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Sample records for replicon assays ic50

  1. ICECAP: an integrated, general-purpose, automation-assisted IC50/EC50 assay platform.

    Science.gov (United States)

    Li, Ming; Chou, Judy; King, Kristopher W; Jing, Jing; Wei, Dong; Yang, Liyu

    2015-02-01

    IC50 and EC50 values are commonly used to evaluate drug potency. Mass spectrometry (MS)-centric bioanalytical and biomarker labs are now conducting IC50/EC50 assays, which, if done manually, are tedious and error-prone. Existing bioanalytical sample preparation automation systems cannot meet IC50/EC50 assay throughput demand. A general-purpose, automation-assisted IC50/EC50 assay platform was developed to automate the calculations of spiking solutions and the matrix solutions preparation scheme, the actual spiking and matrix solutions preparations, as well as the flexible sample extraction procedures after incubation. In addition, the platform also automates the data extraction, nonlinear regression curve fitting, computation of IC50/EC50 values, graphing, and reporting. The automation-assisted IC50/EC50 assay platform can process the whole class of assays of varying assay conditions. In each run, the system can handle up to 32 compounds and up to 10 concentration levels per compound, and it greatly improves IC50/EC50 assay experimental productivity and data processing efficiency. © 2014 Society for Laboratory Automation and Screening.

  2. Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

    International Nuclear Information System (INIS)

    Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

    2015-01-01

    Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC 50 values in WST-1 assays. The IC 50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles

  3. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    International Nuclear Information System (INIS)

    Filone, Claire Marie; Heise, Mark; Doms, Robert W.; Bertolotti-Ciarlet, Andrea

    2006-01-01

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion

  4. Zika Virus Replicons for Drug Discovery

    Directory of Open Access Journals (Sweden)

    Xuping Xie

    2016-10-01

    Full Text Available The current epidemic of Zika virus (ZIKV has underscored the urgency to establish experimental systems for studying viral replication and pathogenesis, and countermeasure development. Here we report two ZIKV replicon systems: a luciferase replicon that can differentiate between viral translation and RNA synthesis; and a stable luciferase replicon carrying cell line that can be used to screen and characterize inhibitors of viral replication. The transient replicon was used to evaluate the effect of an NS5 polymerase mutation on viral RNA synthesis and to analyze a known ZIKV inhibitor. The replicon cell line was developed into a 96-well format for antiviral testing. Compare with virus infection-based assay, the replicon cell line allows antiviral screening without using infectious virus. Collectively, the replicon systems have provided critical tools for both basic and translational research.

  5. Nonlinear concentration gradients regulated by the width of channels for observation of half maximal inhibitory concentration (IC50) of transporter proteins.

    Science.gov (United States)

    Abe, Yuta; Kamiya, Koki; Osaki, Toshihisa; Sasaki, Hirotaka; Kawano, Ryuji; Miki, Norihisa; Takeuchi, Shoji

    2015-08-21

    This paper describes a simple microfluidic device that can generate nonlinear concentration gradients. We changed the "width" of channels that can drastically shorten the total microfluidic channel length and simplify the microfluidic network design rather than the "length" of channels. The logarithmic concentration gradients generated by the device were in good agreement with those obtained by simulation. Using this device, we evaluated a probable IC50 value of the ABC transporter proteins by the competitive transport assays at five different logarithmic concentrations. This probable IC50 value was in good agreement with an IC50 value (0.92 μM) obtained at the diluted concentrations of seven points.

  6. A novel application of t-statistics to objectively assess the quality of IC50 fits for P-glycoprotein and other transporters.

    Science.gov (United States)

    O'Connor, Michael; Lee, Caroline; Ellens, Harma; Bentz, Joe

    2015-02-01

    Current USFDA and EMA guidance for drug transporter interactions is dependent on IC50 measurements as these are utilized in determining whether a clinical interaction study is warranted. It is therefore important not only to standardize transport inhibition assay systems but also to develop uniform statistical criteria with associated probability statements for generation of robust IC50 values, which can be easily adopted across the industry. The current work provides a quantitative examination of critical factors affecting the quality of IC50 fits for P-gp inhibition through simulations of perfect data with randomly added error as commonly observed in the large data set collected by the P-gp IC50 initiative. The types of errors simulated were (1) variability in replicate measures of transport activity; (2) transformations of error-contaminated transport activity data prior to IC50 fitting (such as performed when determining an IC50 for inhibition of P-gp based on efflux ratio); and (3) the lack of well defined "no inhibition" and "complete inhibition" plateaus. The effect of the algorithm used in fitting the inhibition curve (e.g., two or three parameter fits) was also investigated. These simulations provide strong quantitative support for the recommendations provided in Bentz et al. (2013) for the determination of IC50 values for P-gp and demonstrate the adverse effect of data transformation prior to fitting. Furthermore, the simulations validate uniform statistical criteria for robust IC50 fits in general, which can be easily implemented across the industry. A calibration of the t-statistic is provided through calculation of confidence intervals associated with the t-statistic.

  7. Chromosomal replicons of higher plants

    International Nuclear Information System (INIS)

    Van't Hof, J.

    1987-01-01

    This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs

  8. Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug- Drug Interaction Risk Assessment Decision Criterias

    NARCIS (Netherlands)

    Bentz, J.; O'Connor, M.P.; Bednarczyk, D.; Coleman, J.; Lee, C.; Palm, J.; Pak, Y.A.; Perloff, E.S.; Reyner, E.; Balimane, P.; Brännström, M.; Chu, X.; Funk, C.; Guo, A.; Hanna, I.; Herédi-Szabó, K.; Hillgren, K.; Li, L.; Hollnack-Pusch, E.; Jamei, M.; Lin, X.; Mason, A.K.; Neuhoff, S.; Patel, A.; Podila, L.; Plise, E.; Rajaraman, G.; Salphati, L.; Sands, E.; Taub, M.E.; Taur, J.-S.; Weitz, D.; Wortelboer, H.M.; Xia, C.Q.; Xiao, G.; Yabut, J.; Yamagata, T.; Zhang, L.; Ellens, H.

    2013-01-01

    A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC 50 determinations. Each laboratory followed its in-house protocol to determine in vitro

  9. In vitro neuraminidase inhibitory concentration (IC50) of four neuraminidase inhibitors in the Japanese 2016-17 season: Comparison with the 2010-11 to 2015-16 seasons.

    Science.gov (United States)

    Ikematsu, Hideyuki; Kawai, Naoki; Iwaki, Norio; Kashiwagi, Seizaburo; Ishikawa, Yusuke; Yamaguchi, Hiroki; Shiosakai, Kazuhito

    2018-05-11

    To assess the extent of susceptibility to the four most commonly used neuraminidase inhibitors (NAIs) in the viruses epidemic in the 2016-17 Japanese influenza season, we measured the 50% inhibitory concentration (IC 50 ) of these NAIs for influenza virus isolates from patients and compared them with the results from the 2010-11 to 2015-16 seasons. Viral isolation was done with specimens obtained prior to treatment, and the type and subtype was determined by RT-PCR using type- and subtype-specific primers. The IC 50 was determined by a neuraminidase inhibition assay using a fluorescent substrate. A total of 276 virus isolates, 6 A (H1N1)pdm09 (2.2%), 249 A (H3N2) (90.2%), and 21 B (7.6%), had the IC 50 measured for the four NAIs. B isolates included 11 (52.4%), 9 (42.9%), and one (4.8%) of the Victoria, Yamagata, and undetermined strains, respectively. No A (H1N1)pdm09 with highly reduced sensitivity for oseltamivir was found in the 2016-17 season. No isolate with highly reduced sensitivity to the four NAIs have been found for A (H3N2) or B from the 2010-11 to 2016-17 seasons. No significant trend of increase or decrease was found in the geometric mean IC 50 s of the four NAIs during the seven studied seasons. These results indicate that the sensitivity to the four commonly used NAIs has been maintained and that any change in the effectiveness of these NAIs would be minute. Common usage of NAIs for patient treatment has not been a driving force in the selection of NAI resistant viruses. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  10. Validation and Clinical Utility of the hERG IC50:Cmax Ratio to Determine the Risk of Drug-Induced Torsades de Pointes: A Meta-Analysis.

    Science.gov (United States)

    Lehmann, David F; Eggleston, William D; Wang, Dongliang

    2018-03-01

    Use of the QT interval corrected for heart rate (QTc) on the electrocardiogram (ECG) to predict torsades de pointes (TdP) risk from culprit drugs is neither sensitive nor specific. The ratio of the half-maximum inhibitory concentration of the hERG channel (hERG IC50) to the peak serum concentration of unbound drug (C max ) is used during drug development to screen out chemical entities likely to cause TdP. To validate the use of the hERG IC50:C max ratio to predict TdP risk from a culprit drug by its correlation with TdP incidence. Medline (between 1966 and March 2017) was accessed for hERG IC50 and C max values from the antihistamine, fluoroquinolone, and antipsychotic classes to identify cases of drug-induced TdP. Exposure to a culprit drug was estimated from annual revenues reported by the manufacturer. Inclusion criteria for TdP cases were provision of an ECG tracing that demonstrated QTc prolongation with TdP and normal serum values of potassium, calcium, and magnesium. Cases reported in patients with a prior rhythm disturbance and those involving a drug interaction were excluded. The Meta-Analysis of Observational Studies in Epidemiology checklist was used for epidemiological data extraction by two authors. Negligible risk drugs were defined by an hERG IC50:C max ratio that correlated with less than a 5% chance of one TdP event for every 100 million exposures (relative risk [RR] 1.0). The hERG IC50:C max ratio correlated with TdP risk (0.312; 95% confidence interval 0.205-0.476, pratio of 80 (RR 1.0). The RR from olanzapine is on par with loratadine; ziprasidone is comparable with ciprofloxacin. Drugs with an RR greater than 50 include astemizole, risperidone, haloperidol, and thioridazine. The hERG IC50:C max ratio was correlated with TdP incidence for culprit drugs. This validation provides support for the potential use of the hERG IC50:C max ratio for clinical decision making in instances of drug selection where TdP risk is a concern. © 2018

  11. Characterization of cell lines stably transfected with rubella virus replicons

    International Nuclear Information System (INIS)

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-01-01

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  12. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  13. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  14. Construction and characterization of poliovirus subgenomic replicons.

    Science.gov (United States)

    Kaplan, G; Racaniello, V R

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid

  15. Construction and characterization of poliovirus subgenomic replicons

    International Nuclear Information System (INIS)

    Kaplan, G.; Racaniello, V.R.

    1988-01-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of [ 35 S-labeled] viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A pro , 2C, and 3D pol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs

  16. Engineering a CTL-Tailored Replicon RNA Vaccine against PRRSV

    DEFF Research Database (Denmark)

    Welner, Simon; Werder, Simea; Nielsen, Morten

    The development of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) has been hampered by the high mutation rate and the multiple immunoevasive strategies of the virus. With the overall aim of designing a broad coverage vaccine that induces an effective CTL response aga...... will be available for IVIS. This study exemplifies how bioinformatics epitope prediction, recombinant SLA molecules and RNA virus replicon design can be used to engineer a replicating non-propagating vaccine tailored to deliver conserved and immunogenic CTL epitopes....... against PRRSV, we have used a bioinformatics approach to identify common PRRSV type 2 epitopes predicted to react broadly with predominant swine MHC (SLA) alleles. All possible 9- and 10-mer peptides derived from 104 wild-type strains were analyzed in silico for their predicted binding affinity to 3...... cloned into a classical swine fever virus (CSFV)-derived replicon vector. Virus replicon particles (VRP) were rescued by transfection of a complementing cell line with replicon RNA. Polyepitope expression and subsequent proteasomal degradation was confirmed indirectly by increased FLAG-tagged protein...

  17. Alphavirus replicon approach to promoterless analysis of IRES elements.

    Science.gov (United States)

    Kamrud, K I; Custer, M; Dudek, J M; Owens, G; Alterson, K D; Lee, J S; Groebner, J L; Smith, J F

    2007-04-10

    Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

  18. Hepatitis C virus replicons: dinosaurs still in business?

    Science.gov (United States)

    Woerz, I; Lohmann, V; Bartenschlager, R

    2009-01-01

    Since the molecular cloning of the hepatitis C virus (HCV) genome for the first time in 1989, there has been tremendous progress in our understanding of the multiple facets of the replication cycle of this virus. Key to this progress has been the development of systems to propagate the virus in cell culture, which turned out to be a notoriously difficult task. A major breakthrough has been the construction of subgenomic replicons that self-amplify in cultured human hepatoma cells. These RNAs recapitulate the intracellular steps of the HCV replication cycle and have been instrumental to decipher details of the RNA amplification steps including the identification of key host cell factors. However, reproduction of the complete viral replication cycle only became possible with the advent of a particular molecular HCV clone designated JFH-1 that replicates to very high levels and supports the production of infectious virus particles. The availability of this new culture system raises the question, whether the use of replicons is still justified. In this review, we will discuss the pros and cons of both systems.

  19. Dual Incorporation of the in vitro Data (IC50) and in vivo (Cmax) Data for the Prediction of Area Under the Curve (AUC) for Statins using Regression Models Developed for Either Pravastatin or Simvastatin.

    Science.gov (United States)

    Srinivas, N R

    2016-08-01

    Linear regression models utilizing a single time point (Cmax) has been reported for pravastatin and simvastatin. A new model was developed for the prediction of AUC of statins that utilized the slopes of the above 2 models, with pharmacokinetic (Cmax) and a pharmacodynamic (IC50 value) components for the statins. The prediction of AUCs for various statins (pravastatin, atorvastatin, simvastatin and rosuvastatin) was carried out using the newly developed dual pharmacokinetic and pharmacodynamic model. Generally, the AUC predictions were contained within 0.5 to 2-fold difference of the observed AUC suggesting utility of the new models. The root mean square error predictions wereAUC for statins. Such a new concept as described in the work may have utility in both drug discovery and development stages. © Georg Thieme Verlag KG Stuttgart · New York.

  20. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    Science.gov (United States)

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells

    DEFF Research Database (Denmark)

    Saeed, Mohsan; Scheel, Troels K H; Gottwein, Judith M

    2012-01-01

    culture adaptive mutations originally reported for genotype 1b replicons. RNA replication was confirmed by quantitative reverse transcription-PCR and detection of viral protein. Sequencing of multiple independent replicon clones revealed the presence of additional nonsynonymous mutations. Interestingly......, all potentially adaptive mutations mapped to the NS3 protein. These mutations, when introduced back into original constructs, substantially increased colony formation efficiency. To make these replicons useful for high-throughput screening and evaluation of antiviral compounds, they were modified...

  2. Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1978-01-01

    A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [ 3 H]thymidine ([ 3 H]TdR) and bromodeoxyuridine (BrDUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, L/sub obs/, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of x rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions L/sub obs/ continues to increase only until operating replicons have completed their replication. This value for L/sub obs/ then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before x irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 μM, whereas for Chinese hamster ovary cells, the average replicon size is about 42 μM

  3. Analysis of classical swine fever virus RNA replication determinants using replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Fahnøe, Ulrik; Gullberg, Maria

    2013-01-01

    Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome (BAC), was modified by targeted...... recombination within E. coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate......), as well as by detection of the CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for the replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain...

  4. Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

    Science.gov (United States)

    Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

    2015-01-01

    Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106

  5. Robustness encoded across essential and accessory replicons of the ecologically versatile bacterium Sinorhizobium meliloti

    Science.gov (United States)

    Walker, Graham C.; Finan, Turlough M.; Mengoni, Alessio; Griffitts, Joel S.

    2018-01-01

    Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone. PMID:29672509

  6. Construction of self-replicating subgenomic West Nile virus replicons for screening antiviral compounds.

    Science.gov (United States)

    Alcaraz-Estrada, Sofia L; Reichert, Erin Donohue; Padmanabhan, Radhakrishnan

    2013-01-01

    Mosquito-borne flavivirus RNA genomes encode one long open reading frame flanking 5'- and 3'-untranslated regions (5'- and 3'-UTRs) which contain cis-acting RNA elements playing important roles for viral RNA translation and replication. The viral RNA encodes a single polyprotein, which is processed into three structural proteins and seven nonstructural (NS) proteins. The regions coding for the seven NS proteins are sufficient for replication of the RNA. The sequences encoding the structural genes can be deleted except for two short regions. The first one encompasses 32 amino acid (aa) residues from the N-terminal coding sequence of capsid (C) and the second, 27 aa region from the C-terminus of envelope (E) protein. The deleted region can be substituted with a gene coding for a readily quantifiable reporter to give rise to a subgenomic reporter replicon. Replicons containing a variety of reporter genes and marker genes for construction of stable mammalian cell lines are valuable reagents for studying the effects of mutations in translation and/or replication in isolation from processes like the entry and assembly of the virus particles. Here we describe the construction of two West Nile virus (WNV) replicons by overlap extension PCR and standard recombinant DNA techniques. One has a Renilla luciferase (Rluc) reporter gene followed by an internal ribosome entry site (element) for cap-independent translation of the open reading frame encompassing the carboxy-terminal sequence of E to NS5. The second replicon has in tandem the Rluc gene, foot and mouth disease virus 2A, and neomycin phosphotransferase gene that allows establishment of a stable mammalian cell line expressing the Rluc reporter in the presence of the neomycin analog, G418. The stable replicon-expressing Vero cell line has been used for cell-based screening and determination of EC50 values for antiviral compounds that inhibited WNV replication.

  7. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    Science.gov (United States)

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-05-01

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

    Science.gov (United States)

    Zhang, Xiuren; Mason, Hugh

    2006-02-05

    A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

  9. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons

    International Nuclear Information System (INIS)

    Jackson, Cheryl A.; Messinger, Jeff; Palmer, Matthew T.; Peduzzi, Jean D.; Morrow, Casey D.

    2003-01-01

    The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or

  10. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

    Science.gov (United States)

    A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

  11. Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

    Science.gov (United States)

    Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Tian, Chang Fu; Chen, Wen Xin

    2014-02-01

    In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

  12. Establishment and mitotic stability of an extra-chromosomal mammalian replicon

    Directory of Open Access Journals (Sweden)

    Jackson Dean A

    2007-08-01

    Full Text Available Abstract Background Basic functions of the eukaryotic nucleus, like transcription and replication, are regulated in a hierarchic fashion. It is assumed that epigenetic factors influence the efficiency and precision of these processes. In order to uncouple local and long-range epigenetic features we used an extra-chromosomal replicon to study the requirements for replication and segregation and compared its behavior to that of its integrated counterpart. Results The autonomous replicon replicates in all eukaryotic cells and is stably maintained in the absence of selection but, as other extra-chromosomal replicons, its establishment is very inefficient. We now show that following establishment the vector is stably associated with nuclear compartments involved in gene expression and chromosomal domains that replicate at the onset of S-phase. While the vector stays autonomous, its association with these compartments ensures the efficiency of replication and mitotic segregation in proliferating cells. Conclusion Using this novel minimal model system we demonstrate that relevant functions of the eukaryotic nucleus are strongly influenced by higher nuclear architecture. Furthermore our findings have relevance for the rational design of episomal vectors to be used for genetic modification of cells: in order to improve such constructs with respect to efficiency elements have to be identified which ensure that such constructs reach regions of the nucleus favorable for replication and transcription.

  13. The Anti-trichomonas Activity of Berberine by Applying the Bromocresol Purple Colorimetric Spectroscopic Assay in vitro%溴甲酚紫法测定黄连素的体外抗阴道毛滴虫作用

    Institute of Scientific and Technical Information of China (English)

    陈熙; 周本江

    2013-01-01

    目的 溴甲酚紫法体外测试黄连素的抗阴道毛滴虫活性.方法 设计实验组、已知疗效对照组和阴性对照组,溴甲酚紫比色法体外测定药物作用于阴道毛滴虫后不同时间、不同药物浓度的培养基OD值,经线性回归分析,绘制IC50曲线图,进行药效学比较分析.结果 药物作用早期(2 h),黄连素IC50值高于甲硝唑;4~6h2种药物的IC50值较接近,12 h时,甲硝唑的IC50值为11.64 μg/mL,黄连素的IC50值为10.58 μg/mL,24 h,黄连素、甲硝唑的IC50值继续下降,在9.53~9.61 μg/mL之间.48 h,两药的IC50值在5.38~6.03 μg/mL之间,黄连素IC50值稍高.结论 黄连素体外抗阴道毛滴虫的活性与甲硝唑相当,作为治疗阴道毛滴虫感染或治疗耐甲硝唑虫株的替代药物,具有很好的开发利用前景.%Objective To test in vitro the anti-trichomonas vaginalis activity of berberine by bromocresol purple assay.Methods The Trichmonas vaginals in experimental group,known-efficacy group and negative group,were tested by applying the bromocresol purple colorimetric spectroscopic assay.Before and after the treatment,the OD values of culture medium were measured at different times to make the linear regression analysis and draw IC50 curves.Results Compared at the earlier incubation period (2 h),the IC50 value of berberine was higher than that of metronidazole.During the 4 h and 6 h,the IC50 values of two medicaments were similar.At 12 h,the IC50 value of metronidazole was 11.64 μg/mL and that of berberine was 10.58 μg/mL.At 24 h,the the IC50 values of two medicaments kept decreasing,were between 9.53 and 9.61 μg/mL.At 48 h,the IC50 values of two medicaments were between 5.38 and 6.03 μg/mL while the IC50 value of berberine was higher.Conclusion The berberine has similar anti-trichomonas vaginalis activity with metronidazole,and can substitute metronidazole in treating the trichomonas vaginalis infection and the metronidazole-resistant strains infection

  14. Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

    Directory of Open Access Journals (Sweden)

    Luftig Ronald

    2007-09-01

    Full Text Available Abstract Background Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. Results HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b. Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. Conclusion This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

  15. Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

    International Nuclear Information System (INIS)

    Herd, Karen A.; Harvey, Tracey; Khromykh, Alexander A.; Tindle, Robert W.

    2004-01-01

    The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses

  16. Inhibition of replicon initiation and DNA elongation in Chinese hamster ovary cells by treatment at 45.5 degrees C

    International Nuclear Information System (INIS)

    Wong, R.S.; Dewey, W.C.

    1982-01-01

    Heat treatment of Chinese hamster ovary cells at 45.5 degrees C for 15 minutes resulted in the inhibition of both the replicon initiation and the DNA elongation processes. Analysis of the DNA made after treatment showed that for up to 30 minutes after hyperthermia, there was a significant increase (45-80% above control level) in the amount of labeled DNA less than or equal to 40S in size and having a distinct peak of 20S. Therefore, elongation of 20S molecules into larger molecules was inhibited or slowed down. These small molecules did not accumulate when recovery times were longer than 30 minutes. The DNA made after 120 and 240 minutes postheat incubation was larger than control size and indicated that, although replicon initiation was still inhibited, elongation between replicons into 120S molecules could take place. However, their subsequent elongation into parental-size molecules was inhibited. The same delay in DNA elongation seen in cells examined immediately after treatment was still observed in cells heated and allowed to recover for 30 minutes. Also, after 30 minutes of recovery, heated cells still had more newly synthesized DNA in the single-stranded fraction than did control cells, which indicates that DNA elongation within a replicon is delayed for at least 30 minutes after heating. Furthermore, at 4 hours after heating, the inhibition of elongation of clusters of replicons into parental molecules prevailed

  17. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. Crown Copyright © 2012. Published by Elsevier India Pvt Ltd. All rights reserved.

  18. Development of expression vectors for Escherichia coli based on the pCR2 replicon

    Directory of Open Access Journals (Sweden)

    Deb J K

    2007-05-01

    Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

  19. Venezuelan Equine Encephalitis Replicon Particles Can Induce Rapid Protection against Foot-and-Mouth Disease Virus

    OpenAIRE

    Diaz-San Segundo, Fayna; Dias, Camila C. A.; Moraes, Mauro P.; Weiss, Marcelo; Perez-Martin, Eva; Owens, Gary; Custer, Max; Kamrud, Kurt; de los Santos, Teresa; Grubman, Marvin J.

    2013-01-01

    We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/β) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice a...

  20. Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

    Directory of Open Access Journals (Sweden)

    Gerosolimo Germano

    2008-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system. Results First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis. Conclusion Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful

  1. DNA replication in ultraviolet light irradiated Chinese hamster cells: the nature of replicon inhibition and post-replication repair

    International Nuclear Information System (INIS)

    Doniger, J.

    1978-01-01

    DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons were observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m 2 at 254 nm the rate of fork progression was the same as in unirradiated cells, while the rate of replication was reduced by 50%. After a dose of 10J/m 2 the rate of fork progression was reduced 40%, while the replication rate was only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label was first incorporated into short nascent strands, the average size of which was approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands were eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of 'gapped synthesis' and rule out a major role for 'replicative bypass'. (author)

  2. A Polyprotein-Expressing Salmonid Alphavirus Replicon Induces Modest Protection in Atlantic Salmon (Salmo Salar Against Infectious Pancreatic Necrosis

    Directory of Open Access Journals (Sweden)

    Azila Abdullah

    2015-01-01

    Full Text Available Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN in farmed Atlantic salmon (Salmo salar in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV, was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214 and epithelioma papulosum cyprini (EPC cells. The replicon constructs pSAV/polyprotein (pSAV/PP and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar by a single intramuscular injection and tested in a subsequent IPN virus (IPNV challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

  3. Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome

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    George C. diCenzo

    2018-05-01

    Full Text Available Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT. Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.

  4. Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome.

    Science.gov (United States)

    diCenzo, George C; Wellappili, Deelaka; Golding, G Brian; Finan, Turlough M

    2018-05-04

    Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome. Copyright © 2018 diCenzo, et al.

  5. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

    Science.gov (United States)

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans

    2003-05-01

    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  6. Development of a heavy metals enzymatic-based assay using papain

    International Nuclear Information System (INIS)

    Shukor, Yunus; Baharom, Nor Azlan; Rahman, Fadhil Abd.; Abdullah, Mohd. Puad; Shamaan, Nor Aripin; Syed, Mohd. Arif

    2006-01-01

    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC 50 (concentration of toxicant giving 50% inhibition) of Hg 2+ , Ag 2+ , Pb 2 , Zn 2+ is 0.39, 0.40, 2.16, 2.11 mg l -1 , respectively. For Cu 2+ and Cd 2+ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l -1 , respectively. The IC 50 and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox TM , 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis

  7. Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

    Science.gov (United States)

    Ahmad, Muhammad Khairi; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Sandai, Doblin Anak; Mohamed, Rafeezul; Ismail, Ida Shazrina; Zulkiflie, Nurulisa; Yunus, Muhammad Amir

    2017-12-01

    A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

  8. Copper (II) and zinc (II) complexes with flavanone derivatives: Identification of potential cholinesterase inhibitors by on-flow assays.

    Science.gov (United States)

    Sarria, André Lucio Franceschini; Vilela, Adriana Ferreira Lopes; Frugeri, Bárbara Mammana; Fernandes, João Batista; Carlos, Rose Maria; da Silva, Maria Fátima das Graças Fernandes; Cass, Quezia Bezerra; Cardoso, Carmen Lúcia

    2016-11-01

    Metal chelates strongly influence the nature and magnitude of pharmacological activities in flavonoids. In recent years, studies have shown that a promising class of flavanone-metal ion complexes can act as selective cholinesterase inhibitors (ChEIs), which has led our group to synthesize a new series of flavanone derivatives (hesperidin, hesperetin, naringin, and naringenin) complexed to either copper (II) or zinc (II) and to evaluate their potential use as selective ChEIs. Most of the synthesized complexes exhibited greater inhibitory activity against acetylcholinesterase (AChE) than against butyrylcholinesterase (BChE). Nine of these complexes constituted potent, reversible, and selective ChEIs with inhibitory potency (IC 50 ) and inhibitory constant (K i ) ranging from 0.02 to 4.5μM. Copper complexes with flavanone-bipyridine derivatives afforded the best inhibitory activity against AChE and BChE. The complex Cu(naringin)(2,2'-bipyridine) (11) gave IC 50 and K i values of 0.012±0.002 and 0.07±0.01μM for huAChE, respectively, which were lower than the inhibitory values obtained for standard galanthamine (IC 50 =206±30.0 and K i =126±18.0μM). Evaluation of the inhibitory activity of this complex against butyrylcholinesterase from human serum (huBChE) gave IC 50 and K i values of 8.0±1.4 and 2.0±0.1μM, respectively. A Liquid Chromatography-Immobilized Capillary Enzyme Reactor by UV detection (LC-ICER-UV) assay allowed us to determine the IC 50 and K i values and the type of mechanism for the best inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Applicability of the DPPH assay for evaluating the antioxidant capacity of food additives - inter-laboratory evaluation study -.

    Science.gov (United States)

    Shimamura, Tomoko; Sumikura, Yoshihiro; Yamazaki, Takeshi; Tada, Atsuko; Kashiwagi, Takehiro; Ishikawa, Hiroya; Matsui, Toshiro; Sugimoto, Naoki; Akiyama, Hiroshi; Ukeda, Hiroyuki

    2014-01-01

    An inter-laboratory evaluation study was conducted in order to evaluate the antioxidant capacity of food additives by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Four antioxidants used as existing food additives (i.e., tea extract, grape seed extract, enju extract, and d-α-tocopherol) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were used as analytical samples, and 14 laboratories participated in this study. The repeatability relative standard deviation (RSD(r)) of the IC50 of Trolox, four antioxidants, and the Trolox equivalent antioxidant capacity (TEAC) were 1.8-2.2%, 2.2-2.9%, and 2.1-2.5%, respectively. Thus, the proposed DPPH assay showed good performance within the same laboratory. The reproducibility relative standard deviation (RSD(R)) of IC50 of Trolox, four antioxidants, and TEAC were 4.0-7.9%, 6.0-11%, and 3.7-9.3%, respectively. The RSD(R)/RSD(r) values of TEAC were lower than, or nearly equal to, those of IC50 of the four antioxidants, suggesting that the use of TEAC was effective for reducing the variance among the laboratories. These results showed that the proposed DPPH assay could be used as a standard method to evaluate the antioxidant capacity of food additives.

  10. Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

    Science.gov (United States)

    Heath, Tahirah K; Lutz, Marlon R; Reidl, Cory T; Guzman, Estefany R; Herbert, Claire A; Nocek, Boguslaw P; Holz, Richard C; Olsen, Kenneth W; Ballicora, Miguel A; Becker, Daniel P

    2018-01-01

    A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

  11. Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

    Directory of Open Access Journals (Sweden)

    Tahirah K Heath

    Full Text Available A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18 is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I(COD(S,S-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

  12. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Kenneth C. McCullough

    2014-10-01

    Full Text Available Dendritic cells (DC play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

  13. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

    Science.gov (United States)

    Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S.; Chen, Qiang

    2011-01-01

    Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing. PMID:21883868

  14. Alphavirus replicon particles containing the gene for HER2/neu inhibit breast cancer growth and tumorigenesis

    International Nuclear Information System (INIS)

    Wang, Xiaoyan; Wang, Jian-Ping; Maughan, Maureen F; Lachman, Lawrence B

    2005-01-01

    Overexpression of the HER2/neu gene in breast cancer is associated with an increased incidence of metastatic disease and with a poor prognosis. Although passive immunotherapy with the humanized monoclonal antibody trastuzumab (Herceptin) has shown some effect, a vaccine capable of inducing T-cell and humoral immunity could be more effective. Virus-like replicon particles (VRP) of Venezuelan equine encephalitis virus containing the gene for HER2/neu (VRP-neu) were tested by an active immunotherapeutic approach in tumor prevention models and in a metastasis prevention model. VRP-neu prevented or significantly inhibited the growth of HER2/neu-expressing murine breast cancer cells injected either into mammary tissue or intravenously. Vaccination with VRP-neu completely prevented tumor formation in and death of MMTV-c-neu transgenic mice, and resulted in high levels of neu-specific CD8 + T lymphocytes and serum IgG. On the basis of these findings, clinical testing of this vaccine in patients with HER2/neu + breast cancer is warranted

  15. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

    Directory of Open Access Journals (Sweden)

    Olson Daniel G

    2012-05-01

    Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

  16. Antioxidant activities of different solvent extracts of Piper retrofractum Vahl. using DPPH assay

    Science.gov (United States)

    Jadid, Nurul; Hidayati, Dewi; Hartanti, Sylviana Rosyda; Arraniry, Byan Arasyi; Rachman, Rizka Yuanita; Wikanta, Wiwi

    2017-06-01

    Piper retrofractum Vahl., which belongs to the family Piperaceae, is geographically dispersed in tropical region including Indonesia. They are well-known spice possessing high medicinal properties. This study aimed to determine the antioxidant activity of P. retrofractum fruit, extracted with different solvents (methanol, ethyl acetate, n-hexane) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. This research was carried out using different concentrations of methanol, ethyl acetate, and n-hexane extracts, (0, 5, 15, 30, 45, 60 ppm). Ascorbic acid was also used as positive antioxidant control. The percentage of inhibition and IC50 were measured. The results showed that the DPPH free radicals were scavenged by all plant extracts in a concentration dependent manner. Moreover, the IC50 values for DPPH radicals with methanol, ethyl acetate and n-hexane extract of the P. retrofractum Vahl. were found to be 101.74; 66.12 and 57.66 ppm, respectively. Interestingly, the IC50 value of n-hexane extract (57.66 ppm) was lower than ascorbic acid (66.12 ppm), indicating that n-hexane extract was a more potent scavenger of free radicals than methanol and ethyl acetate extracts. Taken together, our results suggested that n-hexane extract of P. Retrofractum Vahl. might contain potential antioxidant compounds.

  17. Venezuelan Equine Encephalitis Replicon Particles Can Induce Rapid Protection against Foot-and-Mouth Disease Virus

    Science.gov (United States)

    Diaz-San Segundo, Fayna; Dias, Camila C. A.; Moraes, Mauro P.; Weiss, Marcelo; Perez-Martin, Eva; Owens, Gary; Custer, Max; Kamrud, Kurt; de los Santos, Teresa

    2013-01-01

    We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/β) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 107 or 108 infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV. PMID:23468490

  18. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

    Directory of Open Access Journals (Sweden)

    Loy John Dustin

    2013-01-01

    Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

  19. Differences in replicon behavior between x-irradiation-sensitive L5178Y mouse lymphoma cells and A-T fibroblasts using DNA fiber autoradiography

    International Nuclear Information System (INIS)

    Ockey, C.H.

    1983-01-01

    Replicon behavior in radiosensitive Ataxia telangiectasia (A-T) fibroblasts and mouse lymphoma L5178Y (LS) cells was studied by DNA fiber autoradiography. LS cells, irradiated at 13 Gy, showed a similar reduction in rate of DNA chain growth and initiation of replicons as did resistant (LR) cells. A progressive increase in the intensity of [ 3 H]TdR labeling of many replicons was observed after irradition in the LS cells, but not in LR cells. This indicated a reduced or absent endogenous dTTP supply after irradiation in the LS cells, implicating a defect in nucleoside precursor production. Irradiated normal human and A-T cells did not show this effect. After 2 Gy, the frequency of initiation of replicons into synthesis was temporarily reduced in the normal human but not in the A-T cells. After 20 Gy, the rate of DNA chain growth was preferentially reduced in the normal human cells, but an increase was observed in the A-T cells. This increased rate could be explained in terms of a normal supply of complexes involved in chain elongation being distributed over a reduced number of initiated replicon clusters in the A-T cells

  20. ParABS Systems of the Four Replicons of Burkholderia cenocepacia: New Chromosome Centromeres Confer Partition Specificity†

    Science.gov (United States)

    Dubarry, Nelly; Pasta, Franck; Lane, David

    2006-01-01

    Most bacterial chromosomes carry an analogue of the parABS systems that govern plasmid partition, but their role in chromosome partition is ambiguous. parABS systems might be particularly important for orderly segregation of multipartite genomes, where their role may thus be easier to evaluate. We have characterized parABS systems in Burkholderia cenocepacia, whose genome comprises three chromosomes and one low-copy-number plasmid. A single parAB locus and a set of ParB-binding (parS) centromere sites are located near the origin of each replicon. ParA and ParB of the longest chromosome are phylogenetically similar to analogues in other multichromosome and monochromosome bacteria but are distinct from those of smaller chromosomes. The latter form subgroups that correspond to the taxa of their hosts, indicating evolution from plasmids. The parS sites on the smaller chromosomes and the plasmid are similar to the “universal” parS of the main chromosome but with a sequence specific to their replicon. In an Escherichia coli plasmid stabilization test, each parAB exhibits partition activity only with the parS of its own replicon. Hence, parABS function is based on the independent partition of individual chromosomes rather than on a single communal system or network of interacting systems. Stabilization by the smaller chromosome and plasmid systems was enhanced by mutation of parS sites and a promoter internal to their parAB operons, suggesting autoregulatory mechanisms. The small chromosome ParBs were found to silence transcription, a property relevant to autoregulation. PMID:16452432

  1. Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

    Science.gov (United States)

    Steele, J C P; Phelps, R J; Simmonds, M S J; Warhurst, D C; Meyer, D J

    2002-07-01

    Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) 40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

  2. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Directory of Open Access Journals (Sweden)

    Juana M Sánchez-Puig

    Full Text Available Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  3. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Science.gov (United States)

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  4. Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

    Science.gov (United States)

    Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

    2017-07-01

    Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir

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

    Directory of Open Access Journals (Sweden)

    Adam R Brown

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

  6. A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

    Science.gov (United States)

    Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

    2004-12-01

    With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

  7. Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

    Science.gov (United States)

    Dahan-Meir, Tal; Filler-Hayut, Shdema; Melamed-Bessudo, Cathy; Bocobza, Samuel; Czosnek, Henryk; Aharoni, Asaph; Levy, Avraham A

    2018-04-18

    Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a mean to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double strand break (DSB) in the target gene, via homologous recombination. Mutagenesis experiments, performed in the Micro-Tom variety achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting experiments, our target was a fast-neutron-induced crtiso allele that contained a 281bp deletion. This deletion was repaired with the wildtype sequence through homologous recombination between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient gene targeting can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

    Science.gov (United States)

    Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

    2013-05-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

  9. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value...... culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause...... for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay....

  10. Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon

    DEFF Research Database (Denmark)

    Chen, Zhengjun; Lin, Jinzhong; Ma, Chengjie

    2014-01-01

    . These plasmids showed distinct properties: pEL5.7 was capable of replicating in L. casei MCJΔ1 and Lactobacillus delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas pEL5.6 replicated in three different strains, including L. casei MCJΔ1, L. casei NJ, Lactobacillus......Many lactic acid bacteria carry different plasmids, particularly those that replicate via a theta mechanism. Here we describe Lactobacillus casei MCJ(CCTCC AB20130356), a new isolate that contains pMC11, carrying two distinct theta-type replicons. Each replicon contained an iteron in the origin...... of replication (oriV1 or oriV2) and a gene coding for the replicase (RepA_1 or RepB_1), both of which are essential for plasmid replication. Escherichia coli/Lactobacillus shuttle vectors were constructed with each replicon, yielding pEL5.7 and pEL5.6 that are based on oriV2 and oriV1 replicons, respectively...

  11. Development of an enzyme-linked immunosorbent assay for the detection of dicamba.

    Science.gov (United States)

    Clegg, B S; Stephenson, G R; Hall, J C

    2001-05-01

    A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.

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

    Science.gov (United States)

    Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

    2009-01-01

    Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing

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

    Directory of Open Access Journals (Sweden)

    Eva Maria Fenyö

    Full Text Available Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1 vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet involving 18 independent participants was organized to compare different assays.Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4 at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs (virus infectivity assays, VI assays, or their Env-pseudotyped (gp160 derivatives produced in 293T cells (PSV assays from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression.PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent.The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of

  14. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    Science.gov (United States)

    2013-07-12

    assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979, 16:710–718. 3. Noedl H, Attlmayr B...40:685–691. 32. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM, Ward SA: Relationship between antimalarial drug activity , accumulation, and...success rate when testing DHA, AS, MQ, QN, CQ, and PPQ activities . A “successful” IC50 assay result for each P. falciparum clinical isolate was defined as

  15. 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

  16. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Stefan J Halbherr

    Full Text Available Highly pathogenic avian influenza viruses (HPAIV of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade. Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  17. A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever.

    Science.gov (United States)

    Sun, Yuan; Li, Hong-Yu; Tian, Da-Yong; Han, Qiu-Ying; Zhang, Xin; Li, Na; Qiu, Hua-Ji

    2011-10-26

    Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.

    Science.gov (United States)

    Chain, Patrick S G; Denef, Vincent J; Konstantinidis, Konstantinos T; Vergez, Lisa M; Agulló, Loreine; Reyes, Valeria Latorre; Hauser, Loren; Córdova, Macarena; Gómez, Luis; González, Myriam; Land, Miriam; Lao, Victoria; Larimer, Frank; LiPuma, John J; Mahenthiralingam, Eshwar; Malfatti, Stephanie A; Marx, Christopher J; Parnell, J Jacob; Ramette, Alban; Richardson, Paul; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V; Ulrich, Luke E; Zhulin, Igor B; Tiedje, James M

    2006-10-17

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  19. Burkholderia xernovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

    Energy Technology Data Exchange (ETDEWEB)

    Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Denef, Vincent [University of California, Berkeley; Konstantinidis, Konstantinos T [Michigan State University, East Lansing; Vergez, Lisa [Lawrence Livermore National Laboratory (LLNL); Agullo, Loreine [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Reyes, Valeria Latorre [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Hauser, Loren John [ORNL; Cordova, Macarena [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Gomez, Luis [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Gonzalez, Myriam [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Land, Miriam L [ORNL; Lao, Victoria [Lawrence Livermore National Laboratory (LLNL); Larimer, Frank W [ORNL; LiPuma, John J [University of Michigan; Mahenthiralingam, Eshwar [Cardiff University, Wales; Malfatti, Stephanie [Lawrence Livermore National Laboratory (LLNL); Marx, Christopher J [Harvard University; Parnell, J Jacob [Michigan State University, East Lansing; Ramette, Alban [Michigan State University, East Lansing; Richardson, P M [U.S. Department of Energy, Joint Genome Institute; Seeger, Michael [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Smith, Daryl [University of British Columbia, Vancouver; Spilker, Theodore [University of Michigan; Sul, Woo Jun [Michigan State University, East Lansing; Tsoi, Tamara V [Michigan State University, East Lansing; Zhulin, Igor B [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Tiedje, James M. [Michigan State University, East Lansing

    2006-01-01

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven 'central aromatic' and twenty 'peripheral aromatic' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  20. Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

    Directory of Open Access Journals (Sweden)

    Shoufeng Ren

    2018-01-01

    Full Text Available Ebola virus (EBOV causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP is the major protective antigen of EBOV, and can generate virus-like particles (VLPs by co-expression with matrix protein (VP40. In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV replicon vector DREP to express EBOV GP and matrix viral protein (VP40. EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40. Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

  1. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Science.gov (United States)

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  2. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

    Full Text Available A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10% were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  3. Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease

    International Nuclear Information System (INIS)

    Fultz, Patricia N.; Stallworth, Jackie; Porter, Donna; Novak, Miroslav; Anderson, Marie J.; Morrow, Casey D.

    2003-01-01

    In the search for an effective vaccine against the human immunodeficiency virus (HIV), novel ways to deliver viral antigens are being evaluated. One such approach is the use of nonreplicating viral vectors encoding HIV and/or SIV genes that are expressed after infection of host cells. Nonreplicating poliovirus vectors, termed replicons, that expressed HIV-1/HXB2 and SIVmac239 gag and various HIV-1 env genes from different clades were tested for immunogenicity and protective efficacy against intravenous challenge of pig-tailed macaques with SHIV-89.6P. To maximize both cellular and humoral immune responses, a prime-boost regimen was used. Initially, macaques were immunized four times over 35 weeks by either the intranasal and intrarectal or the intramuscular (im) route with mixtures of poliovirus replicons expressing HIV-1 gag and multiple env genes. Immunization with replicons alone induced both serum antibodies and lymphocyte proliferative responses. After boosting with purified Env protein, neutralizing antibodies to SHIV-89.6P were induced in four of five immunized animals. In a second experiment, four macaques were immunized im three times over 27 weeks with replicons expressing the SIVmac239 gag and HIV-1/HXB2 env genes. All immunized animals were then boosted twice with purified HIV-1-89.6 rgp140-Env and SIVmac239 p55-Gag proteins. Four control animals received only the two protein inoculations. Immunized and control animals were then challenged intravenously with the pathogenic SHIV-89.6P. After challenge the animals were monitored for virus isolation from peripheral blood mononuclear cells and plasma viremia and for changes in virus-specific antibody titers. Naieve pig-tailed macaques experienced rapid loss of CD4 + T cells and died between 38 and 62 weeks after infection. In contrast, macaques immunized with replicons and proteins rapidly cleared plasma virus and did not experience sustained loss of CD4 + lymphocytes. Furthermore, two of the four macaques

  4. High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis.

    Science.gov (United States)

    Halladay, Jason S; Delarosa, Erlie Marie; Tran, Daniel; Wang, Leslie; Wong, Susan; Khojasteh, S Cyrus

    2011-08-01

    Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.

  5. Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays.

    Science.gov (United States)

    Klopčič, Ivana; Skledar, Darja Gramec; Mašič, Lucija Peterlin; Dolenc, Marija Sollner

    2016-10-01

    The anti-androgenic and anti-thyroid hormonal activities of the two novel brominated flame retardants, TBB and TBPH and of their metabolites TBBA and TBMEPH have been compared using the luciferase reporter gene assays. Only the parent compounds TBB and TBPH exhibited anti-glucocorticoid activity with IC50 values of 1.9 μM and 0.3 μM. Furthermore, mode of action for these two compounds is by direct competing to the glucocorticoid receptor (GR) with IC50 values of 0.03 μM and 0.002 μM. All four tested compounds possess anti-androgenic and anti-thyroid hormonal activities, without agonist activities on the respective receptors. Anti-androgenic activities with IC50 values of 43.5 μM, 0.1 μM, 47.5 μM and 1.3 μM were found for TBB, TBPH, TBBA and TBMEPH. The anti-thyroid hormonal IC50 values of 37.5 μM, 0.1 μM, 22.8 μM and 32.3 μM for TBB, TBPH, TBBA and TBMEPH, together with the above quoted results, indicate that metabolism can modify anti-androgenic, anti-glucocorticoid and anti-thyroid hormonal effects of these novel brominated flame retardants. Furthermore, the parent flame retardants are shown to be able to disrupt the function of the GR as antagonists by direct competition to the receptor. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  7. Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14 - insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons.

    Science.gov (United States)

    Romaniuk, Krzysztof; Dziewit, Lukasz; Decewicz, Przemyslaw; Mielnicki, Sebastian; Radlinska, Monika; Drewniak, Lukasz

    2017-01-01

    Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Opposite Effects of Two Human ATG10 Isoforms on Replication of a HCV Sub-genomic Replicon Are Mediated via Regulating Autophagy Flux in Zebrafish

    Directory of Open Access Journals (Sweden)

    Yu-Chen Li

    2018-04-01

    Full Text Available Autophagy is a host mechanism for cellular homeostatic control. Intracellular stresses are symptoms of, and responses to, dysregulation of the physiological environment of the cell. Alternative gene transcription splicing is a mechanism potentially used by a host to respond to physiological or pathological challenges. Here, we aimed to confirm opposite effects of two isoforms of the human autophagy-related protein ATG10 on an HCV subgenomic replicon in zebrafish. A liver-specific HCV subreplicon model was established and exhibited several changes in gene expression typically induced by HCV infection, including overexpression of several HCV-dependent genes (argsyn, leugpcr, rasgbd, and scaf-2, as well as overexpression of several ER stress related genes (atf4, chop, atf6, and bip. Autophagy flux was blocked in the HCV model. Our results indicated that the replication of the HCV subreplicon was suppressed via a decrease in autophagosome formation caused by the autophagy inhibitor 3MA, but enhanced via dysfunction in the lysosomal degradation caused by another autophagy inhibitor CQ. Human ATG10, a canonical isoform in autophagy, facilitated the amplification of the HCV-subgenomic replicon via promoting autophagosome formation. ATG10S, a non-canonical short isoform of the ATG10 protein, promoted autophagy flux, leading to lysosomal degradation of the HCV-subgenomic replicon. Human ATG10S may therefore inhibit HCV replication, and may be an appropriate target for future antiviral drug screening.

  9. Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

    Science.gov (United States)

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2014-10-01

    Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    International Nuclear Information System (INIS)

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-01-01

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  11. A radioreceptor-assay of a methionine-enkephalin-like substance in human CSF

    International Nuclear Information System (INIS)

    Furui, Tomoo; Kageyama, Naoki; Haga, Tatsuya; Ichiyama, Arata; Fukushima, Masanori.

    1980-01-01

    The purpose of this study was to establish a radioreceptor-assay system of the met-enkephalin-like substance in human CSF. A particulate fraction was prepared from rat brain essentially according to the method of Pasternak and used as a receptor. In order to obtain the most sensitive radioreceptor-assay, displacement curves by met-enkephalin were compared with each other using three kinds of radiolabeled ligand: 3 H-met-enkephalin, 3 H-naloxone and 3 H-dihydromorphine. When 3 H-dihydromorphine was used as the radiolabeled ligand, the concentration of met-enkephalin required to inhibit 50% of specific binding (IC 50 ) was the lowest. The addition of 1 mM EDTA and 2 mM Mg was found to decrease further the IC 50 and enhance the binding. Thus the radioreceptor-assay of the metenkephalin-like substance in CSF was carried out using 3 H-dihydromorphine as the radiolabeled ligand in the presence of 1 mM EDTA and 2 mM Mg. The sensitivity of the assay ranged from about 1 to 100 pmoles of met-enkephalin. The isolation of met-enkephalin from CSF was performed by a Sephadex G 10 gel filtration followed by a SP-Sephadex (H + ) column chromatography. Sodium and non-specific inhibitor (s) of the specific binding of 3 H-dihydromorphine were removed from CSF by the chromatography. The overall recovery of met-enkephalin was about 60%. Human CSF was obtained from 8 patients hospitalized for neurosurgical study and therapy. All assays were duplicated. The met-enkephalin-like substance levels were 3.3 +- 2.1 (mean +- S.D., n=8) pmoles/ml and ranged from 0.7 to 6.7 pmoles of the met-enkephalin equivalents. (J.P.N.)

  12. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    Directory of Open Access Journals (Sweden)

    Raja Elina Raja Aziddin

    2011-08-01

    Full Text Available The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE on human recombinant cytochrome P450 (CYP enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC50 values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC50 of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC50 of CYP2C19 could not be determined, however, because inhibition was < 50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6, ketoconazole (CYP3A4, tranylcypromine (CYP2C19 and furafylline (CYP1A2 were used as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

  13. Comparison of tetrazolium colorimetric and [3H]-uridine assays for in vitro chemosensitivity testing.

    Science.gov (United States)

    Ford, C H; Richardson, V J; Tsaltas, G

    1989-01-01

    We have routinely used a [3H]-uridine microplate assay for assessing chemosensitivity. A colorimetric assay with the advantages of safety, cost and simplicity has previously been described and relies on the ability of living cells to reduce a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MMT), into an insoluble formazan precipitate. We compared the chemosensitivity of 14 human tumour cell lines of colonic, lung and cervical carcinoma origin to doxorubicin, vindesine or vindesine immunoconjugates in both the [3H]-uridine assay and a modified MTT assay to evaluate whether we could change to the non-radiolabelled method. Correlation between the concentration of drug causing 50% inhibition of cell growth (IC50) for these agents between the two assays was very poor. However, taking account of recent reports in the literature, we modified the MTT assay by removing serum-containing medium and using dimethyl sulphoxide to solubilise the formazan precipitate. This considerably improved the correlation between the assays for doxorubicin (r = 0.871; P = 0.001) and vindesine (r = 0.981; P less than 0.001). Our data indicates that the MTT assay can be used to replace the [3H]-uridine assay for chemosensitivity screening, but further modifications are necessary to improve the sensitivity and decrease the problem of cell loss after washing, which was noted with some adherent cell lines.

  14. A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

    2015-03-01

    The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

    Science.gov (United States)

    Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

    2004-12-01

    The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

  16. 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)

  17. 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

  18. Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

    Science.gov (United States)

    Evans, Christopher C; Moorhead, Andrew R; Storey, Bobby E; Blagburn, Byron L; Wolstenholme, Adrian J; Kaplan, Ray M

    2017-11-15

    Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L 3 ). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L 3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC 50 values ranged from 1.57 to 5.56μM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L 3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage. Published by Elsevier B.V.

  19. Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

    Science.gov (United States)

    Jacobs, K R; Guillemin, G J; Lovejoy, D B

    2018-02-01

    Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

  20. Development of an enzyme-linked immunosorbent assay for residue analysis of the insecticide emamectin benzoate in agricultural products.

    Science.gov (United States)

    Kondo, Mika; Yamashita, Hiroshi; Uchigashima, Mikiko; Kono, Takeshi; Takemoto, Toshihide; Fujita, Masahiro; Saka, Machiko; Iwasa, Seiji; Ito, Shigekazu; Miyake, Shiro

    2009-01-28

    A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the analysis of emamectin residues in agricultural products was developed using a prepared mouse monoclonal antibody. The working range was 0.3-3.0 ng/mL, and the 50% inhibition concentration (IC(50)) was 1.0 ng/mL. The assay was sufficiently sensitive for analysis of the maximum residue limits in agricultural products in Japan (>0.1 microg/g). Emamectin residues contain the following metabolites: the 4''-epi-amino analogue, the 4''-epi-(N-formyl)amino analogue, the 4''-epi-(N-formyl-N-methyl)amino analogue, and the 8,9-Z isomer. The dc-ELISA reacted with these compounds at ratios of 113, 55, 38, and 9.1% of the IC(50) value of emamectin benzoate. Seven kinds of vegetables were spiked with emamectin benzoate at concentrations of 15-300 ng/g, and the recoveries were 91-117% in the dc-ELISA. The dc-ELISA results agreed reasonably well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using spiked samples and actual (incurred) samples. The results indicate that the dc-ELISA was useful for the analysis of emamectin benzoate residues in agricultural products.

  1. 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...

  2. Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice.

    Science.gov (United States)

    Lee, John S; Groebner, Jennifer L; Hadjipanayis, Angela G; Negley, Diane L; Schmaljohn, Alan L; Welkos, Susan L; Smith, Leonard A; Smith, Jonathan F

    2006-11-17

    The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.

  3. SH2 modified STAT1 induces HLA-I expression and improves IFN-γ signaling in IFN-α resistant HCV replicon cells.

    Directory of Open Access Journals (Sweden)

    Bret Poat

    2010-09-01

    Full Text Available We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2 domains of STAT1 (at Ala-656 and Asn-658 efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.

  4. An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs.

    Science.gov (United States)

    Dahiya, S S; Saini, M; Kumar, P; Gupta, P K

    2011-01-01

    A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

  5. Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum.

    Science.gov (United States)

    Basco, Leonardo K

    2003-08-01

    In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

  6. Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

    Science.gov (United States)

    O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

    2017-01-27

    Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

  7. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    Science.gov (United States)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-12-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  8. Mean rate of DNA replication and replicon size in the shoot apex of Silence coeli-rosa. During the initial 120 minutes of the first day of floral induction

    International Nuclear Information System (INIS)

    Ormrod, J.C.; Francis, D.

    1986-01-01

    28-day-old plants of Silence coeli-rosa were exposed, at 1700 hours, to long day (LD) conditions comprising light of low fluence rate provided by tungsten bulbs, or maintained in darkness as short day (SD) controls. All plants were exposed at 1700 hours to tritiated-(methyl- 3 H)-thymidine for 30, 45, 60, 90, or 120 minutes. Apical domes were isolated and prepared as fiber autoradiographs from which replicon size and rates of DNA replication, per single replication fork were recorded. In SD, replicon size was between 15-20 μm and exposure to LD conditions altered neither replicon size nor the pattern of deployment of replicons during S-phase relative to the SD controls. However, the mean rate of replication in LD was 8.7 μm h -1 compared with 5.2 μm h -1 in SD. Thus, exposure to LD resulted in a 1.7-fold increase in the rate of DNA replication relative to the SD controls. This rapid increase in replication rate, detectable within 30 minutes of the start of the LD is discussed in relation to changes known to occur to the cell cycle in Silene during the first day of floral induction. (Author)

  9. Synthesis and Heme Polymerization Inhibitory Activity (HPIA Assay of Antiplasmodium of (1-N-(3,4-Dimethoxybenzyl-1,10-Phenanthrolinium Bromide from Vanillin

    Directory of Open Access Journals (Sweden)

    Dhina Fitriastuti

    2014-03-01

    Full Text Available The synthesis of (1-N-(3,4-dimethoxy-benzyl-1,10-phenanthrolinium bromide had been conducted from vanillin. Heme polymerization inhibitory activity assay of the synthesized antiplasmodium has also been carried out. The first step of reaction was methylation of vanillin using dimethylsulfate and NaOH. The mixture was refluxed for 2 h to yield veratraldehyde in the form of light yellow solid (79% yield. Methylation product was reduced using sodium borohydride (NaBH4 with grinding method and yielded veratryl alcohol in the form of yellow liquid (98% yield. Veratryl alcohol was brominated using PBr3 to yield yellowish black liquid (85% yield. The final step was benzylation of 1,10-phenanthroline monohydrate with the synthesized veratryl bromide under reflux condition in acetone for 14 h to afford (1-N-(3,4-dimethoxy-benzyl-1,10-phenanthrolinium bromide (84% as yellow solid with melting point of 166-177 °C. The structures of products were characterized by FT-IR, GC-MS and 1H-NMR spectrometers. The results of heme polymerization inhibitory activity assay of (1-N-(3,4-dimethoxybenzyl-1,10-phenanthrolinium bromide showed that it had IC50 HPIA of 3.63 mM, while chloroquine had IC50 of4.37 mM. These results indicated that (1-N-(3,4-dimethoxybenzyl-1,10-phenanthrolinium bromide was more potential antiplasmodium than chloroquine.

  10. 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.

  11. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    Science.gov (United States)

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. A cost-effective assay for antioxidant using simple cotton thread combining paper based device with mobile phone detection.

    Science.gov (United States)

    Sateanchok, Suphasinee; Wangkarn, Sunanta; Saenjum, Chalermpong; Grudpan, Kate

    2018-01-15

    A cost-effective assay for antioxidant using simple cotton thread combining paper based device with mobile phone detection has been investigated. Standard and sample solutions flow along a bunch of cotton thread treated with sodium hydroxide via microfluidic behaviors without external pumping. The analyte solution reacts with the reagents that have been immobilized on the paper strip fixed at the end of the cotton bunch. The developed platforms were used for the assays of total phenolic content and antioxidant capacity by employing Folin-Ciocalteu and 2, 2-diphenyl-1-picryhydrazyl (DPPH) respectively. Simple detection can be made by employing a mobile phone camera (iPhone 4S) with Image J or Photoshop for image processing and evaluation. Gallic acid was used as a reference standard in this work, as its polyphenol structures can be found in many plants. The total phenolic content is expressed as gallic acid equivalents (GAE) (mg/g material). Inhibition capacity is calculated by the equation: % I = [(I o - I s )/ I o ] × 100, where I s is the relative magenta intensity (CMYK mode) of sample, and I o the relative magenta intensity of DPPH•. IC 50 inhibition can be estimated from the graph and can be used for the antioxidant capacity consideration. Applications to the assay green tea samples were demonstrated. The total phenolic contents in the green tea samples were found to be 48-105mg/g, with %RSD of less than 10 for that of higher 50 GAE mg/g and IC 50 values of the samples studied were 25-50mg/L. The results obtained by the developed methods agree with that of the standard methods. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Antioxidant properties of species from the Brazilian cerrado by different assays

    Directory of Open Access Journals (Sweden)

    K.S. Farias

    2013-01-01

    Full Text Available The purpose of this study was to screen the antioxidant activity of medicinal plant extracts from the Brazilian cerrado, through other methods than the total phenolic content and its correlation with the antioxidant activity. Ethanolic extracts of ten species were evaluated through three antioxidant assays, in vitro, including 2,2-diphenyl-1-picrylhydrazyl (DPPH, total antioxidant activity and reducing power; and by using the Folin-Ciocalteu method the total phenolic content was determined. Ethanolic extracts of Stryphnodendron obovatum, Cecropia pachystachya and Duguetia furfuraceae showed strong antioxidant activity (IC50<5 µg mL-1 in the DPPH free radical scavenging assay; the species Vernonia phosphorea, Hymenaea stignocarpa and Jacaranda ulei may also be highlighted. These results were confirmed in the assays of total antioxidant capacity and reducing power. The extracts of S. obovatum and V. phosphorea showed an abundant phenolic content; therefore, the phenolic content may play a role in the antioxidant activity. These two species, traditionally used in Brazil, showed great power in these assay systems and may be a promising source for the development of natural antioxidants and future candidates for phytochemical and pharmacological studies in related diseases.

  14. Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay.

    Science.gov (United States)

    McDowell, Arlene; Thompson, Scott; Stark, Mirjam; Ou, Zong-Quan; Gould, Kevin S

    2011-12-01

    There is considerable interest in antioxidant dietary components that can be protective against degenerative diseases in humans. Puha (Sonchus oleraceus L.) is a rich source of polyphenols, and exhibits strong antioxidant activity as measured by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. However, the potential of puha to protect against degenerative diseases requires that low molecular weight antioxidants (LMWA) are absorbed by, and active in, human cells. The cellular antioxidant activity (CAA) assay was used to investigate the antioxidant activity of puha leaf extracts. Preparation methods of freezing and freeze-drying reduced the total polyphenolic content compared with fresh puha, but did not affect the LMWA potential as determined by the DPPH assay. The IC(50) values were 0.012 ± 0.003 mg/mL and 0.010 ± 0.005 mg/mL for freeze-dried and fresh puha leaves, respectively. Using the CAA assay, it was shown that LMWAs from foliar extracts of puha were effectively absorbed into HepG2 cells, and exerted antioxidant activity at levels comparable to those of extracts from blueberry fruits, the much-touted antioxidant superfood. Methylene blue staining of HepG2 cells indicated that puha extracts were not cytotoxic at concentrations below 100 mg DW/mL. The data indicate the potential of puha as a nutraceutical supplement for human health. Copyright © 2011 John Wiley & Sons, Ltd.

  15. Genotoxic and cytotoxic potential of whole plant extracts of Kalanchoe laciniata by Ames and MTT assay.

    Science.gov (United States)

    Sharif, Ali; Akhtar, Muhammad Furqan; Akhtar, Bushra; Saleem, Ammara; Manan, Maria; Shabbir, Maryam; Ashraf, Muneeb; Peerzada, Sohaib; Ahmed, Shoaib; Raza, Moosa

    2017-01-01

    Lack of data on safety of herbal medicines have endangered human health and life. The present study evaluated the genotoxic and mutagenic effect of Kalanchoe laciniata to access the safety and usefulness of the medicinal plant. Aqua-methanolic and n-hexane extracts of K. laciniata were evaluated for the genotoxic potential using Ames assay and cytotoxicity was evaluated using MTT assay. Ames assay was conducted using two strains of Salmonella typhimurium TA-100 and TA-102 whereas MTT assay was performed on baby hamster kidney cell line BHK-21. Aqua-methanolic extract of K. laciniata exhibited significant mutagenicity when exposed to TA-102 strain with a mutagenic index of 50.66 and 54.74 at maximum dose 150 mg/plate. The extract was also mutagenic to TA-100 strain but to a lesser extent. M.I of n-hexane extract was 12.15 and 15.51 for TA-100 and TA-102 respectively. n-hexane extract was mutagenic but little difference was observed between results of two strains. Both extracts were found to be cytotoxic with an IC 50 of 321.9 and 638.5 µg/mL for aqua-methanolic and n-hexane extracts respectively. On the basis of results it was concluded that aqua-methanolic and n-hexane extracts of K. laciniata possess mutagenic and cytotoxic potential. It is suggested to explore the plant further to evaluate its safety in rodents and other species.

  16. An HTS-compatible 3D colony formation assay to identify tumor-specific chemotherapeutics.

    Science.gov (United States)

    Horman, Shane R; To, Jeremy; Orth, Anthony P

    2013-12-01

    There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

  17. Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay

    Directory of Open Access Journals (Sweden)

    Kwang Jin Lee

    2015-01-01

    Full Text Available This study investigated the antioxidant activity of one hundred kinds of pure chemical compounds found within a number of natural substances and oriental medicinal herbs (OMH. Three different methods were used to evaluate the antioxidant activity of DPPH radical-scavenging activity, ABTS radical-scavenging activity, and online screening HPLC-ABTS assays. The results indicated that 17 compounds exhibited better inhibitory activity against ABTS radical than DPPH radical. The IC50 rate of a more practical substance is determined, and the ABTS assay IC50 values of gallic acid hydrate, (+-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively. The ABTS assay is more sensitive to identifying the antioxidant activity since it has faster reaction kinetics and a heightened response to antioxidants. In addition, there was a very small margin of error between the results of the offline-ABTS assay and those of the online screening HPLC-ABTS assay. We also evaluated the effects of 17 compounds on the NO secretion in LPS-stimulated RAW 264.7 cells and also investigated the cytotoxicity of 17 compounds using a cell counting kit (CCK in order to determine the optimal concentration that would provide an effective anti-inflammatory action with minimum toxicity. These results will be compiled into a database, and this method can be a powerful preselection tool for compounds intended to be studied for their potential bioactivity and antioxidant activity related to their radical-scavenging capacity.

  18. Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities.

    Science.gov (United States)

    Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

    2017-09-01

    We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC 50 = 111.2μg/ml) in DPPH and IC 50 = 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD 50 = 1.1μg/ml) in brine shrimp lethality test and with (IC 50 = 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC 50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli , Aspergillus niger and Candida albicans. The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

  19. Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT assay and Larvicidal Activities

    Directory of Open Access Journals (Sweden)

    Saeed Tavakoli

    2017-10-01

    Full Text Available Background: We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant.Methods: Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Anti­oxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anoph­eles stephensi was carried out according to the method described by WHO.Results: In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-ver­benol (9.66%, isobutyl acetate (25.73% and E-β-caryophyllene (8.68% were main compounds. The oil showed (IC50= 111.2µg/ml in DPPH and IC50= 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD50= 1.1µg/ml in brine shrimp lethality test and with (IC50= 22.0, 25.0 and 42.55 µg/ml on three cancerous cell lines (MCF-7, A-549 and HT-29 respectively. LC50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli, Asper­gillus niger and Candida albicans.Conclusion: The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

  20. Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

    Science.gov (United States)

    Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

    2017-12-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

  1. Discovery of new angiotensin converting enzyme (ACE) inhibitors from medicinal plants to treat hypertension using an in vitro assay

    Science.gov (United States)

    2013-01-01

    Background and purpose of the study Angiotensin converting enzyme (ACE) inhibitors plays a critical role in treating hypertension. The purpose of the present investigation was to evaluate ACE inhibition activity of 50 Iranian medicinal plants using an in vitro assay. Methods The ACE activity was evaluated by determining the hydrolysis rate of substrate, hippuryl-L-histidyl-L-leucine (HHL), using reverse phase high performance liquid chromatography (RP-HPLC). Total phenolic content and antioxidant activity were determined by Folin-Ciocalteu colorimetric method and DPPH radical scavenging assay respectively. Results Six extracts revealed > 50% ACE inhibition activity at 330 μg/ml concentration. They were Berberis integerrima Bunge. (Berberidaceae) (88.2 ± 1.7%), Crataegus microphylla C. Koch (Rosaceae) (80.9 ± 1.3%), Nymphaea alba L. (Nymphaeaceae) (66.3 ± 1.2%), Onopordon acanthium L. (Asteraceae) (80.2 ± 2.0%), Quercus infectoria G. Olivier. (Fagaceae) (93.9 ± 2.5%) and Rubus sp. (Rosaceae) (51.3 ± 1.0%). Q. infectoria possessed the highest total phenolic content with 7410 ± 101 mg gallic acid/100 g dry plant. Antioxidant activity of Q. infectoria (IC50 value 1.7 ± 0.03 μg/ml) was more than that of BHT (IC50 value of 10.3 ± 0.15 μg/ml) and Trolox (IC50 value of 3.2 ± 0.06 μg/ml) as the positive controls. Conclusions In this study, we introduced six medicinal plants with ACE inhibition activity. Despite the high ACE inhibition and antioxidant activity of Q. infectoria, due to its tannin content (tannins interfere in ACE activity), another plant, O. acanthium, which also had high ACE inhibition and antioxidant activity, but contained no tannin, could be utilized in further studies for isolation of active compounds. PMID:24359711

  2. Antitumor activity of zoledronic acid in primary breast cancer cells determined by the ATP tumor chemosensitivity assay

    International Nuclear Information System (INIS)

    Fehm, Tanja; Zwirner, Manfred; Wallwiener, Diethelm; Seeger, Harald; Neubauer, Hans

    2012-01-01

    The NeoAzure study has demonstrated that the use of the bisphosphonate zoledronic acid (Zol) in the neoadjuvant setting increases the rate of complete response in primary breast cancer and therefore indicates direct antitumor activity. The purpose of this study was to compare the antitumor effect of Zol with standard chemotherapy in primary breast cancer cells using ATP-tumor chemosensitivity assay (ATP-TCA). Breast cancer specimens were obtained from patients with breast cancer who underwent primary breast cancer surgery at the Department of Obstetrics and Gynecology, Tübingen, Germany, between 2006 through 2009. Antitumor effects of Zol, TAC (Docetaxel, Adriamycin, Cyclophosphamide) and FEC (5-Fluorouracil, Epirubicin, Cyclophosphamide) were tested in 116 fresh human primary breast cancer specimens using ATP-TCA. ATP-TCA results were analyzed with different cut-off levels for the half maximal inhibitory concentration (IC50), for IC90 and for the sensitivity index (IndexSUM). Each single agent or combination was tested at six doubling dilutions from 6.25, 12.5, 25, 50, 100, and 200% of test drug concentrations (TDC) derived from the plasma peak concentrations determined by pharmacokinetic data. The assay was carried out in duplicate wells with positive and negative controls. The median IndexSUM value was lower for Zol than for the combined regimen FEC (36.8%) and TAC (12.9%), respectively, indicating increased antitumor activity of Zol in primary breast cancer cells. The difference regarding Zol and FEC was significant (p < 0.05). The median IC50 value for Zol (8.03% TDC) was significantly lower than the IC50 values for FEC (33.5% TDC) and TAC (19.3% TDC) treatment (p < 0.05). However, the median IC90 value for Zol (152.5% TDC) was significantly higher than the IC90 value obtained with TAC (49.5% TDC; p < 0.05), but similar to the IC90 value for FEC (180.9% TDC). In addition a significant positive correlation was observed for the IndexSum of Zol and the ER status

  3. Heme polymerization inhibition activity (HPIA) assay of synthesized xanthone derivative as antimalarial compound

    Science.gov (United States)

    Fitriastuti, Dhina; Jumina, Priatmoko

    2017-03-01

    Xanthone is a phenolic secondary metabolite of Garcinia and Calophyllum herbs which has been clinically proven to display anti malaria activity. In the present paper, 2,3,4-trihydroxy-5-methyl xanthone which has been synthesized from gallic acid and o-cresol in Eaton's reagent was tested for its activity as antimalarial. Thus, HPIA assay of the synthesized xanthones was successfully conducted. The HPIA assay was carried out towards the xanthone, chloroquine diphosphate as positive control and distilled water as negative control in various concentration. The samples were reacted with hematin (ferriprotoporphyrin IX hydroxide) and the absorbance of the precipitate was observed by using Elisa reader. The results of HPIA assay showed that 2,3,4-trihydroxy-5-methyl xanthone and chloroquine have IC50 values of 0.755 and 1.462 mg/mL or 2.92 and 4.57 mM, respectively. 2,3,4-Trihydroxy-5-methyl xanthone displayed better antimalarial activity than chloroquine.

  4. An Inhibitive Enzyme Assay to Detect Mercury and Zinc Using Protease from Coriandrum sativum

    Directory of Open Access Journals (Sweden)

    Gunasekaran Baskaran

    2013-01-01

    Full Text Available Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this study, an inhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum. In this assay, casein was used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis. In the absence of inhibitors, casein was hydrolysed and the solution became brown, while in the presence of metal ions such as Hg2+ and Zn2+, the hydrolysis of casein was inhibited and the solution remained blue. Both Hg2+ and Zn2+ exhibited one-phase binding curve with IC50 values of 3.217 mg/L and 0.727 mg/L, respectively. The limits of detection (LOD and limits of quantitation (LOQ for Hg were 0.241 and 0.802 mg/L, respectively, while the LOD and LOQ for Zn were 0.228 and 0.761 mg/L, respectively. The enzyme exhibited broad pH ranges for activity. The crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of Hg2+ and Zn2+ in the aquatic environments.

  5. CYP1A inhibition in fish gill filaments: A novel assay applied on pharmaceuticals and other chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Beijer, Kristina; Abrahamson, Alexandra; Brunstroem, Bjoern [Department of Environmental Toxicology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden); Brandt, Ingvar, E-mail: ingvar.brandt@ebc.uu.se [Department of Environmental Toxicology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden)

    2010-01-31

    The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from {beta}-naphthoflavone ({beta}NF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 {mu}M the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 {mu}M. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 {mu}M. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

  6. CYP1A inhibition in fish gill filaments: A novel assay applied on pharmaceuticals and other chemicals

    International Nuclear Information System (INIS)

    Beijer, Kristina; Abrahamson, Alexandra; Brunstroem, Bjoern; Brandt, Ingvar

    2010-01-01

    The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from β-naphthoflavone (βNF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 μM the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 μM. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 μM. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

  7. Exploration of the Fluorescent Properties and the Modulated Activities against Sirtuin Fluorogenic Assays of Chromenone-Derived Natural Products

    Directory of Open Access Journals (Sweden)

    Hui Wen

    2018-05-01

    Full Text Available Chromenone-derived natural products include chromones (flavone, isoflavone and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC. Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 μM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.

  8. Transient Expression of Lumbrokinase (PI239 in Tobacco (Nicotiana tabacum Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots

    Directory of Open Access Journals (Sweden)

    Alexia Dickey

    2017-01-01

    Full Text Available Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239 was produced from a plant system. Both wild-type (WT and plant codon-optimized (OP PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

  9. Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

  10. A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

    Directory of Open Access Journals (Sweden)

    Jing Lu

    Full Text Available Polo-like kinase 1 (PLK1, one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

  11. Droplet-based microfluidics for dose-response assay of enzyme inhibitors by electrochemical method.

    Science.gov (United States)

    Gu, Shuqing; Lu, Youlan; Ding, Yaping; Li, Li; Zhang, Fenfen; Wu, Qingsheng

    2013-09-24

    A simple but robust droplet-based microfluidic system was developed for dose-response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose-response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC50 (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

    Directory of Open Access Journals (Sweden)

    Mindy I Davis

    Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

  13. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and anti-tumor effects

    Science.gov (United States)

    Osada, Takuya; Berglund, Peter; Morse, Michael A.; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2013-01-01

    We recently demonstrated that Venezuelan equine encephalitis (VEE) virus-based replicon particles (VRP) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP expressing Interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and anti-tumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)) and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12 and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing anti-tumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted. PMID:22488274

  14. Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

    Science.gov (United States)

    Knudsen, Maria L; Mbewe-Mvula, Alice; Rosario, Maximillian; Johansson, Daniel X; Kakoulidou, Maria; Bridgeman, Anne; Reyes-Sandoval, Arturo; Nicosia, Alfredo; Ljungberg, Karl; Hanke, Tomás; Liljeström, Peter

    2012-04-01

    Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

  15. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA or with HIV gp140 protein antigen.

    Directory of Open Access Journals (Sweden)

    Maria L Knudsen

    Full Text Available Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  16. 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.

  17. 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.)

  18. 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.

  19. An enzyme-linked immunosorbent assay and a gold-nanoparticle based immuno chromatographic test for amatoxins using recombinant antibody

    International Nuclear Information System (INIS)

    He, Kuo; Zhao, Ruiping; Wang, Lixia; Feng, Tingting; Wei, Dong; Zhang, Xiuyuan

    2016-01-01

    The authors describe two kinds of rapid assays for the determination of amatoxins in mushrooms. The first is an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase. The second is a rapid immuno chromatographic assay that uses colloidal gold as a red label (CG-ICA). Both are based on the use of a well-characterized recombinant single chain variable fragment antibody (named scFv-A4). The half-maximum inhibition concentrations (IC50) of α-amanitin, β-amanitin and γ-amanitin are 78, 85 and 90 ng⋅mL"-"1, and the limits of detection (LODs; for IC15) are 1.9, 2.1 and 2.8 ng⋅mL"-"1. The method was applied to the determination of amanitins in mushrooms, and the LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were found to be 4.9, 6.4 and 8.3 ng⋅mL"-"1. The visual minimum detection limits of the optimized CGIA are 4 and 6 ng⋅mL"-"1 for mushroom samples. The test can be performed within 10 min. The results of the analysis of spiked samples showed that the CG-IA can rapidly and semi-quantitatively quantify amatoxins in mushroom samples on site and at low costs. (author)

  20. An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine.

    Science.gov (United States)

    Asciolla, James J; Miele, Matthew M; Hendrickson, Ronald C; Resh, Marilyn D

    2017-08-18

    Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC 50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O -acyl transferase family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. 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.

  2. A simple radiometric in vitro assay for acetylcholinesterase inhibitors

    International Nuclear Information System (INIS)

    Guilarte, T.R.; Burns, H.D.; Dannals, R.F.; Wagner, H.N. Jr.

    1983-01-01

    A radiometric method for screening acetylcholinesterase inhibitors has been described. The method is based on the production of [ 14 C]carbon dioxide from the hydrolysis of acetylcholine. The inhibitory concentration at 50% (IC50) values for several known acetylcholinesterase inhibitors were in agreement with literature values. The new radiometric method is simple, inexpensive, and has the potential for automation

  3. 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)

  4. Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

    Science.gov (United States)

    Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

    2015-10-01

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

  5. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  6. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

    Science.gov (United States)

    Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

    2005-05-31

    High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

  7. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-10-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive. © 2013 Elsevier B.V. All rights reserved.

  8. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

    Science.gov (United States)

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  9. Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors.

    Science.gov (United States)

    Zhang, Qin; Tang, Xiuwen; Hou, Fenghua; Yang, Jianping; Xie, Zhiyong; Cheng, Zhiyi

    2013-10-01

    We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC50 value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. 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 ...

  11. 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 ...

  12. 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 ...

  13. 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 ...

  14. 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

  15. A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus.

    Science.gov (United States)

    Liu, Shaohua; Song, Dongmei; Bai, Han; Lu, Weiwei; Dai, Xinxian; Hao, Chunsheng; Zhang, Zhongyang; Guo, Huijie; Zhang, Yue; Li, Xiuling

    2017-12-01

    With the promotion of inactivated poliomyelitis vaccine (IPV) and live attenuated oral poliomyelitis vaccine (OPV), the global reported cases of poliomyelitis have reduced sharply from 0.35 million in 1988 to 74 in 2015. The Polio Eradication & Endgame Strategic Plan published by WHO in 2013 included the strategy of implementation of poliovirus safe handling and containment measures to minimize the risks of facility-associated reintroduction of virus into the polio-free community to prevent the re-import of poliovirus. Toward this strategy, we produced replication-incompetent pseudovirus of poliovirus type 1, 2, 3 attenuated strains by constructing poliovirus capsid expression vectors and poliovirus replicon then transfecting HEK293T cells and developed a pseudovirus-based neutralization assay (pNA) to determine neutralizing antibody titer which is more secure, time-saving and reliable than conventional neutralization assay (cNA). By using anti-poliovirus rat serum, we demonstrated excellent correlation between neutralizing antibody titers measured by cNA and pNA. It was concluded that pNA can be a potential alternative to replace cNA as a safe and time-saving system for titer determination after live poliovirus's safekeeping. © 2017 Wiley Periodicals, Inc.

  16. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    International Nuclear Information System (INIS)

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang Leyu; Moore, Jennifer; Kuo, Ming-Shang T.; LaMarr, William A.; Ozbal, Can C.; Bhat, B. Ganesh

    2008-01-01

    Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K m = 10.5 μM). The assay was highly reproducible with an average Z' value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC 50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM - 14% conformation rate). Of the confirmed hits 172 had IC 50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal

  17. Actions of four organic acids in radix isatidis on endotoxin-neutralization investigated by kinetic turbidimetric assay.

    Science.gov (United States)

    Ma, Li; He, Ying-jun; Li, You; Gong, Mu-xin

    2012-06-01

    To investigate anti-endotoxin action of four OAs reacted with endotoxin by the LAL assay with KTA. Using a incubating kinetic tube reader and kinetic turbidimetric assay (KTA), the concentration-response time curve of endotoxin reacted with limulus amebocyte lysate (LAL) at 37 degrees C were obtained and the action of four organic acids (OAs) on it were investigated. The four OAs were benzoic acid, salicylic acid, syringic acid and 2-amino-benzoic acid from Radix isatidis. Meanwhile, the temperature variation caused by endotoxin with the four OAs was studied by the rabbit pyrogen test (RPT). It was showed that a low concentration (1 mg/mL) of the four OAs had a little effect of anti-endotoxin, and when the concentrations of the four OAs were 30 mg/mL, the endotoxin was neutralized completely. The relationships between the concentrations of endotoxin and the OAs were all linear with correlation coefficients of greater than 0.9995, indicating that the four OAs all had strong anti-endotoxin action, while syringic acid had the strongest action among the four OAs with IC50 of 12.84 mg/mL. The investigations of KTA agreed well with the results obtained by means of RPT.

  18. Bioluminescent Vibrio fischeri assays in the assessment of seasonal and spatial patterns in toxicity of contaminated river sediments

    Directory of Open Access Journals (Sweden)

    Sergio Jarque

    2016-11-01

    Full Text Available Several bacteria-based assays, notably Vibrio fischeri luminescence assays, are often used as environmental monitoring tool for toxicity in sediments that may serve as both sinks and secondary source of contamination in aquatic ecosystems. In this study, we used 30-s kinetic bioassays based on V. fischeri to evaluate the toxicity associated to sediments from five localities with different contamination inputs (Morava River and its tributary Drevnice River in the south-eastern part of the Czech Republic. Toxicity assessed as half maximal inhibitory concentration (IC50 over the course of a year-long sampling was compared in bottom sediments and freshly trapped particulate material. Standard approach based on testing of aqueous elutriates was compared with toxicity of whole sediments (contact suspension toxicity. Bottom sediments showed lower toxicity compared to freshly trapped suspended materials in all cases. On the other hand, standardized elutriates induced generally weaker effects than suspended sediments likely due to losses during the extraction process. Toxicity generally increased during winter reaching maximum peaks in early spring months in all five sites. Total organic carbon (TOC was found to be highly correlated with toxic effects. Toxicity from sites with direct industrial and agricultural water inputs also correlated with concentrations of metals, polycyclic aromatic hydrocarbons (PAHs and polychlorinated biphenyls (PCBs. Single time point sampling followed by the extraction and testing of elutriates, do not truly reflect the spatial and temporal variability in natural sediments and may lead to underestimation of ecotoxic risks.

  19. A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

    Science.gov (United States)

    Massey, Andrew J

    2018-01-01

    Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

  20. An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

    Directory of Open Access Journals (Sweden)

    Jing-Jing Wang

    2014-08-01

    Full Text Available An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam in the incubates were quantified using LC–MS/MS methods either by an internal standard (IS calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition. Keywords: LC–MS/MS, Cytochrome P450, Cocktail-probe, Inhibition assessment, Drug screenning

  1. DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

    Science.gov (United States)

    Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

    2004-05-01

    Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

  2. Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

    Science.gov (United States)

    Santillo, Michael F; Mapa, Mapa S T

    2018-02-28

    Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC 50 , 0.4-4.0 ng mL -1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g -1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

  3. Development of sensitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for monitoring bisphenol-A in canned foods and beverages.

    Science.gov (United States)

    Lu, Yang; Peterson, Joshua Richard; Gooding, John Justin; Lee, Nanju Alice

    2012-06-01

    Enzyme-linked immunosorbent assays (ELISAs) are investigated in this work for the detection of bisphenol-A (BPA), a plastic monomer and a critical contaminant in food and environment. A series of polyclonal antibodies generated in vivo using BPA-butyrate-protein conjugate and BPA-valerate-protein conjugate were evaluated on direct and indirect competitive assay formats with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate, and BPA-2-valerate). Two indirect ELISAs and one direct ELISA exhibiting high sensitivity and specificity for BPA were developed. The 50 % inhibition of antibody binding (IC(50)) values were 0.78 ± 0.01-1.20 ± 0.26 μg L(-1), and the limits of detection as measured by the IC(20) values were 0.10 ± 0.03-0.20 ± 0.04 μg L(-1). The assays were highly specific to BPA, only displaying low cross-reactivity (3-8 % for the indirect assays and 26 % for the direct assay) for 4-cumylphenol (4-CP), at pH 7.2. The degree of cross-reaction of 4-CP was influenced by the antibody/hapten conjugate combination, assay conditions, and the assay format. The assays were optimized for the analysis of BPA in canned vegetables, bottled water and carbonated drinks. The limits of quantification for these three evaluated sample types, based on the spike and recovery data, were 0.5, 2.5, and 100 μg L(-1), respectively.

  4. 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.)

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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)

  10. 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

  11. 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

  12. 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.).

  13. (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 ...

  14. 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...

  15. 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...

  16. Prediction of Multi-Target Networks of Neuroprotective Compounds with Entropy Indices and Synthesis, Assay, and Theoretical Study of New Asymmetric 1,2-Rasagiline Carbamates

    Directory of Open Access Journals (Sweden)

    Francisco J. Romero Durán

    2014-09-01

    Full Text Available In a multi-target complex network, the links (Lij represent the interactions between the drug (di and the target (tj, characterized by different experimental measures (Ki, Km, IC50, etc. obtained in pharmacological assays under diverse boundary conditions (cj. In this work, we handle Shannon entropy measures for developing a model encompassing a multi-target network of neuroprotective/neurotoxic compounds reported in the CHEMBL database. The model predicts correctly >8300 experimental outcomes with Accuracy, Specificity, and Sensitivity above 80%–90% on training and external validation series. Indeed, the model can calculate different outcomes for >30 experimental measures in >400 different experimental protocolsin relation with >150 molecular and cellular targets on 11 different organisms (including human. Hereafter, we reported by the first time the synthesis, characterization, and experimental assays of a new series of chiral 1,2-rasagiline carbamate derivatives not reported in previous works. The experimental tests included: (1 assay in absence of neurotoxic agents; (2 in the presence of glutamate; and (3 in the presence of H2O2. Lastly, we used the new Assessing Links with Moving Averages (ALMA-entropy model to predict possible outcomes for the new compounds in a high number of pharmacological tests not carried out experimentally.

  17. Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors

    Science.gov (United States)

    Seol, Yeonee; Zhang, Hongliang; Agama, Keli; Lorence, Nicholas; Pommier, Yves; Neuman, Keir C.

    2015-01-01

    Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anti-cancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors. PMID:26351326

  18. Chemosensitivity and radiosensitivity of small cell lung cancer cell lines studied by a newly developed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay

    International Nuclear Information System (INIS)

    Hida, T.; Ueda, R.; Takahashi, T.; Watanabe, H.; Kato, T.; Suyama, M.; Sugiura, T.; Ariyoshi, Y.

    1989-01-01

    The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay was developed by technically combining the human tumor clonogenic assay and the MTT assay to make the most of both assays. This assay was able to estimate the in vitro growth of cultured cell lines and of tumor cells in pleural effusion, suggesting the possibility of its use for assessment of chemosensitivity and radiosensitivity of fresh tumor samples. Multiple cell lines [including morphological and/or phenotypical in vitro converters and cisplatin (CDDP)-resistant lines] were established from three patients with small cell lung cancer at different stages of the disease. Chemosensitivity of these cell lines to four commonly used chemotherapeutic drugs was tested by the MTT hybrid assay. SK1 and SK3 lines were established from Patient S. K. before and after chemotherapy and radiotherapy, respectively. SK3/CDDP, a CDDP-resistant line derived from the SK3 line, was 30-fold more resistant to CDDP [50% inhibiting dose (IC50), 21.5 micrograms/ml] than the SK1 line. In Patient M. O., MOA2/CDDP, a CDDP-resistant line derived from MOA2 (an in vitro converter from the MO line), was 41-fold more resistant to CDDP (IC50, 37 micrograms/ml) than the parent MO line. From Patient T. M., TM1 and TM2 lines were established before and after chemotherapy, respectively. The latter showed 6-fold more resistance to CDDP than the former. Chemosensitivity of these lines to three other drugs, 4-hydroperoxycyclophosphamide, Adriamycin, and etoposide, suggested cross-resistance between CDDP and 4-hydroperoxycyclophosphamide. Radiosensitivity study was also carried out with the MTT hybrid assay. The MOA2 line was more resistant [Do, 3.0 Gy; extrapolation number (n), 4.0] than the parental MO line (Do, 1.6 Gy; n, 2.1). There was no clear difference in radiosensitivity between the cell lines established before and after radiation therapy in Patient S. K

  19. Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

  20. 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)

  1. 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)

  2. Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

    Science.gov (United States)

    Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

    2018-06-26

    Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  4. A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y).

    Science.gov (United States)

    Santillo, Michael F; Liu, Yitong

    2015-01-01

    Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells. Published by Elsevier Inc.

  5. 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.)

  6. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  7. Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay.

    Science.gov (United States)

    Wang, Xiu-Li; Zhu, Ying; Fang, Qun

    2014-01-07

    In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensin I and angiotensin II as model samples, and the repeatabilities of peak area for angiotensin I and angiotensin II were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.

  8. Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase.

    Science.gov (United States)

    Guimarães, Rafaela; Calhelha, Ricardo C; Froufe, Hugo J C; Abreu, Rui M V; Carvalho, Ana Maria; Queiroz, Maria João R P; Ferreira, Isabel C F R

    2016-01-01

    Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

  9. 123I labelled vasoactive intestinal peptide: Optimization of the radioiodination method, in vivo and in vitro assays

    International Nuclear Information System (INIS)

    Pozzi, O.R.; Sajaroff, E.O.; Edreira, M.; Gomez, S.I.; Manzini, A.

    2002-01-01

    In the framework of the CRP, our country has worked on the optimization of synthesis, quality control, in vitro and in vivo evaluation of 123 I radiopharmaceuticals based on peptides. We have worked on selective labelling procedures using prosthetic groups with the goal to create a strong carbon-halogen bond, which will be resistant to in vivo dehalogenation and other catabolic processes. The method utilizes the labelling agent, reactive with ε-amino lysine groups, N-succinimidyl 3-iodobenzoate. This conjugation agent was radiolabelled by using an organometallic intermediate to facilitate the reaction. The organometallic N-succinimidyl 3-(tri-nbutylstannyl) benzoate (ATE) was made in a three-step synthesis pathway. The yields for the reactions of this synthetic pathway were: 56.4% for the first reaction, 67% for the second, and 58% for the ATE (469 mg, 0.92 mmol). Because of only 0.1 μmol of ATE is needed for the labelling of peptides, from one batch of organic synthesis we obtained ATE to make more than 9000 labelling. The N-succinimidyl 3-(tri-n-butylstannyl) benzoate (ATE) was radiolabelled in 55-85% radiochemical yield to obtain the N-succinimidyl 3-iodobenzoate ( [ 131 I]SIB ). Parameters like reactive concentration and isolation method of the labelling agent were studied. The labelling agent [ 131 I]SIB was subsequently conjugated to a human IgG and a peptide. A chemotactic peptide was used as a model peptide. A potent chemotactic peptide N-formyl-norleucyl-leucyl-phenylalanyl-norleucyltyrosyl- lysine (fNleLFNleYK) was derivatized by reaction with the labelling agent in 59-75% of radiochemical yield. This derivatized peptide bound specifically to human polymorphonuclear leukocytes in vitro and exhibited biological activity in a superoxide production assay. Binding affinity IC 50 : 36 nM, in the displacing of [ 3 H]fMLF binding, and IC 50 : 68 nM, in the displacing of the fNleLFNleYK-[ 131 I]SIB conjugate, for the derivatized peptide were obtained. Because

  10. 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.

  11. 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

  12. 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.)

  13. Chromogenic platform based on recombinant Drosophila melanogaster acetylcholinesterase for visible unidirectional assay of organophosphate and carbamate insecticide residues

    International Nuclear Information System (INIS)

    Han Zheng; Chi Chensen; Bai Bing; Liu Gang; Rao Qinxiong; Peng Shaojie; Liu Hong; Zhao Zhihui; Zhang Dabing; Wu Aibo

    2012-01-01

    Highlight: ► A visible chromogenic platform for rapid analysis of OP and CM insecticide residues was developed. ► The assay has the capabilities of both qualitative measurement and quantitative analysis. ► The sensitivity, capabilities of resisting interferences and storage stability were desirable. ► Matrix effects were acceptable and detection performance was satisfactory in real application. - Abstract: In this study we propose a chromogenic platform for rapid analysis of organophosphate (OP) and carbamate (CM) insecticide residues, based on recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) as enzyme and indoxyl acetate as substrate. The visible chromogenic strip had the advantages identical to those of commonly used lateral flow assays (LFAs) with utmost simplicity in sample loading and result observation. After optimization, depending on the color intensity (CI) values, the well-established assay has the capabilities of both qualitative measurement via naked eyes and quantitative analysis by colorimetric reader with the desirable IC 50 values against the tested six insecticides (0.06 μg mL −1 of carbofuran, 0.28 μg mL −1 of methomyl, 0.03 μg mL −1 of dichlorvos, 31.6 μg mL −1 of methamidophos, 2.0 μg mL −1 of monocrotophos, 6.3 μg mL −1 of omethoate). Acceptable matrix effects and satisfactory detection performance were confirmed by in-parallel LC–MS/MS analysis in different vegetable varieties at various spiked levels of 10 −3 to 10 1 μg g −1 . Overall, the testified suitability and applicability of this novel platform meet the requirements for practical use in food safety management and environmental monitoring, especially in the developing world.

  14. Influence of standard and novel LTB4 analogs on human neutrophil chemotaxis measured by the multiwell cap assay.

    Science.gov (United States)

    Psychoyos, S; Uziel-Fusi, S; Bhagwat, S; Morrissey, M M

    1989-11-30

    Standard and novel LTB4 analogs were tested for neutrophil chemoattractant activity using the multiwell cap assay (Evans et al. (1986) Biosc. Rep. 6, 1041). The assay uses disposable equipment and measures chemotaxis by the number of cells able to migrate across the full thickness of cellulose nitrate filters. Under standard conditions (90 min incubation at 37 degrees C in buffer containing 2% bovine albumin), LTB4 and 6-cis-LTB1 had EC50 values of 3.5 and 15,000 nM, respectively. 20-hydroxy-LTB4 was equipotent with LTB4 and exhibited a similar biphasic chemotactic response, however, only one third of the number of cells migrated through the filter. 20-carboxy-LTB4 was inactive up to 1,000 nM. 5-desoxy-((6,7)-cis-cyclopropyl)-LTB2, (6,7)-benzo-LTB2 and 5-desoxy-(8,10)-LTB2 had EC50 values of 11,300, 50,000 and 84,000 nM, respectively. Checkerboard analysis indicated a chemokinetic component of 42% for LTB4 at a concentration causing peak chemotaxis. Reduction of albumin in the buffer to 0.5% increased the apparent potencies of LTB4 and 6-cis-LTB1 five-fold. Since LTB4 is a mediator of inflammation, various anti-inflammatory agents were tested at peak concentrations observed in vivo for in vitro inhibition of LTB4-stimulated chemotaxis in the presence of 0.5% albumin. Under the conditions of the assay, chloroquine diphosphate, dexamethasone, indomethacin, penicillamine, piroxicam and diclofenac sodium were inactive; gold sodium thiomalate was inhibitory (IC50 = 20 microM).

  15. Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil.

    Science.gov (United States)

    Xu, Jing; Zhang, Yuanyang; Yi, Jian; Meng, Meng; Wan, Yuping; Feng, Caiwei; Wang, Shanliang; Lu, Xiao; Xi, Rimo

    2010-10-01

    Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 μg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 μg L(-1) and 19.6 μg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

  16. Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1

    Science.gov (United States)

    Ku, Hye-Jin; Park, Myeong Soo; Lee, Ju-Hoon

    2015-01-01

    A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2’, repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2’, and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 105 CFU/μg, 1.3 × 101 CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria. PMID:25691882

  17. Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-enzymatic Properties against Urease by an ESI-MS Based Assay

    Directory of Open Access Journals (Sweden)

    Sherif T. S. Hassan

    2017-04-01

    Full Text Available For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS and its bioactive constituent protocatechuic acid (PCA, have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC50 values of 0.92 and 1.43 µg∙mL−1, respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC50 value of 82.4 µg∙mL−1. This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

  18. Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-enzymatic Properties against Urease by an ESI-MS Based Assay.

    Science.gov (United States)

    Hassan, Sherif T S; Švajdlenka, Emil; Berchová-Bímová, Kateřina

    2017-04-30

    For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC 50 values of 0.92 and 1.43 µg∙mL -1 , respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC 50 value of 82.4 µg∙mL -1 . This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

  19. Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

    Science.gov (United States)

    Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

    2015-01-01

    Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

  20. Characterization of the commercially-available fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a marker for chloroquine resistance and uptake in a 96-well plate assay.

    Directory of Open Access Journals (Sweden)

    Cheryl C Y Loh

    Full Text Available Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.

  1. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

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

  2. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

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

  3. PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501-and pHT beta-related replicons associated with glycopeptide resistance and stabilizing toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Rosvoll, T.C.S.; Pedersen, T.; Sletvold, H.

    2010-01-01

    A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHT beta (n=14) were observed in 83% of the strains, while pS86, pCF10, pA...

  4. Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

    Science.gov (United States)

    Gul, Sheraz; Brown, Richard; May, Earl; Mazzulla, Marie; Smyth, Martin G; Berry, Colin; Morby, Andrew; Powell, David J

    2004-11-01

    DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

  5. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

    Science.gov (United States)

    Shabani, R; Ashtari, K; Behnam, B; Izadyar, F; Asgari, H; Asghari Jafarabadi, M; Ashjari, M; Asadi, E; Koruji, M

    2016-06-01

    Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture. © 2015 Blackwell Verlag GmbH.

  6. Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.

    Science.gov (United States)

    Bradford, Seth S; Ross, Martin James; Fidai, Insiya; Cowan, James A

    2014-06-01

    The complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all-D-amino acid form of this metallopeptide, complex 2-Cu, are reported and compared with those of complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =7.9 ± 1.2 μM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 μM and cytotoxicity exceeding 100 μM. RT-PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three-day dosing increments. This study shows the influence that the α-carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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.

  8. In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors

    International Nuclear Information System (INIS)

    Toomey, R.E.; Goode, R.L.; Petrow, V.; Neubauer, B.L.

    1991-01-01

    An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of [3H]-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for [3H]-T metabolites. Intraprostatic [3H]-T and [3H]-DHT reached peak levels within 5 min after injection of [3H]-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of [3H]-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of [3H]-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat

  9. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    Directory of Open Access Journals (Sweden)

    Ruchy Jain

    2014-05-01

    Full Text Available Avicennia marina (AM exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT causing androgenic alopecia (AGA. An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs, the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1 inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C.

  10. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2009-01-01

    Full Text Available To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95, which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

  11. Antihypertensive Effects, Molecular Docking Study, and Isothermal Titration Calorimetry Assay of Angiotensin I-Converting Enzyme Inhibitory Peptides from Chlorella vulgaris.

    Science.gov (United States)

    Xie, Jingli; Chen, Xujun; Wu, Junjie; Zhang, Yanyan; Zhou, Yan; Zhang, Lujia; Tang, Ya-Jie; Wei, Dongzhi

    2018-02-14

    The aim of this work is to explore angiotensin I-converting enzyme (ACE) inhibitory peptides from Chlorella vulgaris (C. vulgaris) and discover the inhibitory mechanism of the peptides. After C. vulgaris proteins were gastrointestinal digested in silico, several ACE inhibitory peptides with C-terminal tryptophan were screened. Among them, two novel noncompetitive ACE inhibitors, Thr-Thr-Trp (TTW) and Val-His-Trp (VHW), exhibited the highest inhibitory activity indicated by IC 50 values 0.61 ± 0.12 and 0.91 ± 0.31 μM, respectively. Both the peptides were demonstrated stable against gastrointestinal digestion and ACE hydrolysis. The peptides were administrated to spontaneously hypertensive rats (SHRs) in the dose 5 mg/kg body weight, and VHW could decrease 50 mmHg systolic blood pressure of SHRs (p < 0.05). Molecular docking displayed that both TTW and VHW formed six hydrogen bonds with active site pockets of ACE. Besides, isothermal titration calorimetry assay discovered that VHW could form more stable complex with ACE than TTW. Therefore, VHW was an excellent ACE inhibitor.

  12. Rapid determination of ractopamine residues in edible animal products by enzyme-linked immunosorbent assay: development and investigation of matrix effects.

    Science.gov (United States)

    Zhang, Yan; Wang, Fengxia; Fang, Li; Wang, Shuo; Fang, Guozhen

    2009-01-01

    To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC(50) of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 mug/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R(2) > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

  13. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Directory of Open Access Journals (Sweden)

    Helton J Wiggers

    Full Text Available A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i in the low micromolar range (3-60 µM acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM, yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE of 0.33 kcal mol(-1 atom(-1

  14. Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

    Science.gov (United States)

    Wiggers, Helton J; Rocha, Josmar R; Fernandes, William B; Sesti-Costa, Renata; Carneiro, Zumira A; Cheleski, Juliana; da Silva, Albérico B F; Juliano, Luiz; Cezari, Maria H S; Silva, João S; McKerrow, James H; Montanari, Carlos A

    2013-01-01

    A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound

  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. 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.)

  18. Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

    Science.gov (United States)

    Cui, Xiping; Vasylieva, Natalia; Wu, Panpan; Barnych, Bogdan; Yang, Jun; Shen, Ding; He, Qiyi; Gee, Shirley J; Zhao, Suqing; Hammock, Bruce D

    2017-10-17

    Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC 50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

  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. Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

    Science.gov (United States)

    Limame, Ridha; Wouters, An; Pauwels, Bea; Fransen, Erik; Peeters, Marc; Lardon, Filip; De Wever, Olivier; Pauwels, Patrick

    2012-01-01

    Background Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Methodology/Principal Findings Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. Conclusions/Significance The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different

  1. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

    Directory of Open Access Journals (Sweden)

    Michael J Smout

    Full Text Available BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

  2. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Directory of Open Access Journals (Sweden)

    Ridha Limame

    Full Text Available BACKGROUND: Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (pathobiological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer and A549 (lung cancer cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964. Cytotoxic action by paclitaxel (0-100 nM correlated well with SRB (Rho>0.95 with similar IC(50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90 and optical density (OD measurement of extracted dye (Rho>0.95. Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95. Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE: The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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)

  11. 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

  12. Discriminative power of an assay for automated in vitro screening of teratogens

    DEFF Research Database (Denmark)

    Walmod, Peter S; Gravemann, Ute; Nau, Heinz

    2004-01-01

    -trans-retinoic acid, pentyl-4-yn-valproic acid, saccharin, salicylic acid and valproic acid. All compounds, with the exception of dimethadione inhibited proliferation in a linear dose-dependent manner, and there were statistically significant compound class-dependent differences between the IC(50)-values...... to teratogenicity were: 5-bromo-2(')-deoxyuridine, 6-aminonicotinamide, acrylamide, boric acid, D-(+)-camphor, dimethadione, dimethyl phthalate, diphenhydramine, hydroxyurea, isobutyl-ethyl-valproic acid, lithium chloride, methyl mercury chloride, methotrexate, methoxyacetic acid, penicillin G, all...

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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...

  18. 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...

  19. Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays, Especially on Different Superoxide Anion Radical Generation Systems.

    Science.gov (United States)

    Sugahara, Shintaro; Ueda, Yuto; Fukuhara, Kumiko; Kamamuta, Yuki; Matsuda, Yasushi; Murata, Tatsuro; Kuroda, Yasuhiro; Kabata, Kiyotaka; Ono, Masateru; Igoshi, Keiji; Yasuda, Shin

    2015-11-01

    Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot-water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot-water extract was similarly demonstrated among these extracts. Varying concentrations of the hot-water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC50 28.1 μg/mL), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (EC50 23.7 μg/mL), galvinoxyl radical (EC50 3.06 μg/mL), and chlorpromazine cation radical (EC50 475 μg/mL). The yacon tea-leaf extract further demonstrated superoxide anion (O2(-)) radical scavenging effects in the phenazine methosulfate-NADH-nitroblue tetrazolium (EC50 64.5 μg/mL) and xanthine oxidase assay systems (EC50 20.7 μg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea-leaf extract could suppress the cellular O2(-) radical generation (IC50 65.7 μg/mL) in a phorbol 12-myristate 13-acetate-activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders. Yacon has been considered to be a potent alternative food source for patients who require a dietary cure in regional area, while the leaf part has been provided and consumed as an herbal tea in local markets. We demonstrated here potent antioxidative effects of the tea

  20. 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

  1. 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)

  2. 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

  3. Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin

    Directory of Open Access Journals (Sweden)

    Fang Zhao

    2016-02-01

    Full Text Available Voltage-gated sodium channels (VGSCs are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1–Nav1.9, Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR membrane potential (FMP blue dye. In HEK-293 cells heterologously expressing hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNav1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.

  4. Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.

    Science.gov (United States)

    Zhao, Fang; Li, Xichun; Jin, Liang; Zhang, Fan; Inoue, Masayuki; Yu, Boyang; Cao, Zhengyu

    2016-02-16

    Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1-Nav1.9), Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNav1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.

  5. Novel hydroxyamides and amides containing D-glucopyranose or D-fructose units: Biological assays in MCF-7 and MDST8 cell lines.

    Science.gov (United States)

    Carreiro, Elisabete P; Costa, Ana R; Cordeiro, Maria M; Martins, Rute; Pires, Tiago O; Saraiva, Mafalda; Antunes, Célia M; Burke, Anthony J

    2016-02-01

    A novel library of 15 compounds, hydroxyamides and amides containing a β-D-glucopyranose (D-Gluc) or a β-D-fructose (D-Fruc) units was designed and synthesized for antiproliferative assays in breast (MCF-7) and colon (MDST8) cancer cell lines. Twelve of them were hydroxyamides and were successfully synthesized from β-D-glucuronic acid (D-GluA). Six of these hydroxyamides which were acetylated hydroxy-β-D-glucopyranuronamide 2a-2f (1st Family) and the other six were their respective isomers, that is, hydroxy-β-D-fructuronamide 3a-3f (2nd Family), obtained by acid-base catalyzed isomerization. These compounds have the general structure, D-Gluc-C=ONH-CHR-(CH2)n-OH and D-Fruc-C=ONH-CHR-(CH2)n-OH, where R=an aromatic, alkyl or a hydrogen substituent, with n=0 or 1. Eight of these contained a chiral aminoalcohol group. Three compounds were amides containing a D-glucopyranose unit (3rd Family). SAR studies were conducted with these compounds. Antiproliferative studies showed that compound 4a, the bromo-amide containing the β-D-glucopyranose ring, potently inhibits the proliferation of the MDST8 cells. Five compounds (2e, 2f, 3d, 3e, and 3f) were shown to potently selectively inhibit the proliferation of the MCF-7 cells. Compound 4b was the only one showing inhibition in both cell lines. In general, the more active compounds were the amides and hydroxyamides containing the β-D-fructose moiety, and containing an alkyl group or hydrogen. Half-inhibitory concentrations (IC50) of between 0.01 and 10 μM, were observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Synthesis of novel amides based on acridone scaffold with interesting antineoplastic activity.

    Science.gov (United States)

    Mahajan, Anand A; Rane, Rajesh A; Amritkar, Anish A; Naphade, Shital S; Miniyar, Pankaj B; Bangalore, Pavan Kumar; Karpoormath, Rajshekhar

    2015-01-01

    In search of novel cytotoxic agents based on acridone scaffold, twenty five derivatives of acridone-2- carboxamide were synthesized and evaluated against a panel of eleven cancer cell lines by using MTT assay. Amides, A5 and A8 (IC50 = 0.3 µM) exhibited good cytotoxicity against MCF7. Compound A22 (IC50 = 4.3 µM) was found to be selectively cytotoxic against cancer cell line MCF7 and KB403. Particularly, promising cytotoxic activities were shown by amides A6 (IC50 = 0.7 µM), A16 (IC50 = 6.3 µM), A8 (IC50 = 0.9 µM ), A21 (IC50 = 1.3 µM), A5 (IC50 = 2.9 µM), A8 (IC50 = 2.8 µM), A14 (IC50 = 0.8 µM), A9 (IC50 = 0.8 µM) and A8 (IC50 = 0.4 µM) against cell lines; PA1, WRL68, CaCO2, TK-10, K-562, PC-3, HOP-92, ECV-304 and UACC-257, respectively. The favorable cytotoxic profile and non-toxicity towards normal human cells displayed by the derivative revealed their potential for further anticancer drug developments.

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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...

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin A.

    Science.gov (United States)

    Köhn, Maja; Benito, Juan M; Ortiz Mellet, Carmen; Lindhorst, Thisbe K; García Fernández, José M

    2004-06-07

    The affinities of the mannose-specific lectin concanavalin A (Con A) towards D-glucose-centred mannosyl clusters differing in the anomeric configuration of the monosaccharide core, nature of the bridging functional groups and valency, have been measured by a competitive enzyme-linked lectin assay. Pentavalent thioether-linked ligands (5 and 7) were prepared by radical addition of 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranose to the corresponding penta-O-allyl-alpha- or -beta-D-glucopyranose, followed by deacetylation. The distinct reactivity of the anomeric position in the D-glucose scaffold was exploited in the preparation of a tetravalent cluster (10) that keeps a reactive aglyconic group for further manipulation, including incorporation of a reporter group or attachment to a solid support. Hydroboration of the double bonds in the penta-O-allyl-alpha-D-glucopyranose derivative and replacement of the hydroxy groups with amine moieties gave a suitable precursor for the preparation of pentavalent and 15-valent mannosides through the thiourea-bridging reaction (17 and 20, respectively). The diastereomeric 1-thiomannose-coated clusters 5 and 7 were demonstrated to be potent ligands for Con A, with IC(50) values for the inhibition of the Con A-yeast mannan association indicative of 6.4- and 5.5-fold increases in binding affinity (valency-corrected values), respectively, relative to the value for methyl alpha-D-mannopyranoside. The tetravalent cluster 10 exhibited a valency-corrected relative lectin-binding potency virtually identical to that of the homologous pentavalent mannoside 7. In sharp contrast, replacement of the 1-thiomannose wedges of 5 with alpha-D-mannopyranosylthioureido units (17) virtually abolished any multivalent or statistic effects, with a dramatic decrease of binding affinity. The 15-valent ligand 20, possessing classical O-glycosidic linkages, exhibited a twofold increase in lectin affinity relative to the penta-O-(thioglycoside) 5; it is

  5. Identification of high-affinity anti-IL-1 α autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1 α

    International Nuclear Information System (INIS)

    Mae, N.; Liberato, D.J.; Chizzonite, R.; Satoh, H.

    1991-01-01

    A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 α (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 α added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 α added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 α in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 α binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 α-specific IgG. The binding of 125I-labeled IL-1 α to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 x 10(-11) M) but not by unlabeled IL-1 β. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 α-specific IgGs was 4.7 x 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 α in human serum by various assay systems and also could be a regulator of circulating IL-1 α

  6. Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

    Science.gov (United States)

    Caillaud, A; Eixarch, H; de la Iglesia, P; Rodriguez, M; Dominguez, L; Andree, K B; Diogène, J

    2012-01-01

    The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

  7. 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)

  8. 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

  9. 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.

  10. 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...

  11. 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...

  12. Polyphenols and Red Wine as Antioxidants against Peroxynitrite and other Oxidants

    Directory of Open Access Journals (Sweden)

    LAURA B VALDEZ

    2004-01-01

    Full Text Available The antioxidant capacity of polyphenols (+-catechin, (--epicatechin and myricetin, and of different types of red wines (Cabernet Sauvignon, Malbec and blended wine was evaluated by three assays. (a NADH oxidation by peroxynitrite (ONOO-: the ONOO- scavenging activity was higher for myricetin (IC50=35 µM than for (+-catechin (IC50=275 µM and (--epicatechin (IC50=313 µM. (b Peroxynitrite initiated chemiluminescence in rat liver homogenate: (--epicatechin (IC50=7.0 µM and (+-catechin (IC50=13 µM were more potent than myricetin (IC50=20 µM in inhibiting the chemiluminescence signal. (c Lucigenin chemiluminescence in aortic rings: (--epicatechin (IC50=15 µM and (+-catechin (IC50=18 µM showed higher antioxidant capacity than myricetin (IC50=32 µM. All the assayed red wines were able to scavenge the oxidants and free radical species that generate the signal in each assay. Cabernet Sauvignon was the red wine with the highest antioxidant capacity in comparison with Malbec and blended wine. It is concluded that the use of sensitive biological systems (as the aortic ring chemiluminescence provides important information in addition to the results from chemical (NADH oxidation by peroxynitrite and biochemical (homogenate chemiluminescence assays and offers advances in the physiological role of polyphenols

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    Science.gov (United States)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  20. Analysis of pirlimycin residues in beef muscle, milk and honey by a biotin-streptavidin-amplified enzyme-linked immunosorbent assay

    Science.gov (United States)

    Food contamination caused by veterinary drug residues is a world-wide public health concern and requires continuous monitoring. In this paper, we describe a biotin–streptavidin-amplified ELISA (BA-ELISA) for detecting pirlimycin residues in beef, milk, and honey. The IC50 value of the BA-ELISA was...

  1. 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

  2. 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.

  3. A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

    Directory of Open Access Journals (Sweden)

    Matthew Brecher

    2017-05-01

    Full Text Available The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2 in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV, West Nile virus (WNV, and Yellow fever virus (YFV on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and

  4. 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,.

  5. 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.

  6. 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...

  7. 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

  8. 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.

  9. 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)

  10. 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

  11. 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

  12. 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

  13. Anti-HIV-1 integrase activity of medicinal plants used as self medication by AIDS patients

    Directory of Open Access Journals (Sweden)

    Sopa Kummee

    2006-07-01

    Full Text Available The extracts of selected medicinal plants used as self medication by AIDS patients were investigated for their inhibitory activities against HIV-1 integrase (HIV-1 IN using the multiplate integration assay (MIA. Of these, the water extract of Eclipta prostrata (whole plant exhibited the most potent inhibitory activity with an IC50 value of 4.8 μg/ml, followed by the methanol extract of Eclipta prostrata (whole plant, IC50 = 21.1 μg/ ml, the water extract of Barleria lupulina (stem, IC50 = 26.4 μg/ml, the chloroform extract of Barleria lupulina (stem, IC50 = 33.0 μg/ml, the methanol extract of Barleria lupulina (stem, IC50 = 38.2 μg/ml and the chloroform extract of Piper betle (leaf, IC50 = 39.3 μg/ml, respectively.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.)

  20. 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

  1. 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)

  2. 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

  3. 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

  4. 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

  5. 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...

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Robust Synthesis of Ciprofloxacin-Capped Metallic Nanoparticles and Their Urease Inhibitory Assay.

    Science.gov (United States)

    Nisar, Muhammad; Khan, Shujaat Ali; Qayum, Mughal; Khan, Ajmal; Farooq, Umar; Jaafar, Hawa Z E; Zia-Ul-Haq, Muhammad; Ali, Rashid

    2016-03-25

    The fluoroquinolone antibacterial drug ciprofloxacin (cip) has been used to cap metallic (silver and gold) nanoparticles by a robust one pot synthetic method under optimized conditions, using NaBH₄ as a mild reducing agent. Metallic nanoparticles (MNPs) showed constancy against variations in pH, table salt (NaCl) solution, and heat. Capping with metal ions (Ag/Au-cip) has significant implications for the solubility, pharmacokinetics and bioavailability of fluoroquinolone molecules. The metallic nanoparticles were characterized by several techniques such as ultraviolet visible spectroscopy (UV), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) methods. The nanoparticles synthesized using silver and gold were subjected to energy dispersive X-ray tests in order to show their metallic composition. The NH moiety of the piperazine group capped the Ag/Au surfaces, as revealed by spectroscopic studies. The synthesized nanoparticles were also assessed for urease inhibition potential. Fascinatingly, both Ag-cip and Au-cip NPs exhibited significant urease enzyme inhibitory potential, with IC50 = 1.181 ± 0.02 µg/mL and 52.55 ± 2.3 µg/mL, compared to ciprofloxacin (IC50 = 82.95 ± 1.62 µg/mL). MNPs also exhibited significant antibacterial activity against selected bacterial strains.

  13. 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)

  14. 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.

  15. 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 ...

  16. 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...

  17. 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

  18. 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…

  19. 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.)

  20. 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)

  1. 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.

  2. 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...

  3. 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...

  4. 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...

  5. 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.)

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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)

  15. 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)

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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;}

  2. 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

  3. 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).

  4. 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

  5. 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.

  6. 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%.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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)

  12. Synthesis and DPPH scavenging assay of reserpine analogues, computational studies and in silico docking studies in AChE and BChE responsible for Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Muhammad Yar

    2015-03-01

    Full Text Available Alzheimer's disease (AD is a fast growing neurodegenerative disorder of the central nervous system and anti-oxidants can be used to help suppress the oxidative stress caused by the free radicals that are responsible for AD. A series of selected synthetic indole derivatives were biologically evaluated to identify potent new antioxidants. Most of the evaluated compounds showed significant to modest antioxidant properties (IC50 value 399.07 140.0±50 µM. Density Functional Theory (DFT studies were carried out on the compounds and their corresponding free radicals. Differences in the energy of the parent compounds and their corresponding free radicals provided a good justification for the trend found in their IC50 values. In silico, docking of compounds into the proteins acetylcholinesterase (AChE and butyrylcholinesterase (BChE, which are well known for contributing in AD disease, was also performed to predict anti-AD potential.

  13. 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.

  14. 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

  15. 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.

  16. Ex Vivo Drug Sensitivity Profiles of Plasmodium falciparum Field Isolates from Cambodia and Thailand, 2005 to 2010, Determined by a Histidine-Rich Protein-2 Assay

    Science.gov (United States)

    2012-06-13

    References 1. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative assessment of antimalarial activity in vitro by a semiautomated...potentially concerning. As before, un- regulated availability of antimalarial medications during that period may have been a contributing factor. Not- ably...et al. observed a similar dip in IC50 values for a range of antimalarial drugs in 2006, and attributed the observa- tion to sampling bias since most

  17. 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.

  18. 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

  19. 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.)

  20. 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 ...

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  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. 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

  11. 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)

  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. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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...

  4. 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...

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    Science.gov (United States)

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

  12. Organization of human replicon: singles or zipping couples?

    Czech Academy of Sciences Publication Activity Database

    Ligasová, Anna; Raška, Ivan; Koberna, Karel

    2009-01-01

    Roč. 165, č. 3 (2009), s. 204-213 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA304/05/0374; GA ČR GA204/07/0133; GA AV ČR KJB500390701; GA AV ČR KAN200520801 Grant - others:MŠk(CZ) LC535; Wellcome Trus(XE) 075834/Z/04/Z Program:LC Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50110509 Keywords : Chromatin * Replication * Replisomes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.673, year: 2009

  13. 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

  14. 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.

  15. 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)

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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)

  5. Synthesis and biological activities of substituted N ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... hydroxyl radical (OH) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays and the IC50 values ... good polydentate- chelating agents, which can form a ..... iron transport and storage due to various exogenous and.

  6. Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems.

    Science.gov (United States)

    Satoh, Kanako; Nonaka, Ryouichi; Ohyama, Ken-ichi; Nagai, Fumiko; Ogata, Akio; Iida, Mitsuru

    2008-03-01

    Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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)

  13. 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.

  14. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    Science.gov (United States)

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

  15. 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)

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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)

  6. 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.

  7. 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.)

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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)

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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)

  19. 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.

  20. 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

  1. 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

  2. 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.)

  3. Rapid quantitative assay for chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  4. Neutron Generators for Spent Fuel Assay

    International Nuclear Information System (INIS)

    Ludewigt, Bernhard A.

    2010-01-01

    The Next Generation Safeguards Initiative (NGSI) of the U.S. DOE has initiated a multi-lab/university collaboration to quantify the plutonium (Pu) mass in, and detect the diversion of pins from, spent nuclear fuel (SNF) assemblies with non-destructive assay (NDA). The 14 NDA techniques being studied include several that require an external neutron source: Delayed Neutrons (DN), Differential Die-Away (DDA), Delayed Gammas (DG), and Lead Slowing-Down Spectroscopy (LSDS). This report provides a survey of currently available neutron sources and their underlying technology that may be suitable for NDA of SNF assemblies. The neutron sources considered here fall into two broad categories. The term 'neutron generator' is commonly used for sealed devices that operate at relatively low acceleration voltages of less than 150 kV. Systems that employ an acceleration structure to produce ion beam energies from hundreds of keV to several MeV, and that are pumped down to vacuum during operation, rather than being sealed units, are usually referred to as 'accelerator-driven neutron sources.' Currently available neutron sources and future options are evaluated within the parameter space of the neutron generator/source requirements as currently understood and summarized in section 2. Applicable neutron source technologies are described in section 3. Commercially available neutron generators and other source options that could be made available in the near future with some further development and customization are discussed in sections 4 and 5, respectively. The pros and cons of the various options and possible ways forward are discussed in section 6. Selection of the best approach must take a number of parameters into account including cost, size, lifetime, and power consumption, as well as neutron flux, neutron energy spectrum, and pulse structure that satisfy the requirements of the NDA instrument to be built.

  5. BROMATOMATRIC ASSAY OF GATIFLOXACIN IN PHARMACEUTICALS

    Directory of Open Access Journals (Sweden)

    KALSANG THARPA

    2008-09-01

    Full Text Available Three new, simple, and cost-effective visible spectrophotometric methods are proposed for determination of gatifloxacin (GTF using bromate-bromide mixture, and three dyes, methyl orange, indigocarmine and thymol blue, as reagents.The methods engross the addition of a known excess of bromate-bromide mixture to GTF in hydrochloric acid medium followed by determination of residual bromine by reacting with a fixed amount of either methyl orange andmeasuring the absorbance at 520 nm (method A or indigo carmine and measuring the absorbance at 610 nm (method B or thymol blue and measuring the absorbance at 550 nm (method C. In all the methods, the amount of brominereacted corresponds to the amount of GTF, and the absorbance is found to increase linearly with the concentration of GTF. Under the optimum conditions, GTF could be assayed in the concentration range 0.25-1.5, 0.5-6.0, and 0.5-10μg/mL by method A, method B and method C, respectively. The apparent molar absorptivities are calculated to be 1.6x105, 4.0x104 and 3.2x104 L mol-1 cm-1 for the method A, method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0025, 0.010 and 0.012 μg/cm2. The intra-day and inter-day precision, and the accuracy of the methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the determination of GTF in pharmaceutical preparations without the interference from any of the pharmaceutical adjuvants.

  6. 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....

  7. Measurement of thyroid-stimulating autoantibodies using a radioreceptor-assay (TRAK-assay)

    International Nuclear Information System (INIS)

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

    1983-01-01

    Autoantibodes against TSH-receptors were determined with a new radioreceptorassay (TRAK-assay) in 37 patients with untreated toxic diffus goitre and in 73 patients with non immunogenic thyroid diseases (41 healthy controls, respectively patients with euthyroid diffus or nodular goitres as well as 32 patients with toxic nodular goitre). The upper limit of normal is a TRAK-Titer of 20% without considerable fluctuations during titers in follow-up. According to this preliminary results it seems to be recommendable to treat patients with TRAK-titer F 20% (expected risk of relapse) primarily by operation or radioiodine. (orig.) [de

  8. The glucose oxidase-peroxidase assay for glucose

    Science.gov (United States)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  9. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  10. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Science.gov (United States)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  11. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...

  12. Developing a yeast-based assay protocol to monitor total ...

    African Journals Online (AJOL)

    A yeast-based assay protocol developed for detecting oestrogenic activity in activated sludge (AS) supernatant is described. The protocol used Saccharomyces cerevisiae construct RMY/ER-ERE with human oestrogen receptor (ERα) and lacZ reporter genes, and was developed by modifying existing assays for use with AS ...

  13. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    International Nuclear Information System (INIS)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

    2015-01-01

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges

  14. a positive control plasmid for reporter gene assay

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-04

    Jul 4, 2008 ... qualification as a positive control for luciferase reporter gene assays. Key words: Reporter gene plasmid, luciferase assay, cytomegalovirus promoter/enhancer, human melanoma cell line. INTRODUCTION. Reporter genes, often called reporters, have become a precious tool in studies of gene expression ...

  15. 40 CFR 79.64 - In vivo micronucleus assay.

    Science.gov (United States)

    2010-07-01

    ... micronucleus assay. (a) Purpose. The micronucleus assay is an in vivo cytogenetic test which uses erythrocytes... that, because it contains RNA, can be differentiated by appropriate staining techniques from a normochromatic erythrocyte (NCE), which lacks RNA. In one to two days, a PCE matures into a NCE. (c) Test method...

  16. An improved plating assay for determination of phage titer | Yang ...

    African Journals Online (AJOL)

    In this study, an improved plating assay was developed for detection of the number of recombinant phage Cap-T7 present in a test solution at a certain dilution point by counting the plaque forming units. The data demonstrated that the improved plating assay is fast, useful, and convenient for the determination of the phage ...

  17. A competitive enzyme linked immunosorbent assay for the ...

    African Journals Online (AJOL)

    A competitive enzyme linked immunosorbent assay for the determination of diminazene residues in animal tissues. ... After six washes with buffer, enzyme activity was determined by adding tetramethyl-benzidine and hydrogen peroxide as substrate. The assay detection limits for diminazene were 2.4 ng/g in muscle, 2.5 ...

  18. Radioenzymatic assay for trimethoprim in very small serum samples.

    OpenAIRE

    Yogev, R; Melick, C; Tan-Pong, L

    1985-01-01

    A modification of the methotrexate radioassay kit (supplied by New England Enzyme Center) enabled determination of trimethoprim levels in 5-microliter serum samples. An excellent correlation between this assay and high-pressure liquid chromatography assay was found. These preliminary results suggest that with this method rapid determination of trimethoprim levels in very small samples (5 to 10 microliters) can be achieved.

  19. Radioenzymatic assay for trimethoprim in very small serum samples

    International Nuclear Information System (INIS)

    Yogev, R.; Melick, C.; Tan-Pong, L.

    1985-01-01

    A modification of the methotrexate radioassay kit (supplied by New England Enzyme Center) enabled determination of trimethoprim levels in 5-microliter serum samples. An excellent correlation between this assay and high-pressure liquid chromatography assay was found. These preliminary results suggest that with this method rapid determination of trimethoprim levels in very small samples (5 to 10 microliters) can be achieved

  20. Metabolism Retrofit Strategies for ToxCast Assays (BOSC)

    Science.gov (United States)

    The EPA’s ToxCast program utilizes a wide variety of high-throughput screening assays (HTS) to assess chemical perturbations of molecular and cellular endpoints. A limitation of many HTS assays used for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes...

  1. Reagents for the assay of cardenolide glycosides and aglycones

    International Nuclear Information System (INIS)

    Wilkinson, S.

    1976-01-01

    Some novel reagents are described for use in the radioimmunoassay of the 3-glycone derivatives of cardenolides (cardiac glycosides) and more especially digoxin, digitoxin, gitoxin, periplocin and lanatosides. Using these reagents these cardenolides and their derivatives may be assayed both in aqueous solution and in urine. A method is also described for performing such assays, including a suitable kit. (U.K.)

  2. Assay of spent fuel by a simple reactivity method

    International Nuclear Information System (INIS)

    Lee, D.M.; Lindquist, L.O.

    1982-01-01

    A new method for the assay of spent-fuel assemblies has been developed that eliminates the need for external isotopic neutron sources, yet retains the advantages of an active interrogation system. The assay is accomplished by changing the reactivity of the system and correlating the measurements to burnup. 7 figures

  3. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

    2015-01-15

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  4. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    Science.gov (United States)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

    2015-01-01

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  5. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

  6. 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

  7. A radioreceptor assay for measurement of plasma glucocorticoid binding activity

    International Nuclear Information System (INIS)

    Fan Jie

    1990-01-01

    A radioreceptor assay (RRA) for plasma glucocorticoid binding activity (GCBA) has been developed using glucocorticoid receptor in rat thymocytes. Unlike other assays for natural and certain synthetic corticosteroids, RRA measures the GCBA of all natural and synthetic GC in plasma. The range of standard curve was 0 ∼ 1.00 mg/L. The sensitivity was 0.01 mg/l. The recovery rate was 92.1%, and the intra and inter assay CV was 0.7% (n = 3) and 4.4% (n = 3) respectively. The level of corticosterone in 9 rat plasma samples was determined by RRA and CBG-isotope binding assay. There was a general correlation over a wide range between the values determined by the two assays (r = 0.95; P < 0.001). The measuring condition was described in detail

  8. Establishment of immunoradiometric assay for free prostate-specific antigen

    International Nuclear Information System (INIS)

    Ma Lianxue

    2009-01-01

    An immunoradiometric assay (IRMA) of free prostate specific antigen (F-PSA) in serum was established. One monoclonal antibody against total PSA (T-PSA) was coated on the plastic tubes, the other against F-PSA was labeled with 125 I. The sensitivity of assay was 0.04 μg/L (n=20, +2s), the CVs were 2.9%-4.0% for the intra-assay and 3.5%-10.5% for the inter-assay and the average recovery was 102.7%. The correlative equation comparing with the FPSA-RIA (CIS BIO) is y=0.965 1 χ -0.001 1, and r=0.996 4. This F-PSA IRMA is a sensitive and precise method in detecting F-PSA and fit for the vitro assay. (authors)

  9. Substrate coated with receptor and labelled ligand for assays

    International Nuclear Information System (INIS)

    1980-01-01

    Improvements in the procedures for assaying ligands are described. The assay consists of a polystyrene tube on which receptors are present for both the ligand to be assayed and a radioactively labelled form of the ligand. The receptors on the bottom portion of the tube are also coated with labelled ligands, thus eliminating the necessity for separate addition of the labelled ligand and sample during an assay. Examples of ligands to which this method is applicable include polypeptides, nucleotides, nucleosides and proteins. Specific examples are given in which the ligand to be assayed is digoxin, the labelled form of the ligand is 3-0-succinyl digoxyigenin tyrosine ( 125 I) and the receptor is digoxin antibody. (U.K.)

  10. Nondestructive assay system development for a plutonium scrap recovery facility

    International Nuclear Information System (INIS)

    Hsue, S.T.; Baker, M.P.

    1984-01-01

    A plutonium scrap recovery facility is being constructed at the Savannah River Plant (SRP). The safeguards groups of the Los Alamos National Laboratory have been working since the early design stage of the facility with SRP and other national laboratories to develop a state-of-the-art assay system for this new facility. Not only will the most current assay techniques be incorporated into the system, but also the various nondestructive assay (NDA) instruments are to be integrated with an Instrument Control Computer (ICC). This undertaking is both challenging and ambitious; an entire assay system of this type has never been done before in a working facility. This paper will describe, in particular, the effort of the Los Alamos Safeguards Assay Group in this endeavor. Our effort in this project can be roughly divided into three phases: NDA development, system integration, and integral testing. 6 references

  11. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

    Full Text Available The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  12. Radioisotopic 51Cr-leukocyte adherence inhibition (LAI) assay. I

    International Nuclear Information System (INIS)

    Tsang, P.H.; Tangnavarad, K.; Lesnick, G.; Holland, J.F.; Bekesi, J.G.; Perloff, M.

    1980-01-01

    A simplified radioisotopic leukocyte adherence inhibition assay ( 51 Cr-LAI assay) was used to determine tumor-directed immune responses in patients with cancer of the breast. Essential steps in development of this assay are the standardization of conditions for optimal 51 Cr uptake by peripheral blood lymphocytes (PBL) and the inclusion of autologous or normal AB serum in the incubation media. A dextrose salt mixture (GNK) was found to enhance intracellular uptake of 51 Cr significantly (8-fold) without affecting viability of the cells or without causing selective loss of lymphocyte subpopulations. The presence of 10% autologous or normal AB serum prevented non-specific LAI responses to unrelated tumor antigens. Experimental results are presented and it is seen that this short term (4 h) 51 Cr-LAI assay provides reproducible and specific results analogous to those using tube-LAI assay. The test has the advantages of being accurate, sensitive and free from technical bias. (Auth.)

  13. 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)

  14. Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

    NARCIS (Netherlands)

    van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

    2007-01-01

    A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

  15. Bulk media assay using backscattered neutron spectrometry

    International Nuclear Information System (INIS)

    Csikai, J.

    2000-01-01

    This paper summarized a systematic study of bulk media assay using backscattered neutron spectrometry. The source-sample-detector geometry used for the measurements of leakage and elastically backscattered (EBS) spectra of neutrons is shown. Neutrons up to about 14 MeV were produced via 2 H (d,n) and 9 Be (d,n) reactions using different deuteron beam energies between 5 and 10 MeV at the MGC-20E cyclotron of ATOMKI (Debrecen). Neutron yields of the Pu-Be and 252 Cf sources were 5.25 x 10 6 n/s and 1.8 x 10 6 n/s, respectively. Flux density distributions of thermal and primary 14 MeV neutrons were measured for graphite, water and coal samples in various moderator (M)-sample (S)-reflector (R) geometries. Relative fractions and integrated yields of 252 Cf, Pu-Be and 14 MeV neutrons above the (n,n'γ) reaction thresholds for 12 C, 16 O and 28 Si isotopes vs sample thickness have also been determined. It was found that the integrated reaction rate vs sample thickness decreasing exponentially with different attenuation coefficients depending on the neutron spectrum and the composition of the sample. The spectra of neutrons from sources passing through slabs of water, graphite, sand, Al, Fe and Pb up to 20 cm in thickness have been measured by a PHRS system in the 1.2 to 1.5 MeV range. The leakage neutron spectra from a Pu-Be source placed in the center of 30 cm diameter sphere filled with water, paraffin oil, SiO 2 , zeolite and river sand were also measured. The measured spectra have been compared with the calculated results obtained by the three dimensional Monte-Carlo code MCNP-4A and point-wise cross sections from the ENDF/B-4, ENDF/B-6, ENDF/E-1, BROND-2 and JENDL-3.1 data files. New results were obtained for validation of different data libraries from a comparison on the measured and the calculated spectra. Some typical results for water, Al, sand and Fe are shown. A combination of the backscattered neutron spectrometry with the surface gauge used both for the

  16. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

  17. New low-viscosity overlay medium for viral plaque assays

    Directory of Open Access Journals (Sweden)

    Garten Wolfgang

    2006-08-01

    Full Text Available Abstract Background Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CL™ as overlay media in the plaque and plaque-inhibition assay of influenza viruses. Results Significantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC overlay media. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3% ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances. Conclusion Plaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs.

  18. Complementing in vitro screening assays with in silico ...

    Science.gov (United States)

    High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive under assay conditions but that can generate active metabolites under in vivo conditions. To address this potential issue, a case study will be presented to demonstrate the use of in silico tools to identify inactive parents with the ability to generate active metabolites. This case study used the results from an orthogonal assay designed to improve confidence in the identification of active chemicals tested across eighteen estrogen receptor (ER)-related in vitro assays by accounting for technological limitations inherent within each individual assay. From the 1,812 chemicals tested within the orthogonal assay, 1,398 were considered inactive. These inactive chemicals were analyzed using Chemaxon Metabolizer software to predict the first and second generation metabolites. From the nearly 1,400 inactive chemicals, over 2,200 first-generation (i.e., primary) metabolites and over 5,500 second-generation (i.e., secondary) metabolites were predicted. Nearly 70% of primary metabolites were immediately detoxified or converted to other metabolites, while over 70% of secondary metabolites remained stable. Among these predicted metabolites, those that are most likely to be produced and remain

  19. Evaluation of the Abbott ARCHITECT Toxo IgM assay.

    Science.gov (United States)

    Sickinger, Eva; Braun, Hans-Bertram; Praast, Gerald; Stieler, Myriam; Gundlach, Cordelia; Birkenbach, Claudia; Prostko, John; Palafox, Mary Ann; Frias, Edwin; Hsu, Stephen; Matias, Matthew; Pucci, Dominick; Hausmann, Michael; Sagel, Ulrich; Smith, Darwin

    2009-07-01

    Development of the ARCHITECT Toxo IgM assay has been done to assist the clinician in acute Toxoplasma gondii infection detection, especially in pregnant women. Its use, in conjunction with ARCHITECT Toxo IgG and Toxo Avidity assays, will provide an array of assays particularly useful in the monitoring of pregnant females to determine the risk of maternal transmission of the parasite. Specificity results from 2 testing sites, using populations of pregnant females, hospital patients, and blood donors, demonstrated that the assay has an overall resolved relative specificity of 99.89% (confidence interval, 99.68-99.98%). Relative specificity for pregnant female specimens was 99.95% (n = 2031). Excellent seroconversion sensitivity was observed for the ARCHITECT Toxo IgM assay, which was similar to the Abbott AxSYM Toxo IgM assay (Abbott Laboratories, Abbott Park, IL). In more than 90% of the panels tested, the 1st bleed detected in the serial bleeds was the same for both assays.

  20. Pathology consultation on anticoagulation monitoring: factor X-related assays.

    Science.gov (United States)

    Wool, Geoffrey D; Lu, Chuanyi M

    2013-11-01

    To review various anticoagulation therapies and related laboratory monitoring issues, with a focus on factor X-related chromogenic assays. A case-based approach is used to review pertinent published literatures and product inserts of anticoagulation drugs and to look back on clinical use of factor X-related chromogenic assays. The number of anticoagulants available to clinicians has increased greatly in the past decade. Whether and how these anticoagulants should be monitored are areas of uncertainty for clinicians, which can lead to misuse of laboratory assays and suboptimal patient management. Factor X-related assays are of particular concern because of the similar and often confusing test names. Based on a common clinical case scenario and literature review regarding anticoagulant monitoring, an up-to-date discussion and review of the various factor X-related assays are provided, focusing on the differences in test designs and clinical utilities between the chromogenic anti-Xa and chromogenic factor X activity assays. Anticoagulation therapy and related laboratory monitoring are rapidly evolving areas of clinical practices. A good knowledge of relevant laboratory assays and their clinical applications is necessary to help optimize patient care.

  1. 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.

  2. Quantitative Fissile Assay In Used Fuel Using LSDS System

    Science.gov (United States)

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

    2017-09-01

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

  3. 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.

  4. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  5. 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.

  6. A novel clot lysis assay for recombinant plasminogen activator.

    Science.gov (United States)

    Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

    2015-03-01

    Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

  7. 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.

  8. A highly sensitive and specific assay for vertebrate collagenase

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  9. 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.

  10. A fluorescence sedimentation assay for dsDNA antibodies

    DEFF Research Database (Denmark)

    Duus, K; Draborg, A H; Güven, E

    2017-01-01

    The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other...... on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

  11. Opportunities and Challenges of Multiplex Assays: A Machine Learning Perspective.

    Science.gov (United States)

    Chen, Junfang; Schwarz, Emanuel

    2017-01-01

    Multiplex assays that allow the simultaneous measurement of multiple analytes in small sample quantities have developed into a widely used technology. Their implementation spans across multiple assay systems and can provide readouts of similar quality as the respective single-plex measures, albeit at far higher throughput. Multiplex assay systems are therefore an important element for biomarker discovery and development strategies but analysis of the derived data can face substantial challenges that may limit the possibility of identifying meaningful biological markers. This chapter gives an overview of opportunities and challenges of multiplexed biomarker analysis, in particular from the perspective of machine learning aimed at identification of predictive biological signatures.

  12. A Functional Assay for GPR55: Envision Protocol.

    Science.gov (United States)

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

  13. 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

  14. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  15. Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.; Bruckner-Lea, Cindy J.; Marks, James D.

    2010-08-04

    Methods to detect botulinum toxin, the most poisonous substance known, are reviewed. Current assays are being developed with two main objectives in mind: 1) to obtain sufficiently low detection limits to replace the mouse bioassay with an in vitro assay, and 2) to develop rapid assays for screening purposes that are as sensitive as possible while requiring an hour or less to process the sample an obtain the result. This review emphasizes the diverse analytical approaches and devices that have been developed over the last decade, while also briefly reviewing representative older immunoassays to provide background and context.

  16. The glycophorin A assay for somatic cell mutations in humans

    International Nuclear Information System (INIS)

    Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

    1989-01-01

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

  17. Mouse Assay for Determination of Arsenic Bioavailability in Contaminated Soils

    Science.gov (United States)

    Background: Accurate assessment of human exposure estimates from arsenic-contaminated soils depends upon estimating arsenic (As) soil bioavailability. Development of bioavailability assays provides data needed for human health risk assessments and supports development and valida...

  18. Application of neutron multiplicity counting to waste assay

    Energy Technology Data Exchange (ETDEWEB)

    Pickrell, M.M.; Ensslin, N. [Los Alamos National Lab., NM (United States); Sharpe, T.J. [North Carolina State Univ., Raleigh, NC (United States)

    1997-11-01

    This paper describes the use of a new figure of merit code that calculates both bias and precision for coincidence and multiplicity counting, and determines the optimum regions for each in waste assay applications. A {open_quotes}tunable multiplicity{close_quotes} approach is developed that uses a combination of coincidence and multiplicity counting to minimize the total assay error. An example is shown where multiplicity analysis is used to solve for mass, alpha, and multiplication and tunable multiplicity is shown to work well. The approach provides a method for selecting coincidence, multiplicity, or tunable multiplicity counting to give the best assay with the lowest total error over a broad spectrum of assay conditions. 9 refs., 6 figs.

  19. Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango ...

    African Journals Online (AJOL)

    Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango ( Mangifera indica ) and Bush Mango ( Irvingia gabonensis ) Kernels. ... Nigerian Food Journal ... Phenolic constituents (total phenols, flavonoids, tannins, and anthocyanins), comparative antiradical potency and cytotoxicity of processed mango (Mangifera ...

  20. Development of novel IVD assays: a manufacturer's perspective.

    Science.gov (United States)

    Metcalfe, Thomas A

    2010-01-01

    IVD manufacturers are heavily reliant on novel IVD assays to fuel their growth and drive innovation within the industry. They represent a key part of the IVD industry's value proposition to customers and the healthcare industry in general, driving product differentiation, helping to create demand for new systems and generating incremental revenue. However, the discovery of novel biomarkers and their qualification for a specific clinical purpose is a high risk undertaking and the large, risky investments associated with doing this on a large scale are incompatible with IVD manufacturer's business models. This article describes the sources of novel IVD assays, the processes for discovering and qualifying novel assays and the reliance of IVD manufacturers on collaborations and in-licensing to source new IVD assays for their platforms.