia cordifolia L. is a perennial, herbaceous climbing plant belonging to family Rubiaceae. This plant contain substantial amounts of anthraquinones, especially in the roots. The present study deals with the bioactivity evaluation of phytoconstituents viz. alizarin and purpurin from Rubia cordifolia. Methods: The DNA protective and antioxidant potential of alizarin and purpurin was evaluated using different in vitro assays viz. DNA protection assay, ABTS assay, DPPH assay, Ferric ion reduction potential and Phosphomolybdenum assay. Results: Alizarin and purpurin exhibited good free radical scavenging activity in various assays. In DNA protection assay, alizarin showed more DNA protection against hydroxyl radicals generated by Fenton ’s reagent in comparison to purpurin. Conclusions: Being potent antioxidants, these natural coloring compounds can be boon to the food industry as nutraceuticals. Further, these phytochemicals can be explored for their anticancer activity and may serve as potent cancer chemopreventive molecules.

  13. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  14. Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.

    Science.gov (United States)

    Ali, Yousuf O; Escala, Wilfredo; Ruan, Kai; Zhai, R Grace

    2011-03-11

    Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination. Here we use behavioral assays, including the negative geotaxis assay and the aversive phototaxic suppression assay (APS assay), to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

  15. Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

    Science.gov (United States)

    Schuller, Marion; Höfner, Georg; Wanner, Klaus T

    2017-10-09

    MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kalus, Ulrich; Lombardi, Alessandra; Marcos, Maria Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel W

    2017-07-01

    The analytical performance of the Veris HIV-1 assay for use on the new, fully automated Beckman Coulter DxN Veris molecular diagnostics system was evaluated at 10 European virology laboratories. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log 10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log 10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log 10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.). Copyright © 2017 American Society for Microbiology.

  17. A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-10-01

    Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS ® TaqMan ® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT ® HBV Assay (Versant), and 74 specimens tested with Veris and artus ® HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log 10 IU/mL between Veris and Cobas, -0.46 log 10 IU/mL between Veris and RealTime, -0.36 log 10 IU/mL between Veris and Versant, and -0.12 log 10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation.

    Science.gov (United States)

    Rahman, Md Mahfujur; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Mustafa, Shuhaimi; Hashim, Uda; Hanapi, Ummi Kalthum

    2014-08-01

    A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

    Science.gov (United States)

    Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

    2014-08-05

    Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

  20. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  1. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1984-01-01

    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

  2. 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.

  3. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  4. International network for comparison of HIV neutralization assays: the NeutNet report II.

    Directory of Open Access Journals (Sweden)

    Leo Heyndrickx

    Full Text Available BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s can provide measures of protective immunity. An international collaboration (NeutNet involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs and soluble CD4 (Phase I study. METHODS: In the present study (Phase II, polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs (Virus Infectivity Assays, VIA, or Env (gp160-pseudotyped viruses (pseudoviruses, PSV produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014. Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus

  5. International network for comparison of HIV neutralization assays: the NeutNet report II.

    Science.gov (United States)

    Heyndrickx, Leo; Heath, Alan; Sheik-Khalil, Enas; Alcami, Jose; Bongertz, Vera; Jansson, Marianne; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Ramaswamy, Meghna; Sattentau, Quentin; Tolazzi, Monica; Schuitemaker, Hanneke; Willems, Betty; Wrin, Terri; Fenyö, Eva Maria; Scarlatti, Gabriella

    2012-01-01

    Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend

  6. A high-throughput assay of NK cell activity in whole blood and its clinical application

    International Nuclear Information System (INIS)

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-01-01

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as 51 Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer

  7. A laboratory-scale pretreatment and hydrolysis assay for determination of reactivity in cellulosic biomass feedstocks.

    Science.gov (United States)

    Wolfrum, Edward J; Ness, Ryan M; Nagle, Nicholas J; Peterson, Darren J; Scarlata, Christopher J

    2013-11-14

    The rapid determination of the release of structural sugars from biomass feedstocks is an important enabling technology for the development of cellulosic biofuels. An assay that is used to determine sugar release for large numbers of samples must be robust, rapid, and easy to perform, and must use modest amounts of the samples to be tested.In this work we present a laboratory-scale combined pretreatment and saccharification assay that can be used as a biomass feedstock screening tool. The assay uses a commercially available automated solvent extraction system for pretreatment followed by a small-scale enzymatic hydrolysis step. The assay allows multiple samples to be screened simultaneously, and uses only ~3 g of biomass per sample. If the composition of the biomass sample is known, the results of the assay can be expressed as reactivity (fraction of structural carbohydrate present in the biomass sample released as monomeric sugars). We first present pretreatment and enzymatic hydrolysis experiments on a set of representative biomass feedstock samples (corn stover, poplar, sorghum, switchgrass) in order to put the assay in context, and then show the results of the assay applied to approximately 150 different feedstock samples covering 5 different materials. From the compositional analysis data we identify a positive correlation between lignin and structural carbohydrates, and from the reactivity data we identify a negative correlation between both carbohydrate and lignin content and total reactivity. The negative correlation between lignin content and total reactivity suggests that lignin may interfere with sugar release, or that more mature samples (with higher structural sugars) may have more recalcitrant lignin. The assay presented in this work provides a robust and straightforward method to measure the sugar release after pretreatment and saccharification that can be used as a biomass feedstock screening tool. We demonstrated the utility of the assay by

  8. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    International Nuclear Information System (INIS)

    Morton, M.J.; Armstrong, D.; Abi Gerges, N.; Bridgland-Taylor, M.; Pollard, C.E.; Bowes, J.; Valentin, J.-P.

    2014-01-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility

  9. A high-throughput assay of NK cell activity in whole blood and its clinical application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: kylee117@yuhs.ac [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: jkim63@yuhs.ac [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  10. Neuromyelitis optica immunoglobulin G in Chinese patients detected by immunofluorescence assay on a monkey brain substrate.

    Science.gov (United States)

    Long, Youming; Hu, Xueqiang; Peng, Fuhua; Lu, Zhengqi; Wang, Yuge; Yang, Yu; Qiu, Wei

    2012-01-01

    Serum neuromyelitis optica immunoglobulin G (NMO-IgG) is used as a biomarker to differentiate between neuromyelitis optica (NMO) and multiple sclerosis (MS). However, the original assay is expensive and complex and shows low sensitivity. Here, we investigated the potential of NMO-IgG detection using an indirect immunofluorescence (IIF) assay on monkey brains. NMO-IgG seroprevalence was determined in 168 samples by an IIF assay on a monkey brain substrate. The data were compared with those from a standard mouse brain IIF assay using McNemar and kappa tests. Thirty-one of 50 (62%) NMO patients, 7 of 18 (38.9%) longitudinally extensive transverse myelitis patients, 6 of 57 (10.5%) MS patients, and 5 of 10 (50%) optic neuritis patients were seropositive for NMO-IgG. None of the acute partial transverse myelitis patients (n = 3) or healthy controls (n = 20) was positive. Thus, the sensitivity of the test was 62% for the patients with clinically definite NMO. The specificity was 89.5%, considering the 57 MS patients as the control group. The modified IIF assay on monkey brains and the standard IIF assay based on mouse brains were not significantly different (McNemar test; p = 1.000). The two assays were concordant in 39 seropositive samples and 100 seronegative samples (kappa test; kappa = 0.592, p monkey brain assay was no better than the standard mouse brain IIF assay, we affirmed that NMO-IgG is a sensitive and specific biomarker to differentiate between NMO and MS. Copyright © 2011 S. Karger AG, Basel.

  11. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Jennifer R. McCall

    2014-09-01

    Full Text Available Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/ veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

  12. Radiometric assays for the measurement of PSA

    International Nuclear Information System (INIS)

    Venkatesh, M.

    1997-01-01

    Prostate Specific Antigen, a serine protease enzyme, of M.W. ∼ 26-33 kDa, is widely considered to be a very useful marker for prostate cancer. It satisfies nearly all the requirements of an ideal 'Tumour Marker' and has hence attracted a lot of attention in the past decade. PSA is present in multiple forms in serum, with an appreciable fraction bound to the protease inhibitor α-1-antichymotrypsin (ACT) and to a small extent to other proteins such as α-2-macroglobulin (AMG) leaving the rest in the free form. The total PSA levels have been reported to have 80% sensitivity and 60% specificity towards the detection of prostate cancer. The lack of specificity occurs mainly due to the high levels of t-PSA in benign prostatic hypertrophy(BPH) apart from the cancer. The concept of free PSA has been introduced in the recent past and the ratio of free/total PSA levels have been shown to be advantageous in the differential diagnosis of BPH from prostate cancer. The f/t ratio is considered to be particularly useful in the grey zones of decision making (t-PSA levels 4-20 ng/mL). The need for the development of assays for total and free PSA is felt due to: a. the high incidence of prostate cancers being detected currently; b. the high cost of tests (higher for free PSA assay, and the cost becomes an important parameter when a patient has to be regularly monitored after therapy) that is not affordable for many patients; c. the potential for research in the area of prostate cancer management where the PSA (total and free) assays will be of great help

  13. Results of in vitro chemosensitivity assays

    International Nuclear Information System (INIS)

    Tanigawa, Nobuhiko; Morimoto, Hideki; Akita, Toshiaki; Inoue, Hiroshi; Tanaka, Takeo.

    1986-01-01

    The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60 % of specimens in HTCA and 58 % in SA produced significant growth in vitro. HTCA was 52 % (13/25) reliable for predicting in vivo sensitivity, and 95 % (36/38) reliable for in vivo resistance, whereas SA was 40 % (8/20) reliable for in vivo sensitivity and 88 % (21/24) for in vivo resistance. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48 % and 60 % in HTCA, and 46 % and 68 % in SA, respectively). (p < 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between : 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors. (J.P.N.)

  14. ARIES nondestructive assay system operation and performance

    International Nuclear Information System (INIS)

    Cremers, Teresa L.; Hansen, Walter J.; Herrera, Gary D.; Nelson, David C.; Sampson, Thomas E.; Scheer, Nancy L.

    2000-01-01

    The ARIES (Advanced Recovery and Integrated Extraction System) Project is an integrated system at the Los Alamos Plutonium Facility for the dismantlement of nuclear weapons. The plutonium produced by the ARIES process was measured by an integrated nondestructive assay (NDA) system. The performance of the NDA systems was monitored by a measurement control program which is a part of a nuclear material control and accountability system. In this paper we will report the results of the measurements of the measurement control standards as well as an overview of the measurement of the ARIES process materials

  15. Test procedure for boxed waste assay system

    International Nuclear Information System (INIS)

    Wachter, J.

    1994-01-01

    This document, prepared by Los Alamos National Laboratory's NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system's embedded operating and data reduction software

  16. Assaying gene function by growth competition experiment.

    Science.gov (United States)

    Merritt, Joshua; Edwards, Jeremy S

    2004-07-01

    High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

  17. Automatic titrator for high precision plutonium assay

    International Nuclear Information System (INIS)

    Jackson, D.D.; Hollen, R.M.

    1986-01-01

    Highly precise assay of plutonium metal is required for accountability measurements. We have developed an automatic titrator for this determination which eliminates analyst bias and requires much less analyst time. The analyst is only required to enter sample data and start the titration. The automated instrument titrates the sample, locates the end point, and outputs the results as a paper tape printout. Precision of the titration is less than 0.03% relative standard deviation for a single determination at the 250-mg plutonium level. The titration time is less than 5 min

  18. Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

    Science.gov (United States)

    Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

    2017-10-01

    Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

  19. Radioactive waste package assay facility. Volume 1. Application of assay technology

    International Nuclear Information System (INIS)

    Findlay, D.J.S.; Green, T.H.; Molesworth, T.V.; Staniforth, D.; Strachan, N.R.; Rogers, J.D.; Wise, M.O.; Forrest, K.R.

    1992-01-01

    This report, in three volumes, covers the work carried out by Taylor Woodrow Construction Ltd., and two major sub-contractors: Harwell Laboratory (AEA Technology) and Siemens Plessey Controls Ltd., on the development of a radioactive waste package assay facility, for cemented 500 litre intermediate level waste drums. In volume 1, the reasons for assay are considered together with the various techniques that can be used, and the information that can be obtained. The practical problems associated with the use of the various techniques in an integrated assay facility are identified, and the key parameters defined. Engineering and operational features are examined and provisional designs proposed for facilities at three throughput levels: 15,000, 750 and 30 drums per year respectively. The capital and operating costs for such facilities have been estimated. A number of recommendations are made for further work. 16 refs., 14 figs., 13 tabs

  20. Do detour tasks provide accurate assays of inhibitory control?

    Science.gov (United States)

    Whiteside, Mark A.; Laker, Philippa R.; Beardsworth, Christine E.

    2018-01-01

    Transparent Cylinder and Barrier tasks are used to purportedly assess inhibitory control in a variety of animals. However, we suspect that performances on these detour tasks are influenced by non-cognitive traits, which may result in inaccurate assays of inhibitory control. We therefore reared pheasants under standardized conditions and presented each bird with two sets of similar tasks commonly used to measure inhibitory control. We recorded the number of times subjects incorrectly attempted to access a reward through transparent barriers, and their latencies to solve each task. Such measures are commonly used to infer the differential expression of inhibitory control. We found little evidence that their performances were consistent across the two different Putative Inhibitory Control Tasks (PICTs). Improvements in performance across trials showed that pheasants learned the affordances of each specific task. Critically, prior experience of transparent tasks, either Barrier or Cylinder, also improved subsequent inhibitory control performance on a novel task, suggesting that they also learned the general properties of transparent obstacles. Individual measures of persistence, assayed in a third task, were positively related to their frequency of incorrect attempts to solve the transparent inhibitory control tasks. Neophobia, Sex and Body Condition had no influence on individual performance. Contrary to previous studies of primates, pheasants with poor performance on PICTs had a wider dietary breadth assayed using a free-choice task. Our results demonstrate that in systems or taxa where prior experience and differences in development cannot be accounted for, individual differences in performance on commonly used detour-dependent PICTS may reveal more about an individual's prior experience of transparent objects, or their motivation to acquire food, than providing a reliable measure of their inhibitory control. PMID:29593115

  1. Antifungal chemical compounds identified using a C. elegans pathogenicity assay.

    Directory of Open Access Journals (Sweden)

    Julia Breger

    2007-02-01

    Full Text Available There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2% that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.

  2. An improved in vitro micronucleus assay to biological dosimetry

    International Nuclear Information System (INIS)

    Ocampo, Ivette Z.; Okazaki, Kayo; Vieira, Daniel P.

    2013-01-01

    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 (3x10 4 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)

  3. Effective implementation of novel MET pharmacodynamic assays in translational studies.

    Science.gov (United States)

    Srivastava, Apurva K; Navas, Tony; Herrick, William G; Hollingshead, Melinda G; Bottaro, Donald P; Doroshow, James H; Parchment, Ralph E

    2017-01-01

    MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control pre-analytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.

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

    Science.gov (United States)

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

    2007-07-01

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

  5. Statistical precision of delayed-neutron nondestructive assay techniques

    International Nuclear Information System (INIS)

    Bayne, C.K.; McNeany, S.R.

    1979-02-01

    A theoretical analysis of the statistical precision of delayed-neutron nondestructive assay instruments is presented. Such instruments measure the fissile content of nuclear fuel samples by neutron irradiation and delayed-neutron detection. The precision of these techniques is limited by the statistical nature of the nuclear decay process, but the precision can be optimized by proper selection of system operating parameters. Our method is a three-part analysis. We first present differential--difference equations describing the fundamental physics of the measurements. We then derive and present complete analytical solutions to these equations. Final equations governing the expected number and variance of delayed-neutron counts were computer programmed to calculate the relative statistical precision of specific system operating parameters. Our results show that Poisson statistics do not govern the number of counts accumulated in multiple irradiation-count cycles and that, in general, maximum count precision does not correspond with maximum count as first expected. Covariance between the counts of individual cycles must be considered in determining the optimum number of irradiation-count cycles and the optimum irradiation-to-count time ratio. For the assay system in use at ORNL, covariance effects are small, but for systems with short irradiation-to-count transition times, covariance effects force the optimum number of irradiation-count cycles to be half those giving maximum count. We conclude that the equations governing the expected value and variance of delayed-neutron counts have been derived in closed form. These have been computerized and can be used to select optimum operating parameters for delayed-neutron assay devices

  6. Use of the microculture kinetic assay of apoptosis to determine chemosensitivities of leukemias.

    Science.gov (United States)

    Kravtsov, V D; Greer, J P; Whitlock, J A; Koury, M J

    1998-08-01

    Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate-labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell

  7. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  8. The Statistics of wood assays for preservative retention

    Science.gov (United States)

    Patricia K. Lebow; Scott W. Conklin

    2011-01-01

    This paper covers general statistical concepts that apply to interpreting wood assay retention values. In particular, since wood assays are typically obtained from a single composited sample, the statistical aspects, including advantages and disadvantages, of simple compositing are covered.

  9. Use of polymeric dyes in lignin biodegradation assays

    International Nuclear Information System (INIS)

    Gold, M.H.; Alic, M.; Glenn, J.K.

    1988-01-01

    This paper reviews the historical use of various 14 C-radiolabeled and unlabeled substrates to screen for ligninolytic activity. The disadvantages of these assays are presented. The authors describe the development of assays utilizing polymeric dyes

  10. Synthesis of photothermal nanocomposites and their application to antibacterial assays

    Science.gov (United States)

    Yang, Ning; Wang, Chun; Wang, Xiaoyu; Li, Lidong

    2018-04-01

    In this work, we report a novel gold nanorod (AuNR)-based nanocomposite that shows strong binding to bacterium and high antibacterial efficiency. The AuNRs were used as a photothermal material to transform near-infrared radiation (NIR) into heat. We selected poly (acrylic acid) to modify the surface of the AuNRs based on a simple self-assembly method. After conjugation of the bacterium-binding molecule vancomycin, the nanocomposites were capable of efficiently gathering on the cell walls of bacteria. The nanocomposites exhibited a high bacterial inhibition capability owing to NIR-induced heat generation in situ. Therefore, the prepared photothermal nanocomposites show great potential for use in antibacterial assays.

  11. Detection of garlic gamma-irradiated by assay comet

    International Nuclear Information System (INIS)

    Moreno Alvarez, Damaris L.; Miranda, Enrique F. Prieto; Carro, Sandra; Iglesias Enrique, Isora; Matos, Wilberto

    2009-01-01

    The garlic samples were irradiated in a facility with 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)

  12. 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)

  13. Experimental 233U nondestructive assay with a random driver

    International Nuclear Information System (INIS)

    Goris, P.

    1979-01-01

    Nondestructive assay (NDA) of 233 U in quantities up to 15 grams containing 7 ppM 232 U age 2 years was investigated with a random driver. A passive singles counting technique showed a reproducibility within 0.2% at the 95% confidence level. This technique would be applicable throughout a process in which all of the 233 U had the same 232 U content at the same age. Where the 232 U content varies, determination of 233 U fissile content would require active NDA. Active coincidence counting utilizing a 238 Pu, Li neutron source and a plastic scintillator detector system showed a reproducibility limit within 15% at the 95% confidence limit. The active technique was found to be very dependent on the detector system resolving time in order to make proper random coincidence corrections associated with the high gamma activity from the 232 U decay chain

  14. Cytological assay of micronucleus induction by radiation in oral cancer

    International Nuclear Information System (INIS)

    Bhattathiri, V.N.; Bindu, L.; Remani, P.; Chandralekha, B.; Davis, C.A.; Krishnan Nair, M.

    1996-01-01

    A study was conducted to analyze the dose-response relationship of micronucleus (MN) induction by radiation in eighty-three patients with oral cancers. Serial scrape smears were taken before treatment and after delivery of various fractions of a course of radical radiotherapy. The smears were stained with Giemsa's stain and frequency of micronucleated cells (MNC) evaluated. Before treatment 70.5% of tumours showed MNC, the mean value being 4.18 MNC/1000 cells. The frequency of MNC, increased with increasing dose of radiation. As regards relation to treatment duration, there was initially a slight increase, followed by rapid increase and then a plateauing. Radiosensitive and resistant tumours showed differing pattern of change. The MN test by serial cytological assay has potential as a tool to understand the dynamics of radiation induced cell death and predict radiosensitivity. (author). 4 refs., 5 figs

  15. MCNP efficiency calculations of INEEL passive active neutron assay system for simulated TRU waste assays

    International Nuclear Information System (INIS)

    Yoon, W.Y.; Meachum, T.R.; Blackwood, L.G.; Harker, Y.D.

    2000-01-01

    The Idaho National Engineering and Environmental Laboratory Stored Waste Examination Pilot Plant (SWEPP) passive active neutron (PAN) radioassay system is used to certify transuranic (TRU) waste drums in terms of quantifying plutonium and other TRU element activities. Depending on the waste form involved, significant systematic and random errors need quantification in addition to the counting statistics. To determine the total uncertainty of the radioassay results, a statistical sampling and verification approach has been developed. In this approach, the total performance of the PAN nondestructive assay system is simulated using the computer models of the assay system, and the resultant output is compared with the known input to assess the total uncertainty. The supporting steps in performing the uncertainty analysis for the passive assay measurements in particular are as follows: (1) Create simulated waste drums and associated conditions; (2) Simulate measurements to determine the basic counting data that would be produced by the PAN assay system under the conditions specified; and (3) Apply the PAN assay system analysis algorithm to the set of counting data produced by simulating measurements to determine the measured plutonium mass. The validity of this simulation approach was verified by comparing simulated output against results from actual measurements using known plutonium sources and surrogate waste drums. The computer simulation of the PAN system performance uses the Monte Carlo N-Particle (MCNP) Code System to produce a neutron transport calculation for a simulated waste drum. Specifically, the passive system uses the neutron coincidence counting technique, utilizing the spontaneous fission of 240 Pu. MCNP application to the SWEPP PAN assay system uncertainty analysis has been very useful for a variety of waste types contained in 208-ell drums measured by a passive radioassay system. The application of MCNP to the active radioassay system is also feasible

  16. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  17. Human semen assays for workplace monitoring

    International Nuclear Information System (INIS)

    Wyrobek, A.J.; Gledhill, B.L.

    1978-01-01

    Decades of human semen studies have yielded compelling evidence that sperm can be used to access reproductive potential and diagnose pathology. With these studies as background, the small number of detailed semen studies of men exposed to physical and chemical agents point with optimism to the application of human semen assays as efficient, effective means to monitor for reproductive hazards in the workplace. Sperm are the most accessible of human gonadal tissue and provide a means of monitoring exposure induced changes in the human testes, changes which may result in infertility and increased frequencies of genetically abnormal gametes. The focus on semen has precipitated the development of new sperm bioassays which use older conventional andrological methods (i.e., sperm counts, motility, and morphology) as well as recently developed high speed flow and scanning methods for automated cytological analyses. The status of these sperm assays for workplace surveillance is reviewed, procedures are suggested with examples of use, and their effectiveness is evaluated. The available mouse models of induced semen changes are briefly described and the importance of these models for evaluating the genetic implications of findings in human semen is discussed

  18. Rapid multiple immunoenzyme assay of mycotoxins.

    Science.gov (United States)

    Urusov, Alexandr E; Zherdev, Anatoly V; Petrakova, Alina V; Sadykhov, Elchin G; Koroleva, Olga V; Dzantiev, Boris B

    2015-01-27

    Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  19. Rapid Multiple Immunoenzyme Assay of Mycotoxins

    Directory of Open Access Journals (Sweden)

    Alexandr E. Urusov

    2015-01-01

    Full Text Available Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  20. Rapid screening assay for calcium bioavailability studies

    International Nuclear Information System (INIS)

    Luhrsen, K.R.; Hudepohl, G.R.; Smith, K.T.

    1986-01-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium ( 47 Ca) in a whole body retention assay system. In this system, calcium sources are administered by oral gavage and subsequent counts are determined and corrected for isotopic decay. Unlike iron and zinc retention curves, which exhibit a 2-3 day equilibration period, calcium reaches equilibration after 24 hours. Autoradiographic analysis of the femurs indicate that the newly absorbed calcium is rapidly distributed to the skeletal system. Moreover, the isotope is distributed along the entire bone. Comparisons of calcium bioavailability were made using intrinsic/extrinsic labeled milk from two species i.e. rat and goat as well as CaCO 3 . In addition, extrinsic labeled cow milk was examined. In the rat, the extrinsic labeled calcium from milk was better absorbed than the intrinsic calcium. This was not the case in goat milk or the calcium carbonate which exhibited no significant differences. Chromatographic analysis of the labeled milk indicates a difference in distribution of the 47 Ca. From these data, the authors recommend the use of this assay system in calcium bioavailability studies. The labeling studies and comparisons indicate caution should be used, however, in labeling techniques and species milk comparison

  1. Trofile HIV co-receptor usage assay.

    Science.gov (United States)

    Low, Andrew J; McGovern, Rachel A; Harrigan, P Richard

    2009-03-01

    The introduction of CCR5 antagonists increases the options available for constructing therapeutic drug regimens for HIV-positive patients. However, as these drugs do not inhibit HIV variants that use the CXCR4 co-receptor, a pretreatment test is required to determine accurately HIV co-receptor usage (tropism) before initiating CCR5 antagonist-based therapy. To discuss the Monogram Trofile assay as a diagnostic tool for determining HIV tropism by critically reviewing reported literature and available data. Monogram Trofile has become, largely by default, the de facto standard for HIV tropism assay. However, there is significant room for improvement in the speed, cost and availability of the test. Furthermore, the test is not quantitative, requires high-input HIV RNA viral loads, and produces results that are less biologically stable than expected. These technical considerations may limit the use of CCR5 antagonists in therapy. Nevertheless, this test is likely to remain the most widely used tropism diagnostic for the short term. We expect that a more practical and possibly more accurate method for measuring HIV tropism can be developed.

  2. Steroidogenic disruptive effects of the serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol in the H295R cell assay and in a recombinant CYP17 assay

    DEFF Research Database (Denmark)

    Islin, Julie; Munkboel, Cecilie Hurup; Styrishave, Bjarne

    2018-01-01

    The aim of this study was to determine the steroidogenic endocrine disrupting effect of the three most widely used serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol, using two in vitro models, the H295R assay and a recombinant CYP17 enzyme assay. Steroid hormones were...... quantified using LC-MS/MS. Duloxetine showed endocrine disrupting effects at 5-20μM with CYP17 being the main target. Venlafaxine also affected the steroidogenesis, mainly by affecting the CYP17 lyase reaction, although at much higher concentrations i.e. 100μM. Tramadol only exerted minor effects...... on the steroidogenesis with the lowest observed effect at 314μM. Based on the H295R results, the inhibition of CYP17 by duloxetine and venlafaxine was investigated in a recombinant CYP17 assay with the use of the 4 major CYP17 substrates pregnenolone, progesterone, 17α-hydroxypregnenolone and 17α...

  3. Utility and diagnostic performance of Mycobacterium tuberculosis complex by two immunochromatographic assays as compared with the molecular Genotype assay in Nigeria

    Directory of Open Access Journals (Sweden)

    Benjamin Thumamo Pokam

    2013-01-01

    Full Text Available Among the disadvantages of smear microscopy for detection of tuberculosis cases is its inability to differentiate between Mycobacterium tuberculosis (MTB and non-tuberculous mycobacteria (NTM. This study evaluated two, new immunochromatographic assays – Capilia TB-Neo and SD Bioline – on unheated and heated cultures at 80 °C for 30 min respectively for their ability to discriminate between MTB complex and NTM as compared with the molecular Genotype assay. Mycobacteria used in the study were obtained from smear-positive specimens collected from patients at four major hospitals in Cross River State, Nigeria. Capilia TB-Neo and SD Bioline showed sensitivities of 98.8% and 93.8% respectively and 100% specificity for both assays. Heating the isolates did not significantly impact the test performance. Both tests are recommended for use in rapid differentiation of strains isolated in Nigeria.

  4. Multiplexing a high-throughput liability assay to leverage efficiencies.

    Science.gov (United States)

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  5. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Science.gov (United States)

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  6. Clinical validation of the Tempus xO assay

    Science.gov (United States)

    Beaubier, Nike; Tell, Robert; Huether, Robert; Bontrager, Martin; Bush, Stephen; Parsons, Jerod; Shah, Kaanan; Baker, Tim; Selkov, Gene; Taxter, Tim; Thomas, Amber; Bettis, Sam; Khan, Aly; Lau, Denise; Lee, Christina; Barber, Matthew; Cieslik, Marcin; Frankenberger, Casey; Franzen, Amy; Weiner, Ali; Palmer, Gary; Lonigro, Robert; Robinson, Dan; Wu, Yi-Mi; Cao, Xuhong; Lefkofsky, Eric; Chinnaiyan, Arul; White, Kevin P.

    2018-01-01

    We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification. PMID:29899824

  7. 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... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3305 Herpes simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices...

  8. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  9. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  10. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

    2006-01-01

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  11. Time dependent analysis of assay comparability: a novel approach to understand intra- and inter-site variability over time

    Science.gov (United States)

    Winiwarter, Susanne; Middleton, Brian; Jones, Barry; Courtney, Paul; Lindmark, Bo; Page, Ken M.; Clark, Alan; Landqvist, Claire

    2015-09-01

    We demonstrate here a novel use of statistical tools to study intra- and inter-site assay variability of five early drug metabolism and pharmacokinetics in vitro assays over time. Firstly, a tool for process control is presented. It shows the overall assay variability but allows also the following of changes due to assay adjustments and can additionally highlight other, potentially unexpected variations. Secondly, we define the minimum discriminatory difference/ratio to support projects to understand how experimental values measured at different sites at a given time can be compared. Such discriminatory values are calculated for 3 month periods and followed over time for each assay. Again assay modifications, especially assay harmonization efforts, can be noted. Both the process control tool and the variability estimates are based on the results of control compounds tested every time an assay is run. Variability estimates for a limited set of project compounds were computed as well and found to be comparable. This analysis reinforces the need to consider assay variability in decision making, compound ranking and in silico modeling.

  12. Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

    International Nuclear Information System (INIS)

    Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  13. Comparison of Antioxidant Evaluation Assays for Investigating Antioxidative Activity of Gallic Acid and Its Alkyl Esters in Different Food Matrices.

    Science.gov (United States)

    Phonsatta, Natthaporn; Deetae, Pawinee; Luangpituksa, Pairoj; Grajeda-Iglesias, Claudia; Figueroa-Espinoza, Maria Cruz; Le Comte, Jérôme; Villeneuve, Pierre; Decker, Eric A; Visessanguan, Wonnop; Panya, Atikorn

    2017-08-30

    The addition of antioxidants is one of the strategies to inhibit lipid oxidation, a major cause of lipid deterioration in foods leading to rancidity development and nutritional losses. However, several studies have been reported that conventional antioxidant assays, e.g., TPC, ABTS, FRAP, and ORAC could not predict antioxidant performance in several foods. This study aimed to investigate the performance of two recently developed assays, e.g., the conjugated autoxidizable triene (CAT) and the apolar radical-initiated conjugated autoxidizable triene (ApoCAT) assays to predict the antioxidant effectiveness of gallic acid and its esters in selected food models in comparison with the conventional antioxidant assays. The results indicated that the polarities of the antioxidants have a strong impact on antioxidant activities. In addition, different oxidant locations demonstrated by the CAT and ApoCAT assays influenced the overall antioxidant performances of the antioxidants with different polarities. To validate the predictability of the assays, the antioxidative performance of gallic acid and its alkyl esters was investigated in oil-in-water (O/W) emulsions, bulk soybean oils, and roasted peanuts as the lipid food models. The results showed that only the ApoCAT assay could be able to predict the antioxidative performances in O/W emulsions regardless of the antioxidant polarities. This study demonstrated that the relevance of antioxidant assays to food models was strongly dependent on physical similarities between the tested assays and the food structure matrices.

  14. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Science.gov (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  15. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Directory of Open Access Journals (Sweden)

    D Ransom Hardison

    Full Text Available Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs. One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R in certain labs. A fluorescence based receptor binding assay (RBA(F was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2 for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1. Fish (N = 61 of six different species were screened using the RBA(F. Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a correlated well (R2 = 0.71 with those of the RBA(F, given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F, which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F advantages include the long-term (> 5 years stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R. The RBA(F is cost-effective, allows high sample

  16. Identification of irradiated refrigerated pork with the DNA comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, M.M. E-mail: villavic@net.ipen.br; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail: jmancini@usp.br; Delincee, H.; Villavicencio, A.L.C.H. E-mail: henry.delincee@bfe.uni-karlsruhe.de

    2004-10-01

    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells ('Comet Assay') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA 'Comet Assay'. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  17. Identification of irradiated refrigerated pork with the DNA comet assay

    Science.gov (United States)

    Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.; Villavicencio, A. L. C. H.

    2004-09-01

    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells (``Comet Assay'') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sa~o Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6°C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA ``Comet Assay''. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  18. An improved method for staining cell colonies in clonogenic assays.

    Science.gov (United States)

    Guda, Kishore; Natale, Leanna; Markowitz, Sanford D

    2007-06-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

  19. ABTS assay of phenol oxidase activity in soil.

    Science.gov (United States)

    Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

    2007-12-01

    Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

  20. Development of a Calibration Strip for Immunochromatographic Assay Detection Systems.

    Science.gov (United States)

    Gao, Yue-Ming; Wei, Jian-Chong; Mak, Peng-Un; Vai, Mang-I; Du, Min; Pun, Sio-Hang

    2016-06-29

    With many benefits and applications, immunochromatographic (ICG) assay detection systems have been reported on a great deal. However, the existing research mainly focuses on increasing the dynamic detection range or application fields. Calibration of the detection system, which has a great influence on the detection accuracy, has not been addressed properly. In this context, this work develops a calibration strip for ICG assay photoelectric detection systems. An image of the test strip is captured by an image acquisition device, followed by performing a fuzzy c-means (FCM) clustering algorithm and maximin-distance algorithm for image segmentation. Additionally, experiments are conducted to find the best characteristic quantity. By analyzing the linear coefficient, an average value of hue (H) at 14 min is chosen as the characteristic quantity and the empirical formula between H and optical density (OD) value is established. Therefore, H, saturation (S), and value (V) are calculated by a number of selected OD values. Then, H, S, and V values are transferred to the RGB color space and a high-resolution printer is used to print the strip images on cellulose nitrate membranes. Finally, verification of the printed calibration strips is conducted by analyzing the linear correlation between OD and the spectral reflectance, which shows a good linear correlation (R² = 98.78%).

  1. Evaluation of irradiation in foods using DNA comet assay

    International Nuclear Information System (INIS)

    Khawar, Affaf; Bhatti, Ijaz Ahmad; Khan, Q.M.; Ali, T.; Khan, A.I.; Asi, M.R.

    2011-01-01

    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 60 Co 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)

  2. LSDS Development for Isotopic Fissile Content Assay

    International Nuclear Information System (INIS)

    Lee, Yong Deok; Park, Chang Je; Park, Geun Il; Lee, Jung Won; Song, Kee Chan

    2010-01-01

    Concerning the sustainable energy supply and green house effect, nuclear energy became the most feasible option to meet the energy demand in Korea. However, the production of the spent nuclear fuel is the inevitable situation. Since the first nuclear power plant started to produce the electricity in Korea, the accumulated amount of spent fuels exceeded 10k tomes recently. The accumulation of the spent fuels is the big issue in the society. Therefore, as an option which strengthens the nuclear proliferation resistance and reduces the amount of spent fuels, sodium fast reactor (SFR) program linked with pyro-processing is under development to re-use the PWR spent fuel and produce the energy. In the process, the produced metallic material involves uranium and TRU (transuranic; neptunium, plutonium, and americium). The uranium-TRU is used to fabricate SFR fuel. The burning the recycled fuel in the reactor is to solve the current spent fuel storage problem and to minimize the actinides accumulation having long half-life. Generally, the spent fuel from PWR has unburned ∼1 % U235, produced ∼0.5 % plutonium from decay chain, ∼3 % fission products, ∼ 0.1 % minor actinides (MA) and uranium remainder. About 1.5 % fissile materials still exist in the spent fuel. Therefore, spent fuel is not only waste but energy resource. The direct and isotopic fissile content assay is the crucial technology for the spent fuel reuse. Additionally, the fissile content analysis will contribute to the optimum storage design and safe spent fuel management. Several nondestructive technologies have been developed for the spent fuel assay; gamma ray measurement, passive and active neutron measurements. Spent fuel emits intense gamma rays and neutrons by (a, n) and spontaneous fission. This intense background has the limitation on the direct analysis of fissile materials. Recently, to analyze the individual fissile content, leadslowing down spectrometer (LSDS) has been being developed in Korea

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

    International Nuclear Information System (INIS)

    Giorgio, M.D.; Taja, M.R.; Nasazzi, N.B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

    2000-01-01

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources Co 60 , Cs 137 or Ir 192 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

  4. Localized irradiations, evaluation through 'Comet Assay'

    International Nuclear Information System (INIS)

    Di Giorgio, Marina; Taja, Maria R.; Nasazzi, Nora B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

    2000-01-01

    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

  5. Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

    Science.gov (United States)

    2017-07-27

    The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

  6. Assessment of the microscreen phage-induction assay for screening hazardous wastes (1989)

    Energy Technology Data Exchange (ETDEWEB)

    Houk, V.S.; DeMarini, D.M.

    1989-01-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage Lambda in Escherichia coli WP2s(Lambda), was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that were mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  8. Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

    Directory of Open Access Journals (Sweden)

    Hampus Sunner

    2015-09-01

    Full Text Available Research on glucuronoyl esterases (GEs has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.

  9. Serum gonadotropins levels in childhood. Critical comparison between immunoradiometric assays and radioimmunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Hertogh, R. De; Vankrieken, L. (Physiology of Human Reproduction Research Unit, University of Louvain, School of Medicine, Brussels (Belgium)); Wolter, R.; Vliet, G. Van (Hopital Universitaire des Enfants, Reine Fabiola, Free University of Brussels (Belgium))

    1989-01-01

    The complex changes in serum LH and FSH levels from infancy to adulthood are diversely evaluated by radioimmunoassays or bioassays. The relative lack of sensitivity and specificity of radioimmunoassay, using polyclonal antibodies, could possibly be overcome by new immunoradiometric assays, using specific antibodies to LH and FSH. Significant differences were indeed observed between radioimmunoassays and immunoradiometric assays. During the prepubertal period, LH levels, measured by the immunoradiometric assays, were below the sensitivity of the method in the majority of the samples. LH levels were, however, well detectable when measured with radioimmunoassay, showing the heterogeneity of circulating LH structures. At the onset of puberty, LH levels increased at least 3 to 4 times in both sexes, when measured with immunoradiometric assays, whereas their increase was only 20 to 60% with the radioimmunoassays. FSH levels remained well detectable in the prepubertal period whether measured by immunoradiometric or radioimmunoassays. At pubertal onset, FSH increase in both sexes was more important in the immunoradiometric assays. The results obtained with immunoradiometric assays give a better insight into the quantitative and qualitative function of the gonadotropes during childhood. The almost complete absence of LH during the prepubertal period and the steep increase at the onset of puberty better reflects the reported data obtained with bioassays. The persistance of significant levels of FSH in the prepubertal ages, and the lesser increase at the onset of puberty, when compared with LH, illustrates that the individual regulation of LH and FSH secretion vary over time and is influenced by developmental factors. (author).

  10. A novel reporter system for neutralizing and enhancing antibody assay against dengue virus.

    Science.gov (United States)

    Song, Ke-Yu; Zhao, Hui; Jiang, Zhen-You; Li, Xiao-Feng; Deng, Yong-Qiang; Jiang, Tao; Zhu, Shun-Ya; Shi, Pei-Yong; Zhang, Bo; Zhang, Fu-Chun; Qin, E-De; Qin, Cheng-Feng

    2014-02-18

    Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-15

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

  12. Development and evaluation of a nested-PCR assay for Senecavirus A diagnosis.

    Science.gov (United States)

    Feronato, Cesar; Leme, Raquel A; Diniz, Jaqueline A; Agnol, Alais Maria Dall; Alfieri, Alice F; Alfieri, Amauri A

    2018-02-01

    Senecavirus A (SVA) has been associated with vesicular disease in weaned and adult pigs and with high mortality of newborn piglets. This study aimed to establish a nested-PCR assay for the routine diagnosis of SVA infection. Tissue samples (n = 177) were collected from 37 piglets of 18 pig farms located in four different Brazilian states. For the nested-PCR, a primer set was defined to amplify an internal VP1 fragment of 316 bp of SVA genome. Of the 37 piglets, 15 (40.5%) and 23 (62.2%) were positive for the SVA in the RT-PCR and nested-PCR assays, respectively. The SVA RNA was detected in 61/177 (34.5%) samples with the RT-PCR, while the nested-PCR assay showed 84/177 (47.5%) samples with the virus (p PCR and nested-PCR assays, respectively. Nucleotide sequencing analysis revealed similarities of 98.7-100% among SVA Brazilian strains and of 86.6-98% with SVA strains from other countries. The nested-PCR assay in this study was suitable to recover the SVA RNA in biological specimens, piglets, and/or herds that were considered as negative in the RT-PCR assay, and is proposed for the routine investigation of the SVA infection in piglets, especially when other techniques are not available or when a great number of samples has to be examined.

  13. Microbiological assay for the determination of meropenem in pharmaceutical dosage form.

    Science.gov (United States)

    Mendez, Andreas S L; Weisheimer, Vanessa; Oppe, Tércio P; Steppe, Martin; Schapoval, Elfrides E S

    2005-04-01

    Meropenem is a highly active carbapenem antibiotic used in the treatment of a wide range of serious infections. The present work reports a microbiological assay, applying the cylinder-plate method, for the determination of meropenem in powder for injection. The validation method yielded good results and included linearity, precision, accuracy and specificity. The assay is based on the inhibitory effect of meropenem upon the strain of Micrococcus luteus ATCC 9341 used as the test microorganism. The results of assay were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9999) in the range of 1.5-6.0 microg ml(-1), precise (intra-assay: R.S.D.=0.29; inter-assay: R.S.D.=0.94) and accurate. A preliminary stability study of meropenem was performed to show that the microbiological assay is specific for the determination of meropenem in the presence of its degradation products. The degraded samples were also analysed by the HPLC method. The proposed method allows the quantitation of meropenem in pharmaceutical dosage form and can be used for the drug analysis in routine quality control.

  14. Development of an assay for urinary free cortisol determination on the Technicon Immuno 1 system

    International Nuclear Information System (INIS)

    Letellier, M.; Levesque, A.; Daigle, F.

    1997-01-01

    To develop and evaluate a method using an ethyl acetate extraction procedure for the determination of urinary free cortisol on the Technicon Immuno 1 system from Bayer Corporation. We tested the assay precision, linearity, and correlation with the Urinary Kallestad Quanticoat Cortisol radioimmunoassay. We also studied the efficiency of the extraction procedure, performed a cross-reactivity study with different cortisol metabolites, and determined the reference values. The assay shows within-run CVs varying from 1.6 to 5.3% and between-day CVs from 2.7 to 6.1% for urinary free cortisol concentrations from 58 to 1097 nmol/L. The assay demonstrates an excellent linearity and a very good correlation with the Kallestad Quanticoat Cortisol assay (slope 0.94, y-intercept = 29 nmol/L, Sy|x = 54 nmol/L, r = 0.996). The reference values were estimated at 42-281 nmol/d. The extraction procedure shows an average recovery of 99.0% and minimal interference with the cortisol metabolites tested with the exception of cortisone. The evaluation shows that the developed assay has the analytical characteristics required for its utilization in a clinical laboratory. (author)

  15. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    Science.gov (United States)

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

  16. First two-reagent vitamin D assay for general clinical chemistry.

    Science.gov (United States)

    Saida, Fakhri B; Padilla-Chee, Mario; Dou, Chao; Yuan, Chong

    2018-05-01

    Vitamin D is a lipid-soluble molecule that plays key physiological roles in the metabolism of calcium, phosphate and magnesium. Recent studies show that deficiency in vitamin D is linked to cardiovascular diseases, autoimmune diseases and cancer. As a result, regular monitoring of 25-OH vitamin D (the main circulating form of vitamin D) is becoming essential. Current 25-OH vitamin D testing methodologies are cumbersome (too many reagents, long incubation times, phase separation) and are not compatible with general clinical chemistry platforms. Here, we report on a novel method to detect 25-OH vitamin D that is fast (results in 10 min or less), simple (two reagents) and compatible with virtually all general clinical chemistry analyzers. An immunoturbidimetric assay for 25-OH vitamin D (the Diazyme EZ Vitamin D Assay) has been developed using nanoparticles and vitamin D-specific antibodies. The performance of the assay kit, which consists of two reagents and five calibrators, was tested on the Beckman AU680 analyzer (AU680). The new assay was precise, sensitive (LOD = 7.2 nmol/L), linear (up to 390.1 nmol/L) and correlated strongly (R 2  > 0.95) with major commercial 25-OH vitamin D assays. Additionally, the assay was found to be the fastest to date, with the first results obtained within 10 min. Throughput on the AU680 was estimated at over 300 tests per hour. The newly developed 25-OH vitamin D assay is fast, precise and accurate. It can be run on most general chemistry analyzers. This assay aims at providing vitamin D-testing capabilities to all clinical chemistry laboratories. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  17. Isotopic fissile assay of spent fuel in a lead slowing-down spectrometer system

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Deok; Jeon, Ju Young [Dept. of Fuel Cycle Technology, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Park, Chang Je [Dept. of Nuclear Engineering, Sejong University, Seoul (Korea, Republic of)

    2017-04-15

    A lead slowing-down spectrometer (LSDS) system is under development to analyze isotopic fissile content that is applicable to spent fuel and recycled material. The source neutron mechanism for efficient and effective generation was also determined. The source neutron interacts with a lead medium and produces continuous neutron energy, and this energy generates dominant fission at each fissile, below the unresolved resonance region. From the relationship between the induced fissile fission and the fast fission neutron detection, a mathematical assay model for an isotopic fissile material was set up. The assay model can be expanded for all fissile materials. The correction factor for self-shielding was defined in the fuel assay area. The corrected fission signature provides well-defined fission properties with an increase in the fissile content. The assay procedure was also established. The assay energy range is very important to take into account the prominent fission structure of each fissile material. Fission detection occurred according to the change of the Pu239 weight percent (wt%), but the content of U235 and Pu241 was fixed at 1 wt%. The assay result was obtained with 2∼3% uncertainty for Pu239, depending on the amount of Pu239 in the fuel. The results show that LSDS is a very powerful technique to assay the isotopic fissile content in spent fuel and recycled materials for the reuse of fissile materials. Additionally, a LSDS is applicable during the optimum design of spent fuel storage facilities and their management. The isotopic fissile content assay will increase the transparency and credibility of spent fuel storage.

  18. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    International Nuclear Information System (INIS)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L.

    1990-01-01

    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation

  19. Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

    International Nuclear Information System (INIS)

    Xu Qiuwei; Vu, Heather; Liu Liping; Wang, Ting-Chuan; Schaefer, William H.

    2011-01-01

    Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.

  20. Some target assay uncertainties for passive neutron coincidence counting

    International Nuclear Information System (INIS)

    Ensslin, N.; Langner, D.G.; Menlove, H.O.; Miller, M.C.; Russo, P.A.

    1990-01-01

    This paper provides some target assay uncertainties for passive neutron coincidence counting of plutonium metal, oxide, mixed oxide, and scrap and waste. The target values are based in part on past user experience and in part on the estimated results from new coincidence counting techniques that are under development. The paper summarizes assay error sources and the new coincidence techniques, and recommends the technique that is likely to yield the lowest assay uncertainty for a given material type. These target assay uncertainties are intended to be useful for NDA instrument selection and assay variance propagation studies for both new and existing facilities. 14 refs., 3 tabs

  1. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  2. Time-resolved immunofluorometric assay of serum ferritin

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Yao [China Inst. of Atomic Energy, Beijing (China)

    2007-06-15

    This assay is a solid phase, two-site fluoroimmunometric assay based on the direct sandwish technique. Standards or samples containing ferritin are first reacted with immobilized anti-ferritin antibodies. Then the europium-lablled antibodies are reacted with the bound antigen. The range of this assay is 2-1000 ng/mL. The analytical sentivity is better than 0.05 ng/mL. The intra-assay variation and inter-assay variation are both below 5%; This kit was compared with Wallac DELFIA kit. The correlation is r=0.96. (authors)

  3. Nondestructive Assay Options for Spent Fuel Encapsulation

    Energy Technology Data Exchange (ETDEWEB)

    Tobin, Stephen J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Jansson, Peter [Uppsala Univ. (Sweden)

    2014-10-02

    This report describes the role that nondestructive assay (NDA) techniques and systems of NDA techniques may have in the context of an encapsulation and deep geological repository. The potential NDA needs of an encapsulation and repository facility include safeguards, heat content, and criticality. Some discussion of the facility needs is given, with the majority of the report concentrating on the capability and characteristics of individual NDA instruments and techniques currently available or under development. Particular emphasis is given to how the NDA techniques can be used to determine the heat production of an assembly, as well as meet the dual safeguards needs of 1) determining the declared parameters of initial enrichment, burn-up, and cooling time and 2) detecting defects (total, partial, and bias). The report concludes with the recommendation of three integrated systems that might meet the combined NDA needs of the encapsulation/repository facility.

  4. Development of a Radioactive Waste Assay System

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-12-31

    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  5. Benchmark West Texas Intermediate crude assayed

    International Nuclear Information System (INIS)

    Rhodes, A.K.

    1994-01-01

    The paper gives an assay of West Texas Intermediate, one of the world's market crudes. The price of this crude, known as WTI, is followed by market analysts, investors, traders, and industry managers around the world. WTI price is used as a benchmark for pricing all other US crude oils. The 41 degree API < 0.34 wt % sulfur crude is gathered in West Texas and moved to Cushing, Okla., for distribution. The WTI posted prices is the price paid for the crude at the wellhead in West Texas and is the true benchmark on which other US crudes are priced. The spot price is the negotiated price for short-term trades of the crude. And the New York Mercantile Exchange, or Nymex, price is a futures price for barrels delivered at Cushing

  6. Universal fieldable assay with unassisted visual detection

    Science.gov (United States)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

  7. Development of an integrated assay facility

    International Nuclear Information System (INIS)

    Molesworth, T.V.; Bailey, M.; Findlay, D.J.S.; Sene, M.R.; Swinhoe, M.T.

    1990-01-01

    Initial results of active neutron and active gamma-ray interrogation of a 500 liter cemented simulated CAGR intermediate level radioactive waste drum are described. The basis of the interrogation systems was the Harwell electron linear accelerator HELIOS, which was used to produce the interrogating neutrons and gamma-rays. Several sets of neutron detectors were located around the drum to count signature neutrons. The responses of the system were measured by placing known samples at many different locations within the drum. In general, measured responses confirmed calculated responses. Good agreement was obtained for the azimuthal angle dependences. The absolute responses agreed well for gamma-ray interrogation, but the calculations were apparently over-estimates for neutron interrogation. Those aspects requiring consideration in the practical application of assay techniques are identified. 8 refs., 6 figs

  8. COMPARATIVE STUDY ON MILK CASEIN ASSAY METHODS

    Directory of Open Access Journals (Sweden)

    RODICA CĂPRIłĂ

    2008-05-01

    Full Text Available Casein, the main milk protein was determined by different assay methods: the gravimetric method, the method based on the neutralization of the NaOH excess used for the casein precipitate solving and the method based on the titration of the acetic acid used for the casein precipitation. The last method is the simplest one, with the fewer steps, and also with the lowest error degree. The results of the experiment revealed that the percentage of casein from the whole milk protein represents between 72.6–81.3% in experiment 1, between 73.6–81.3% in experiment 2 and between 74.3–81% in experiment 3.

  9. Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Konstantinou, George N

    2017-01-01

    Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

  10. Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma

    Directory of Open Access Journals (Sweden)

    Zhang Kang-Jian

    2012-02-01

    Full Text Available Abstract Background Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells. Methods By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay. Results The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects. Conclusion The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.

  11. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    Science.gov (United States)

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

    International Nuclear Information System (INIS)

    Chen Zhijian; Lou Jianlin; Chen Shijie; Zheng Wei; Wu Wei; Jin Lifen; Deng Hongping; He Jiliang

    2006-01-01

    To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m 3 . All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10 -4 , respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

  13. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    Energy Technology Data Exchange (ETDEWEB)

    Otero, Paz; Alfonso, Amparo [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain); Alfonso, Carmen [CIFGA Laboratorio, Plaza de Santo Domingo, 1, 27001 Lugo (Spain); Araoz, Romulo; Molgo, Jordi [CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie et Developpement UPR3294, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex (France); Vieytes, Mercedes R. [Departamento de Fisiologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo (Spain); Botana, Luis M., E-mail: luis.botana@usc.es [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain)

    2011-09-09

    Highlights: {yields} A direct assay based in the binding of nAChR to spirolide toxins by FP is described. {yields} A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. {yields} FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. {yields} FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples. - Abstract: In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 {mu}g kg{sup -1} meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

  14. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    International Nuclear Information System (INIS)

    Otero, Paz; Alfonso, Amparo; Alfonso, Carmen; Araoz, Romulo; Molgo, Jordi; Vieytes, Mercedes R.; Botana, Luis M.

    2011-01-01

    Highlights: → A direct assay based in the binding of nAChR to spirolide toxins by FP is described. → A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. → FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. → FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples. - Abstract: In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg -1 meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

  15. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    Science.gov (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Antimicrobial Assay of Soil Mold Isolates from Wonorejo Surabaya

    Directory of Open Access Journals (Sweden)

    Septia Arisanti

    2012-11-01

    Full Text Available This study was aimed to an examine antimicrobial activity of 34 soil molds isolates from the Wonorejo Surabaya on the growth of Gram negatif bacteria (Escherichia coli and Coliform Bacteria Group, Gram positif bacteria (Bacillus subtilis and yeast (Saccharomyces cerevisiae. Antimicrobial ability detected with modification of dual culture antagonism assay in Potato Dextrose Agar (PDA medium. The result showed that genus Aspergillus, Scopulariopsis, Penicillium, Paecilomyces, Fusarium, and Trichoderma were able to inhibit E. coli; while genus Aspergillus, Scopulariopsis, Penicillium, Paecilomyces, Exophiala, Stachybotrys, and Acremonium inhibit B. subtilis; further on only genus Aspergillus could inhibit group of Coliform bacteria; and genus Scopulariopsis, Penicillium, Trichoderma, and Absidia inhibited the growth of yeast S. cerevisiae.

  17. Evaluation of Robust Estimators Applied to Fluorescence Assays

    Directory of Open Access Journals (Sweden)

    U. Ruotsalainen

    2007-12-01

    Full Text Available We evaluated standard robust methods in the estimation of fluorescence signal in novel assays used for determining the biomolecule concentrations. The objective was to obtain an accurate and reliable estimate using as few observations as possible by decreasing the influence of outliers. We assumed the true signals to have Gaussian distribution, while no assumptions about the outliers were made. The experimental results showed that arithmetic mean performs poorly even with the modest deviations. Further, the robust methods, especially the M-estimators, performed extremely well. The results proved that the use of robust methods is advantageous in the estimation problems where noise and deviations are significant, such as in biological and medical applications.

  18. Field nondestructive assay measurements as applied to process inventories

    International Nuclear Information System (INIS)

    Westsik, G.A.

    1979-08-01

    An annual process equipment holdup inventory measurement program for a plutonium processing plant was instituted by Rockwell Hanford Operations (Rockwell) at Richland, Washington. The inventories, performed in 1977 and 1978, were designed to improve plutonium accountability and control. The inventory method used field nondestructive assay (NDA) measurement techniques with portable electronics and sodium iodide detectors. Access to and movement of plutonium in work areas was curtailed during the inventory process using administrative controls. Comparison of the two annual inventories showed good reproducibility of results within the calculated error ranges. For items where no plutonium movement occurred and which contained greater than 20 grams plutonium, the average measurement difference between the two inventories was 22%. The procedures and equipment used and the operational experience from the inventories are described

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

    International Nuclear Information System (INIS)

    Hecht, Ariel; Kinnunen, Paivo; McNaughton, Brandon; Kopelman, Raoul

    2011-01-01

    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.

  20. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, s...... flippase activities in the plasma membrane of cells, using yeast as an example.......P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases......, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  1. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  2. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda

    2012-05-01

    Full Text Available The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone, the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  3. DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

    Science.gov (United States)

    Furst, Ariel L; Barton, Jacqueline K

    2015-07-23

    DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Comparative evaluation of genetic toxicity patterns of carcinogens and noncarcinogens: strategies for predictive use of short-term assays

    International Nuclear Information System (INIS)

    Tennant, R.W.; Spalding, J.W.; Stasiewicz, S.; Caspary, W.D.; Mason, J.M.; Resnick, M.A.

    1987-01-01

    The results of a recent comprehensive evaluation of the relationship between four measures of in vitro genetic toxicity and the capacity of the chemicals to induce neoplasia in rodents carry some important implications. The results showed that while the Salmonella mutagenesis assay detected only about half of the carcinogenes as mutagens, the other three in vitro assays (mutagenesis in MOLY cells or induction of aberrations or SCEs in CHO cells) did not complement Salmonella since they failed to effectively discriminate between the carcinogens and noncarcinogens found negative in the Salmonella assay. The specificity of the Salmonella assay for this group of 73 chemicals was relatively high (only 4 of 29 noncarcinogens were positive). Therefore, the authors have begun to evaluate in vivo genetic toxicity assays for their ability to complement Salmonella in the identification of carcinogens

  5. Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

    Directory of Open Access Journals (Sweden)

    Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

    2012-05-01

    Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

  6. Serotype determination of Salmonella by xTAG assay.

    Science.gov (United States)

    Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

    2017-10-01

    Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

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

    2011-01-01

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

  8. The comet assay: ready for 30 more years.

    Science.gov (United States)

    Møller, Peter

    2018-02-24

    During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

  9. A comparative study of PD-L1 immunohistochemical assays with four reliable antibodies in thymic carcinoma.

    Science.gov (United States)

    Sakane, Tadashi; Murase, Takayuki; Okuda, Katsuhiro; Takino, Hisashi; Masaki, Ayako; Oda, Risa; Watanabe, Takuya; Kawano, Osamu; Haneda, Hiroshi; Moriyama, Satoru; Saito, Yushi; Yamada, Takeshi; Nakanishi, Ryoichi; Inagaki, Hiroshi

    2018-01-23

    Currently, four immunohistochemical assays are registered with the US Food and Drug Administration to detect the expression of PD-L1. We investigated the PD-L1 expression in thymic carcinomas using these four diagnostic assays. The cases of 53 patients were reviewed and their specimens were subjected to four PD-L1 assays with different antibodies (SP142, SP263, 22C3, and 28-8). The PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was evaluated. In TCs, the four assays showed similar scores in each case. Histopathologically, high TC scores were observed in squamous cell carcinomas (SqCCs). Meanwhile, there were no significant relationships among the IC scores in the four assays. In SqCCs, the high expression of PD-L1 (defined as ≥50% TC score) in TCs tended to be associated with early stage cancer. The patients with high expression levels of PD-L1 tended to show longer overall survival in the 22C3 assays (p=0.0200). In thymic carcinomas, the staining pattern showed high concordance among the four assays when TCs - rather than ICs - were stained. High PD-L1 positivity in TCs, especially in SqCCs, indicated that PD-1/PD-L1 targeted therapy may be a promising therapeutic approach.

  10. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  11. In vitro assay shows that PCB metabolites completely saturate thyroid hormone transport capacity in blood of wild polar bears (Ursus maritimus)

    NARCIS (Netherlands)

    Gutleb, A.C.; Cenijn, P.H.; van Velzen, P.; Lie, E.; Ropstad, E.; Skaare, J.U.; Malmberg, T.; Bergman, A.; Gabrielsen, G. W.; Legler, J.

    2010-01-01

    Persistent chemicals accumulate in the arctic environment due to their chemical reactivity and physicochemical properties and polychlorinated biphenyls (PCBs) are the most concentrated pollutant class in polar bears (Ursus maritimus). Metabolism of PCB and polybrominated biphenyl ether (PBDE)

  12. In vitro assay shows that PCB metabolites completely saturate thyroid hormone transport capacity in blood of wild polar bears (Ursus maritimus).

    Science.gov (United States)

    Gutleb, Arno C; Cenijn, Peter; Velzen, Martin van; Lie, Elisabeth; Ropstad, Erik; Skaare, Janneche Utne; Malmberg, Tina; Bergman, Ake; Gabrielsen, Geir W; Legler, Juliette

    2010-04-15

    Persistent chemicals accumulate in the arctic environment due to their chemical reactivity and physicochemical properties and polychlorinated biphenyls (PCBs) are the most concentrated pollutant class in polar bears (Ursus maritimus). Metabolism of PCB and polybrominated biphenyl ether (PBDE) flame-retardants alter their toxicological properties and these metabolites are known to interfere with the binding of thyroid hormone (TH) to transthyretin (TTR) in rodents and humans. In polar bear plasma samples no binding of [125I]-T(4) to TTR was observed after incubation and PAGE separation. Incubation of the plasma samples with [14C]-4-OH-CB107, a compound with a higher binding affinity to TTR than the endogenous ligand T(4) resulted in competitive binding as proven by the appearance of a radio labeled TTR peak in the gel. Plasma incubation with T(4) up to 1 mM, a concentration that is not physiologically relevant anymore did not result in any visible competition. These results give evidence that the binding sites on TTR for T(4) in wild living polar bears are completely saturated. Such saturation of binding sites can explain observed lowered levels of THs and could lead to contaminant transport into the developing fetus.

  13. A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds

    Czech Academy of Sciences Publication Activity Database

    Lee, K. P.; Piskurewicz, U.; Turečková, Veronika; Strnad, Miroslav; Lopez-Molina, L.

    2010-01-01

    Roč. 107, č. 44 (2010), s. 19108-19113 ISSN 0027-8424 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : gibberellins * seed dormancy * DELLA Subject RIV: EF - Botanics Impact factor: 9.771, year: 2010

  14. Comparison of five assays for detection of Clostridium difficile toxin.

    Science.gov (United States)

    Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

    2011-07-01

    Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Comparison of Five Assays for Detection of Clostridium difficile Toxin

    Science.gov (United States)

    Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

    2011-01-01

    Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

  16. Principles of validation of diagnostic assays for infectious diseases

    International Nuclear Information System (INIS)

    Jacobson, R.H.

    1998-01-01

    Assay validation requires a series of inter-related processes. Assay validation is an experimental process: reagents and protocols are optimized by experimentation to detect the analyte with accuracy and precision. Assay validation is a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. Assay validation is a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the target population; accurate predictions of the infection status of animals from test results (PV+ and PV-) are conditional upon the estimated prevalence of disease/infection in the target population. Assay validation is an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results; the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status. Assay validation is a continuous process: the assay remains valid only insofar as it continues to provide accurate and precise results as proven through statistical verification. Therefore, the work required for validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples rather, to assure valid test results from an assay requires constant vigilance and maintenance of the assay, along with reassessment of its performance characteristics for each unique population of animals to which it is applied. (author)

  17. Alzheimer's-associated Aβ oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal

    International Nuclear Information System (INIS)

    Pitt, Jason; Roth, William; Lacor, Pascale; Smith, Amos B.; Blankenship, Matthew; Velasco, Pauline; De Felice, Fernanda; Breslin, Paul; Klein, William L.

    2009-01-01

    It now appears likely that soluble oligomers of amyloid-β 1-42 peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimer's disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt Aβ oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble Aβ species, when assayed with both sequence- and conformation-specific Aβ antibodies, indicating changes in oligomer structure. Analysis of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (Aβ-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.

  18. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  19. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    Science.gov (United States)

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

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

    International Nuclear Information System (INIS)

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

    1993-01-01

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

  1. Kinetic Consideration of AFP irma assay

    International Nuclear Information System (INIS)

    Aly, M. A.; Moustafa, K.A.

    2003-01-01

    Alpha-fetoprotein (AFP) is a glycoprotein produced by the yolk sac and later by the fetal liver during pregnancy. When the neural tube is not properly formed, by the fetal liver during pregnancy. When the neural tube is not properly formed, large amounts of AFP pass into the amniotic fluid and reach the mother's blood. During pregnancy, the major interest in AFP determination in maternal serum and amniotic fluid is on the early diagnosis of fetal abnormalities. AFP also used as a tumor marker for hepatocellular carcinoma. There are many different techniques for measuring AFP in blood, but the more accurate one is the immunoassay technique. The kinetics of the interaction between AFP antigen and two matched antibodies, one labeled with radioactive isotope 1 25I (tracer) and the other is unlabelled and attached to a solid support (tube), are studied using the more recently, two sites (sandwich) immunoradiometric assay (IRMA) technique. We present here a method for determining the rate constants, using an advanced computer program (RKY), which based on the nelder-mead optimization principle. The rate constant, at three variable temperatures and three different antigen concentrations, as well as the half time of exchange (t 1/2 ) were calculated

  2. Mouse lung adhesion assay for Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Burns, K A; Freer, J H [Department of Microbiology, Alexander Stone Building, Bearsden, Glasgow, Scotland

    1982-03-01

    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 ..mu..Ci (37 K Bq) of (/sup 14/C)glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation.

  3. Matrix metalloproteinase activity assays: Importance of zymography.

    Science.gov (United States)

    Kupai, K; Szucs, G; Cseh, S; Hajdu, I; Csonka, C; Csont, T; Ferdinandy, P

    2010-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways. Copyright 2010 Elsevier Inc. All rights reserved.

  4. Immunochromatographic assay of cadmium levels in oysters.

    Science.gov (United States)

    Nishi, Kosuke; Kim, In-Hae; Itai, Takaaki; Sugahara, Takuya; Takeyama, Haruko; Ohkawa, Hideo

    2012-08-15

    Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.l mol L(-1) HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Dyes assay for measuring physicochemical parameters.

    Science.gov (United States)

    Moczko, Ewa; Meglinski, Igor V; Bessant, Conrad; Piletsky, Sergey A

    2009-03-15

    A combination of selective fluorescent dyes has been developed for simultaneous quantitative measurements of several physicochemical parameters. The operating principle of the assay is similar to electronic nose and tongue systems, which combine nonspecific or semispecific elements for the determination of diverse analytes and chemometric techniques for multivariate data analysis. The analytical capability of the proposed mixture is engendered by changes in fluorescence signal in response to changes in environment such as pH, temperature, ionic strength, and presence of oxygen. The signal is detected by a three-dimensional spectrofluorimeter, and the acquired data are processed using an artificial neural network (ANN) for multivariate calibration. The fluorescence spectrum of a solution of selected dyes allows discreet reading of emission maxima of all dyes composing the mixture. The variations in peaks intensities caused by environmental changes provide distinctive fluorescence patterns which can be handled in the same way as the signals collected from nose/tongue electrochemical or piezoelectric devices. This optical system opens possibilities for rapid, inexpensive, real-time detection of a multitude of physicochemical parameters and analytes of complex samples.

  6. The synchronous active neutron detection assay system

    International Nuclear Information System (INIS)

    Pickrell, M.M.; Kendall, P.K.

    1994-01-01

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ''lock-in'' amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design

  7. A portable nondestructive assay measurement control system

    International Nuclear Information System (INIS)

    Palmer, M.E.

    1984-01-01

    Portable nondestructive assay (NDA) of plutonium processing hoods, solvent extraction columns, glove boxes, filters, and other items is required for both nuclear materials accountability and criticality control purposes. The Plutonium Finishing Plant has hundreds of such items that require routine portable NDA measurement. Previous recordkeeping of NDA measurements consisted of boxes of papers containing results and notebooks containing notes for each item to be measured. If the notes for any item were lost, new measurement parameters had to be calculated for that item. As a result, subsequent measurements could no longer be directly compared with previous results for that item due to possible changes in measurement parameters. The new portable NDA management system keeps all the necessary information in a computerized data base. Technicians are provided with a computer-generated drawing of each item to be measured, which also contains comments, measurement points, measurement parameters, and a form for filling in the raw data. After the measurements are made, the technician uses the computer to calculate and print out the results

  8. The local lymph node assay (LLNA).

    Science.gov (United States)

    Rovida, Costanza; Ryan, Cindy; Cinelli, Serena; Basketter, David; Dearman, Rebecca; Kimber, Ian

    2012-02-01

    The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA is the only in vivo method that has been subjected to a formal validation process. The original LLNA protocol is based on measurement of the proliferative activity of draining lymph node cells (LNC), as determined by incorporation of radiolabeled thymidine. Several variants to the original LLNA have been developed to eliminate the use of radioactive materials. One such alternative is considered here: the LLNA:BrdU-ELISA method, which uses 5-bromo-2-deoxyuridine (BrdU) in place of radiolabeled thymidine to measure LNC proliferation in draining nodes. © 2012 by John Wiley & Sons, Inc.

  9. Immunoradiometric assay for ferritin in human serum

    International Nuclear Information System (INIS)

    Leyland, M.J.; Ganguli, P.C.; Blower, D.; Delamore, I.W.

    1975-01-01

    A sensitiv specific and precise immunoradiometric assay for ferritin has been developed. Ferritin was measured in the serum of 160 hospital controls, 101 females (118 plus/minus 9 μg/l) and 59 males (189 plus/minus 16 μg/l). This difference was statistically significant. In 28 patients with untreated iron deficiency anemia, serum ferritin concentration (6.1plus/minus 0.7 μg/l) was significantly lower than in the controls, but it was within the normal range in 14 cases of polycythaemia vera treated by repeated phlebotomy. In 4 patients with primary haemachromatosis (2884 plus/minus 56 μg/l), 25 with secondary iron overload states (5702 plus/minus 1235 μg/l) and 8 with haemolytic anaemia (1612 plus/minus 605 μg/l), serum ferritin levels were markedly elevated. In 14 cases of transfusional siderosis there was a highly significant correlation between serum ferritin concentration and units of blood transfused. A circadian rhythmin serum ferritin concentration was observed in 7 healthy subjects. (author)

  10. Phenotypic assays for Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

    2017-10-01

    Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  11. Mouse lung adhesion assay for Bordetella pertussis

    International Nuclear Information System (INIS)

    Burns, K.A.; Freer, J.H.

    1982-01-01

    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 μCi (37 K Bq) of [ 14 C]glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation. (Auth.)

  12. Interpreting clinical assays for histone deacetylase inhibitors

    International Nuclear Information System (INIS)

    Martinet, Nadine; Bertrand, Philippe

    2011-01-01

    As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients

  13. Waste assay measurement integration system user interface

    International Nuclear Information System (INIS)

    Mousseau, K.C.; Hempstead, A.R.; Becker, G.K.

    1995-01-01

    The Waste Assay Measurement Integration System (WAMIS) is being developed to improve confidence in and lower the uncertainty of waste characterization data. There are two major components to the WAMIS: a data access and visualization component and a data interpretation component. The intent of the access and visualization software is to provide simultaneous access to all data sources that describe the contents of any particular container of waste. The visualization software also allows the user to display data at any level from raw to reduced output. Depending on user type, the software displays a menuing hierarchy, related to level of access, that allows the user to observe only those data sources s/he has been authorized to view. Access levels include system administrator, physicist, QA representative, shift operations supervisor, and data entry. Data sources are displayed in separate windows and presently include (1) real-time radiography video, (2) gamma spectra, (3) passive and active neutron, (4) radionuclide mass estimates, (5) total alpha activity (Ci), (6) container attributes, (7) thermal power (w), and (8) mass ratio estimates for americium, plutonium, and uranium isotopes. The data interpretation component is in the early phases of design, but will include artificial intelligence, expert system, and neural network techniques. The system is being developed on a Pentium PC using Microsoft Visual C++. Future generations of WAMIS will be UNIX based and will incorporate more generically radiographic/tomographic, gamma spectroscopic/tomographics, neutron, and prompt gamma measurements

  14. Trace metal assay of uranium silicide fuel

    International Nuclear Information System (INIS)

    Kulkarni, M.J.; Argekar, A.A.; Thulasidas, S.K.; Dhawale, B.A.; Rajeswari, B.; Adya, V.C.; Purohit, P.J.; Neelam, G.; Bangia, T.R.; Page, A.G.; Sastry, M.D.; Iyer, R.H.

    1994-01-01

    A comprehensive trace metal assay of uranium silicide, a fuel for nuclear research reactors that employs low-enrichment uranium, is carried out by atomic spectrometry. Of the list of specification elements, 21 metallic elements are determined by a direct current (dc) arc carrier distillation technique; the rare earths yttrium and zirconium are chemically separated from the major matrix followed by a dc arc/inductively coupled argon plasma (ICP) excitation technique in atomic emission spectrometry (AES); silver is determined by electrothermal atomization-atomic absorption spectrometry (ETA-AAS) without prior chemical separation of the major matrix. Gamma radioactive tracers are used to check the recovery of rare earths during the chemical separation procedure. The detection limits for trace metallics vary in the 0.1- to 40-ppm range. The precision of the determinations as evaluated from the analysis of the synthetic sample with intermediate range analyte concentration is better than 25% relative standard deviation (RSD) for most of the elements employing dc arc-AES, while that for silver determination by ETS-AAS is 10% RSD. The precision of the determinations for four crucially important rare earths by ICP-AES is better than 3% RSD

  15. Reduction of bias in neutron multiplicity assay using a weighted point model

    Energy Technology Data Exchange (ETDEWEB)

    Geist, W. H. (William H.); Krick, M. S. (Merlyn S.); Mayo, D. R. (Douglas R.)

    2004-01-01

    Accurate assay of most common plutonium samples was the development goal for the nondestructive assay technique of neutron multiplicity counting. Over the past 20 years the technique has been proven for relatively pure oxides and small metal items. Unfortunately, the technique results in large biases when assaying large metal items. Limiting assumptions, such as unifoh multiplication, in the point model used to derive the multiplicity equations causes these biases for large dense items. A weighted point model has been developed to overcome some of the limitations in the standard point model. Weighting factors are detemiined from Monte Carlo calculations using the MCNPX code. Monte Carlo calculations give the dependence of the weighting factors on sample mass and geometry, and simulated assays using Monte Carlo give the theoretical accuracy of the weighted-point-model assay. Measured multiplicity data evaluated with both the standard and weighted point models are compared to reference values to give the experimental accuracy of the assay. Initial results show significant promise for the weighted point model in reducing or eliminating biases in the neutron multiplicity assay of metal items. The negative biases observed in the assay of plutonium metal samples are caused by variations in the neutron multiplication for neutrons originating in various locations in the sample. The bias depends on the mass and shape of the sample and depends on the amount and energy distribution of the ({alpha},n) neutrons in the sample. When the standard point model is used, this variable-multiplication bias overestimates the multiplication and alpha values of the sample, and underestimates the plutonium mass. The weighted point model potentially can provide assay accuracy of {approx}2% (1 {sigma}) for cylindrical plutonium metal samples < 4 kg with {alpha} < 1 without knowing the exact shape of the samples, provided that the ({alpha},n) source is uniformly distributed throughout the

  16. Evaluation of automated assays for immunoglobulin G, M, and A measurements in dog and cat serum.

    Science.gov (United States)

    Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Caldin, Marco; Tecles, Fernando; Ceron, Jose J

    2013-09-01

    Measurements of immunoglobulins (Igs) in companion animals can be useful to detect deficiencies of the humoral immune system, that can be associated with opportunistic or chronic infections, or other immune-mediated disorders including B-cell neoplasms. The purpose of this study was to evaluate commercially available automated immunoturbidimetric assays designed for human IgG, M, and A measurements in canine and feline serum using species-specific calibrators. Canine and feline serum samples with different IgG, M, and A concentrations were used for the analytical validation of the assays. Intra- and inter-assay precision, linearity under dilution, spiking recovery, and limit of detection were determined. In addition, effects of lipemia, hemolysis, and bilirubinemia were evaluated. Finally, Ig concentrations were determined in small groups of diseased dogs and cats, and compared with healthy groups. Spiking recovery and linearity under dilution tests showed that the assays measured Igs in canine and feline serum samples precisely and accurately. Intra- and inter-assay imprecisions were lower than 15% in all cases. Significantly higher IgG, IgM, and IgA levels were observed in dogs with leishmaniasis, while dogs with pyometra showed a statistically significant increase in IgM and IgA concentrations in comparison with healthy dogs. Significantly higher IgG and IgM levels were observed in FIV-infected cats compared with healthy ones. The automated human Ig assays showed adequate precision and accuracy with serum samples from dogs and cats. Also, they were able to discriminate different concentrations of Igs in healthy and diseased animals. © 2013 American Society for Veterinary Clinical Pathology.

  17. Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

    Science.gov (United States)

    Hughes, Christine M; Newman, Frances K; Davidson, Whitni B; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Yan, Lihan; Frey, Sharon E; Belshe, Robert B; Karem, Kevin L; Damon, Inger K

    2012-07-01

    Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

  18. Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-05-01

    Occupational exposure to antineoplastic drugs is associated with genotoxic effects, although comet assay analyzed parameters were higher in exposed comparing with controls, were not significant. Also the study of the susceptibility biomarkers did not show statistical significant differences, the small size of our sample hampered the finding of a possible association, let alone a causality relationship.

  19. Evaluation of a MTT assay in measurement of radiosensitizing effect

    International Nuclear Information System (INIS)

    Higuchi, Keiko; Mitsuhashi, Norio; Saitoh, Jun-ichi; Maebayashi, Katsuya; Sakurai, Hideyuki; Akimoto, Tetsuo; Niibe, Hideo

    1999-01-01

    The usefulness of a MTT assay by measuring the radiosensitizing effect of caffeine on rat yolk sac tumor cell line with a mutant-type p53 in vitro was evaluated. A rat yolk sac tumor cell line with a mutant-type p53, NMT-1R, was used in this study. The radiosensitivity of NMT-1R with or without caffeine was measured with a MTT assay. The results were compared with those by a clonogenic assay. Caffeine at a concentration of 2.0 mM which released radiation-induced G 2 block demonstrated a radiosensitizing effect, but caffeine at a concentration of 0.5 mM did not. The radiosensitizing effect of caffeine measured by a MTT assay correlated with that measured by a clonogenic assay. A MTT assay was useful to measure radiosensitivity and/or a radiosensitizing effect in vitro. (author)

  20. 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.

  1. Latest data shows long-term security of uranium supply

    International Nuclear Information System (INIS)

    2010-01-01

    Full text: According to Uranium 2009: Resources, Production and Demand just published by the OECD Nuclear En ergy Agency (NEA) and the International Atomic Energy Agency (IAEA), uranium resources, production and demand are all on the rise. Exploration efforts have increased recently in line with the expected expansion of nuclear energy in the coming years. Total identified resources have grown but so too have costs of production. Worldwide exploration and mine development expenditures have more than doubled since the publication of the previous edition, Uranium 2007: Resources, Production and Demand. These expenditures have increased despite declining uranium market prices since mid- 2007. The uranium resources presented in this edition, reflecting the situation as of 1 January 2009, show that total identified resources amounted to 6 306 300 tU, an increase of about 15% compared to 2007, including those reported in the high-cost category (< USD 260/kgU or < USD 100/lbU O), reintroduced for the first time since the 1980s. This high-cost 3 8 category was used in the 2009 edition in response to the generally increased market prices for uranium in recent years, despite the decline since mid-2007, expectations of increasing demand as new nuclear power plants are being planned and built, and increased mining costs. Although total identified resources have increased overall, there has been a significant reduction in lower-cost resources owing to increased mining costs. At 2008 rates of consumption, total identified resources are sufficient for over 100 years of supply. The recognition by an increasing number of governments that nuclear power can produce competitively priced, baseload electricity that is essentially free of greenhouse gas emissions, coupled with the role that nuclear can play in enhancing security of energy supply, increases the prospects for growth in nuclear generating capacity, although the magnitude of that growth remains to be determined. According to

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

    Science.gov (United States)

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

    2005-07-01

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

  3. Assessing the toxicity of sediments using the medaka embryo-larval assay and 2 other bioassays.

    Science.gov (United States)

    Barhoumi, Badreddine; Clérandeau, Christelle; Landi, Laure; Pichon, Anaïk; Le Bihanic, Florane; Poirier, Dominique; Anschutz, Pierre; Budzinski, Hélène; Driss, Mohamed Ridha; Cachot, Jérôme

    2016-09-01

    Sediments are sinks for aquatic pollutants, and analyzing toxicity in such complex matrices is still challenging. To evaluate the toxicity of bioavailable pollutants accumulated in sediments from the Bizerte lagoon (Tunisia), a novel assay, the medaka embryo-larval assay by sediment contact, was applied. Japanese medaka (Oryzias latipes) embryos were incubated in direct contact with sediment samples up to hatching. Lethal and sublethal adverse effects were recorded in embryos and larvae up to 20 d postfertilization. Results from medaka embryo-larval assay were compared with cytotoxicity (Microtox®), genotoxicity (SOS chromotest), and pollutant content of sediments. The results highlight differences in the contamination profile and toxicity pattern between the different studied sediments. A significant correlation was shown between medaka embryo-larval assay by sediment contact and SOS chromotest responses and concentrations of most organic pollutants studied. No correlation was shown between pollutant levels and Microtox. According to the number of sediment samples detected as toxic, medaka embryo-larval assay by sediment contact was more sensitive than Microtox, which in turn was more sensitive than the SOS chromotest; and medaka embryo-larval assay by sediment contact allowed sediment toxicity assessment of moderately polluted sediments without pollutant extraction and using an ecologically realistic exposure scenario. Although medaka embryo-larval assay by sediment contact should be tested on a larger sample set, the results show that it is sensitive and convenient enough to monitor the toxicity of natural sediments. Environ Toxicol Chem 2016;35:2270-2280. © 2016 SETAC. © 2016 SETAC.

  4. Slight hypercalcemia is not associated with positive responses in the Comet Assay in male rat liver.

    Science.gov (United States)

    Thiel, Anette; Hamel, Annie; Schaefer, Katrien; Cardoso, Renato; Beilstein, Paul

    2017-08-01

    Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines. The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety. Copyright © 2017 DSM Nutritional Products AG. Published by Elsevier B.V. All rights reserved.

  5. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

    Directory of Open Access Journals (Sweden)

    Joanna Jońca

    Full Text Available Butyrylcholinesterase (BChE activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

  6. Verification of the harmonization of human epididymis protein 4 assays.

    Science.gov (United States)

    Ferraro, Simona; Borille, Simona; Carnevale, Assunta; Frusciante, Erika; Bassani, Niccolò; Panteghini, Mauro

    2016-10-01

    Serum human epididymis protein 4 (HE4) has gained relevance as an ovarian cancer (OC) biomarker and new automated methods have replaced the first released manual EIA by tracing results to it. We verified agreement and bias of automated methods vs. EIA as well as possible effects on patients' management. One hundred and fifteen serum samples were measured by Abbott Architect i2000, Fujirebio Lumipulse G1200, Roche Modular E170, and Fujirebio EIA. Passing-Bablok regression was used to compare automated assays to EIA and agreement between methods was estimated by Lin's concordance correlation coefficient (CCC). The bias vs. EIA was estimated and compared to specifications derived from HE4 biological variation. Median (25th-75th percentiles) HE4 concentrations (pmol/L) were 84.5 (60.1-148.8) for EIA, 82.7 (50.3-153.9) for Abbott, 89.1 (55.2-154.9) for Roche, and 112.2 (67.8-194.2) for Fujirebio. Estimated regressions and agreements (95% confidence interval) were: Abbott=1.01(0.98-1.03) EIA-4.8(-7.5/-2.6), CCC=0.99(0.99-1.00); Roche=0.91(0.89-0.93) EIA+5.7(4.2/8.0), CCC=0.98(0.98-0.99); Fujirebio=1.20(1.17-1.24) EIA+ 2.4(-0.6/4.9), CCC=0.97(0.96-0.98). The average bias vs. EIA resulted within the desirable goal for Abbott [-3.3% (-6.1/-0.5)] and Roche [-0.2% (-3.0/2.5)]. However, while for Abbott the bias was constant and acceptable along the measurement concentration range, Roche bias increased up to -28% for HE4 values >250 pmol/L. Lumipulse showed a markedly positive bias [25.3% (21.8/28.8)]. Abbott and Roche assays exhibited a good comparability in the range of HE4 values around the previously recommended 140 pmol/L cut-off. For patient monitoring, however, the assay used for determining serial HE4 must not be changed as results from different systems in lower and higher concentration ranges can markedly differ.

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

    International Nuclear Information System (INIS)

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

    1975-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1975-02-01

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

  9. Absolute nuclear material assay using count distribution (LAMBDA) space

    Science.gov (United States)

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

    2012-06-05

    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.

  10. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

    Science.gov (United States)

    Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. © The Author 2014. Published by

  11. Designs, formats and applications of lateral flow assay: A literature review

    Directory of Open Access Journals (Sweden)

    Muhammad Sajid

    2015-11-01

    Full Text Available This manuscript provides a brief overview of latest research involving the use of lateral flow assay for qualitative and quantitative analysis in different areas. The excellent features and versatility of detection formats make these strips an ideal choice for point of care applications. We outline and critically discuss detection formats, molecular recognition probes, labels, and detection systems used in lateral flow assay. Applications in different fields along with selected examples from the literature have been included to show analytical performance of these devices. At the end, we summarize accomplishments, weaknesses and future challenges in the area of lateral flow strips.

  12. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

  13. Detection of irradiation treatment of foods using DNA 'comet assay'

    International Nuclear Information System (INIS)

    Khan, Hasan M.; Delincee, Henry

    1998-01-01

    Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results

  14. Computer-determined assay time based on preset precision

    International Nuclear Information System (INIS)

    Foster, L.A.; Hagan, R.; Martin, E.R.; Wachter, J.R.; Bonner, C.A.; Malcom, J.E.

    1994-01-01

    Most current assay systems for special nuclear materials (SNM) operate on the principle of a fixed assay time which provides acceptable measurement precision without sacrificing the required throughput of the instrument. Waste items to be assayed for SNM content can contain a wide range of nuclear material. Counting all items for the same preset assay time results in a wide range of measurement precision and wastes time at the upper end of the calibration range. A short time sample taken at the beginning of the assay could optimize the analysis time on the basis of the required measurement precision. To illustrate the technique of automatically determining the assay time, measurements were made with a segmented gamma scanner at the Plutonium Facility of Los Alamos National Laboratory with the assay time for each segment determined by counting statistics in that segment. Segments with very little SNM were quickly determined to be below the lower limit of the measurement range and the measurement was stopped. Segments with significant SNM were optimally assays to the preset precision. With this method the total assay time for each item is determined by the desired preset precision. This report describes the precision-based algorithm and presents the results of measurements made to test its validity

  15. Detection of radiation-induced apoptosis using the comet assay

    International Nuclear Information System (INIS)

    Wada, Seiichi; Kobayashi, Yasuhiko; Funayama, Tomoo; Yamamoto, Kazuo; Khoa, Tran Van; Natsuhori, Masahiro; Ito, Nobuhiko

    2003-01-01

    The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)

  16. Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

    Science.gov (United States)

    Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

    2014-03-26

    A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Radiometric microbiologic assay for the biologically active forms of niacin

    International Nuclear Information System (INIS)

    Kertcher, J.A.; Guilarte, T.R.; Chen, M.F.; Rider, A.A.; McIntyre, P.A.

    1979-01-01

    A radiometric microbiologic assay has been developed for the determination of niacin in biologic fluids. Lactobacillus plantarum produced 14 CO 2 from L-[U- 14 C] malic acid in quantities proportional to the amount of niacin present. The assay is specific for the biologically active forms of niacin in humans. Thirty normal hemolysates were analyzed and the values ranged from 13.0 to 17.8 μg niacin/ml RBC (mean = 15.27 +- 1.33 s.d.). Good recovery and reproducibility studies were obtained with this assay. On thirty blood samples, correlation was excellent between the radiometric and the conventional turbidimetric assays

  18. Radioenzymatic assay of DOPA (3,4-dihydroxyphenylalanine)

    International Nuclear Information System (INIS)

    Johnson, G.A.; Gren, J.M.; Kupiecki, R.

    1978-01-01

    We modified the single-isotope radioenzymatic assay for catecholamines [Life Sci. 21, 625(1977)] to assay 3,4-dihydroxyphenylalanine (DOPA). DOPA decarboxylase is used to convert DOPA to dopamine, which concurrently is converted to [ 3 H]-3-O-methyldopamine in the presence of catechol-O-methyltransferase and [methyl- 3 H]-S-adenosylmethionine and assayed radioenzymatically. For assay of plasma DOPA, 50 μl of untreated plasma is added directly into the incubation mixture. A duplicate mixture containing an internal standard requires a second 50-μl aliquot of plasma. Because the assay measures both DOPA and endogenous dopamine, two additional aliquots of plasma must be assayed for dopamine in the absence of the decarboxylase by the differential assay; DOPA is estimated by difference. The assay is sensitive to 25 pg (500 ng/liter of plasma). Analysis of DOPA (DOPA plus dopamine) and the concurrent differential assay of catecholamines in at least 10 samples can be done in a single working day. Plasma DOPA concentrations for 42 normotensive adults were 1430 +- 19 ng/liter (mean +- SEM). In contrast, dopamine concentrations for these same subjects averaged 23 +- 20 ng/liter. Values for the 24 women subjects (1510 +- 62 ng/liter) significantly (P = 0.04) exceeded those for the men

  19. A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

    Directory of Open Access Journals (Sweden)

    Anli Zhang

    2015-06-01

    Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

  20. Feasibility of nondestructive assay measurements in uranium enrichment plants

    Energy Technology Data Exchange (ETDEWEB)

    Walton, R.B.

    1978-04-01

    Applications of nondestructive assay methods to measurement problems in uranium enrichment facilities are reviewed. The results of a number of test and evaluation projects that were performed over the last decade at ORGDP and Portsmouth are presented. Measurements of the residual holdup in the top enrichment portion of the shut-down K-25 cascade were made with portable neutron and gamma-ray detectors, and inventory estimates based on these data were in good agreement with ORGDP estimates. In the operating cascade, the tests showed that portable NaI detectors are effective for monitoring NaF and alumina media for gaseous effluent traps and that gas phase enrichments and inventories, as well as large deposits of uranium, can be detected with portable neutron and gamma-ray instrumentation. A wide variety of scrap and waste materials, including barrier and compressor blades, incinerator ash and trapping media, and miscellaneous waste, were measured using passive gamma-ray and neutron methods and 14-MeV neutron interrogation. Methods developed for rapid verification of UF/sub 6/ in shipping containers with portable neutron and gamma-ray instruments are now used routinely by safeguards inspectors. Passive assay methods can also be used to measure continuously the enrichments of /sup 235/U and /sup 234/U in the UF/sub 6/ product and tails withdrawals of a gaseous diffusion plant. A system that was developed and installed in the extended-range product withdrawal station of the Portsmouth facility measures enrichment with a relative accuracy of 0.5%. A stand-alone neutron detector has also been successfully evaluated for the measurement of the isotopic abundance of /sup 234/U in UF/sub 6/ in sample cylinders, an application of potential importance to Minor Isotope Safeguards Technology. Recommendations are made on the role of NDA measurements for enrichment plant safeguards, including additional tests and evaluations that may be needed, particularly for advanced uranium