Sample records for based viability assay

  1. Cell viability assays: introduction. (United States)

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  2. Bioluminescence assay for cell viability. (United States)

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N


    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  3. Capacitance-based assay for real-time monitoring of endocytosis and cell viability. (United States)

    Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa


    Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.

  4. A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol. (United States)

    Hassan, Rabeay Y A; Bilitewski, Ursula


    Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S.cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.

  5. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials. (United States)

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang


    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials.

  6. A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay. (United States)

    Emter, Roger; Natsch, Andreas


    The KeratinoSens™ assay was the first cell-based in vitro test in the skin sensitisation adverse outcome pathway to be endorsed by an ECVAM statement. It includes a cell viability assessment, which serves two purposes: It forms part of the prediction model to exclude false-positive irritants and cytotoxicity provides some information on sensitizer potency of chemicals, which can feed into a multivariate potency model. In the KeratinoSens™ protocol, Nrf2-dependent luciferase induction and the MTT-viability assay are performed in parallel plates. Resazurin-based viability assays do not require cell lysis and are compatible with luciferase measurements in the same cells. Here, we performed detailed comparison of the tetrazolium-based MTT assay and the PrestoBlue® assay on 35 reference chemicals tested in the full KeratinoSens™ protocol. Log-transformed IC50 and IC30 values measured with both methods correlate with an R(2) of 0.97 and 0.95. A single chemical showed divergent results and analysis by four different viability assays indicated the PrestoBlue® read-out to be correct. The new more rapid and resource efficient approach has clear advantages: Dose-response curves show lower variability and the two endpoints are measured on the same cells. This approach is a valid addition to or replacement of the MTT-readout in the KeratinoSens™ assay and it is recommended as a general tool for luciferase-based reporter assays.

  7. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. (United States)

    Vistica, D T; Skehan, P; Scudiero, D; Monks, A; Pittman, A; Boyd, M R


    The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.

  8. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M


    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  9. Kinetic viability assays using DRAQ7 probe. (United States)

    Wlodkowic, Donald; Akagi, Jin; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew


    Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy-to-use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death-inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single-color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.

  10. Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials. (United States)

    Qiu, Tian A; Nguyen, Thu Ha Thi; Hudson-Smith, Natalie V; Clement, Peter L; Forester, Dona-Carla; Frew, Hilena; Hang, Mimi N; Murphy, Catherine J; Hamers, Robert J; Feng, Z Vivian; Haynes, Christy L


    Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.

  11. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay. (United States)

    Paciello, Lucia; Falco, Francesco Cristino; Landi, Carmine; Parascandola, Palma


    Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.

  12. A cellular viability assay to monitor drug toxicity. (United States)

    Hansen, Jakob; Bross, Peter


    A central part of the research in protein misfolding and its associated disorders is the development of treatment strategies based on ensuring cellular protein homeostasis. This often includes testing chemical substances or drugs for their ability to counteract protein misfolding processes and to promote correct folding. Such investigations also include assessment of how the tested chemical substances affect cellular viability, that is, their cytotoxic effect. Investigations of cytotoxicity often require testing several different concentrations and drug exposure times using cells in culture. It is therefore attractive to use a viability test that permits the analysis of many samples with little handling time. This protocol describes a simple and fast methodology to analyze viability of lymphoblastoid cells and to test putative cytotoxic effects associated with exposure to a chemical substance, here exemplified by celastrol. The natural substance celastrol has been used for many years in traditional Chinese medicine and has subsequently been shown to induce transcription of genes encoding molecular chaperones (heat shock proteins) that are involved in promoting folding of cellular proteins. The well-described colorimetric tetrazolium salt (MTT) assay, which monitors metabolic activity of cultured cells, was adapted to analyze the viability of cells exposed to celastrol. After having established a suitable cell seeding density, the dose-dependence and time-course of viability reduction of lymphoblastoid cells treated with celastrol were determined. It was found that 4- and 24-h exposure to 0.8 microM celastrol reduced the viability of lymphoblastoid cells, with the most severe effect observed at 24 h with MTT reductions approaching 30% of non-exposed cells. For a series of incubations for 24 h, it was found that concentrations as low as 0.2 microM were sufficient to affect the viability, and celastrol concentrations of 0.5 microM reduced the MTT reduction rate to

  13. A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

    Directory of Open Access Journals (Sweden)

    Avery Vicky M


    Full Text Available Abstract Background Human African Trypanosomiasis (HAT is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS of large compound library collections. Results We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of T.b.brucei. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC50 data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for T.b.brucei. Conclusion Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening.

  14. A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the β-actin promoter and enhancer.

    Directory of Open Access Journals (Sweden)

    Shawn E Lupold

    Full Text Available Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.

  15. A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the β-actin promoter and enhancer. (United States)

    Lupold, Shawn E; Johnson, Tamara; Chowdhury, Wasim H; Rodriguez, Ronald


    Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.

  16. Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations. (United States)

    Alakomi, H-L; Mättö, J; Virkajärvi, I; Saarela, M


    Cell viability in probiotic preparations is traditionally assessed by the plate count technique. Additionally, fluorescent staining combined with epifluorescence microscopy or flow cytometry has been developed for the viability assessment, but the currently available assays are either laborious or require highly sophisticated equipment. The aim of this study was to investigate the applicability of a microplate scale fluorochrome assay for predicting the cell state of freeze-dried Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis preparations. In addition to viability assessment with LIVE/DEAD BacLight Bacterial Viability Kit, DiBAC(4)3 stain was used for the kinetic measurement of changes in bifidobacterial cell membrane functions during exposure to low pH. The microplate scale fluorochrome assay results on the viability and cell numbers of probiotic preparations correlated well with the results obtained with the culture-based technique and (with few exceptions) with epifluorescence microscopy. The assay was applicable also for the viability assessment of stressed (acid-treated) cells provided that the cell density in treatments was adjusted to the optimal measurement level of the fluorometer. The microplate scale fluorochrome assay offers a rapid and robust tool for the viability assessment of probiotic preparations, and enables also kinetic measurements.

  17. Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment. (United States)

    Gaucher, Sonia; Jarraya, Mohamed


    MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

  18. Predictive value of the surface-enhanced resonance Raman scattering-based MTT assay: a rapid and ultrasensitive method for cell viability in situ. (United States)

    Mao, Zhu; Liu, Zhuo; Chen, Lei; Yang, Jin; Zhao, Bing; Jung, Young Mee; Wang, Xu; Zhao, Chun


    SERRS (surface-enhanced resonance Raman scattering) has been used to develop and optimize a novel and quantitative MTT assay for living cell viability. This highly sensitive method derives from two factors for formazan signal enhancing: the addition of Au nanoparticles and the resonance effect by 632.8 nm of excitation. The results show that the background elements, such as excessive MTT residues, serum, and the drug, did not interfere with the detection of formazan. Moreover, the detection limit of formazan is as low as 1 ng/mL. With the use of this method to quantify metabolically viable cells, dose-response curves of treated and untreated cells with the drug were constructed on the human lung cancer cell A549. The results also show that the Raman signal generated is dependent on the degree of activation of the cells. In comparison to the traditional method, the main advantages of this method are its rapidity (30 min), high-selectivity, high-precision, and cost-effectiveness (0.1 mg/mL MTT) without time-consuming steps and any modifying or labeling procedure. This work reports on an improved research tool that may help researchers apply this method for in situ cell assays.

  19. An optimized SYBR Green I/PI assay for rapid viability assessment and antibiotic susceptibility testing for Borrelia burgdorferi. (United States)

    Feng, Jie; Wang, Ting; Zhang, Shuo; Shi, Wanliang; Zhang, Ying


    Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.

  20. Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro. (United States)

    Li, Jie; Zhang, Donglan; Ward, Kathleen M; Prendergast, George C; Ayene, Iraimoudi S


    Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

  1. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter. (United States)

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei


    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  2. Sperm MTT Viability Assay: A New Method for Evaluation of Human Sperm Viability


    Nasr-Esfahani, Mohammad Hossein; Aboutorabi, Roshanak; Esfandiari, Ebrahim; Mardani, Mohammad


    Purpose: MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is commonly used as a cell proliferation assay. The objective of this study was to evaluate the capability of MTT assay to discriminate between viable and nonviable sperms and compare its efficiency with E&N (eosin and nigrosin) and HOST (hypo-osmotic swelling test).

  3. Live tissue viability and chemosensitivity assays using digital holographic motility contrast imaging. (United States)

    An, Ran; Turek, John; Matei, Daniela Elena; Nolte, David


    Holographic optical coherence imaging is an en face form of optical coherence tomography that uses low-coherence digital holography as a coherence gate to select light from a chosen depth inside scattering tissue. By acquiring successive holograms at a high camera frame rate at a fixed depth, dynamic speckle provides information concerning dynamic light scattering from intracellular motility. Motility contrast imaging (MCI) uses living motion as a label-free and functional biomarker. MCI provides a new form of viability assay and also is applicable for proliferation and cytotoxicity assays. The results presented here demonstrate that low-coherence digital holography can extract viability information from biologically relevant three-dimensional (3D) tissue based on multicellular tumor spheroids by moving beyond the format of two-dimensional cell culture used for conventional high-content analysis. This paper also demonstrates the use of MCI for chemosensitivity assays on tumor exgrafts of excised ovarian cancer tumors responding to standard-of-care cisplatin chemotherapy. This ex vivo application extends the applicability of MCI beyond 3D tissue culture grown in vitro.

  4. Liposomes and MTT cell viability assay: an incompatible affair. (United States)

    Angius, Fabrizio; Floris, Alice


    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to evaluate the cytotoxicity potential of drugs vehicled by liposomes. However, liposome delivering drugs could produce inconsistent values of MTT absorbance. On the basis of previous experiments demonstrating the MTT affinity for lipid droplets, this paper aims to show that empty-liposomes interfere, per se, on MTT assay due to its lipidic nature. This brings into question the use of MTT testing cytotoxicity when liposomes are involved in delivering drugs.

  5. A versatile assay for the accurate, time-resolved determination of cellular viability. (United States)

    Amano, Toyoki; Hirasawa, Ken ichi; O'Donohue, Michael J; Pernolle, Jean Claude; Shioi, Yuzo


    A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.

  6. Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria. (United States)

    Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R


    Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

  7. In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites. (United States)

    Heredero-Bermejo, I; Copa-Patiño, J L; Soliveri, J; Gómez, R; de la Mata, F J; Pérez-Serrano, J


    The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

  8. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas


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

  9. Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms. (United States)

    Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary


    Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

  10. Particle-induced artifacts in the MTT and LDH viability assays. (United States)

    Holder, Amara L; Goth-Goldstein, Regine; Lucas, Donald; Koshland, Catherine P


    In vitro testing is a common first step in assessing combustion-generated and engineered nanoparticle-related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose-response data. Nanoparticles, well-known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/mL, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/mL and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations.

  11. A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip. (United States)

    Torisawa, Yu-suke; Takagi, Airi; Nashimoto, Yuji; Yasukawa, Tomoyuki; Shiku, Hitoshi; Matsue, Tomokazu


    We describe a novel multicellular spheroid culture system that facilitates the easy preparation and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of approximately 2 weeks) is available on the chip. Measurement of albumin production in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 weeks. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochemical microscopy. Responses to four different chemical stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells.

  12. Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores. (United States)

    Shafaat, Hannah S; Ponce, Adrian


    We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.

  13. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening. (United States)

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta


    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  14. A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ

    Directory of Open Access Journals (Sweden)

    Schulze Katja


    Full Text Available Abstract Background Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating or preparation-intensive (eg. fluorescent staining. In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation. Results The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism Synechocystis sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis. Conclusions The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.

  15. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay. (United States)

    Silva, Filomena S G; Starostina, Irina G; Ivanova, Vilena V; Rizvanov, Albert A; Oliveira, Paulo J; Pereira, Susana P


    The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

  16. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. (United States)

    Stepanenko, A A; Dmitrenko, V V


    The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended.

  17. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra


    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.



  19. A rapid colorimetric assay for the quantitation of the viability of free-living larvae of nematodes in vitro. (United States)

    James, Catherine E; Davey, Mary W


    With increasing drug resistance in gastrointestinal parasites, identification of new anthelmintics is essential. The non-parasitic nematode Caenorhabditis elegans is used extensively as a model to identify drug targets and potential novel anthelmintics because it can be readily cultured in vitro. Traditionally, the assessment of worm viability has relied on labour-intensive developmental and behavioral assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) colorimetric assay uses metabolic activity as a marker of viability in mammalian cell culture systems and has been applied for use with filarial nematodes. In the present study, this assay has been optimized and validated to rapidly assess the viability of C. elegans after drug treatment. Living, but not dead, C. elegans take up MTT and reduce it to the blue formazan, providing visual, qualitative, and quantitative assessment of viability. MTT at a concentration of 5 mg/ml with 3 h incubation was optimal for detecting changes in viability with drug treatment. We have applied this assay to quantitate the effects of ivermectin and short-chain alcohols on the viability of C. elegans. This assay is also applicable to first-stage larvae of the parasitic nematode Haemonchus contortus. The advantage of this assay is the rapid quantitation in screening drugs to identify potential anthelmintics.

  20. Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability. (United States)

    Comley, J C; Turner, C H


    Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.

  1. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability. (United States)

    Welch, Aaron Z; Koshland, Douglas E


    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities viability.

  2. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    Full Text Available Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  3. A novel multiplex cell viability assay for high-throughput RNAi screening. (United States)

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael


    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  4. 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

  5. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. (United States)

    Repetto, Guillermo; del Peso, Ana; Zurita, Jorge L


    The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

  6. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells. (United States)

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A


    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  7. Assessment of GFP expression and viability using the tali image-based cytometer. (United States)

    Remple, Krissy; Stone, Laurel


    Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements(1). Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations(2). Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer(3). The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis(4-6) and cell viability analysis with propidium iodide (PI)(7-11). Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell

  8. A rapid, sensitive and cost-efficient assay to estimate viability of potato cyst nematodes

    NARCIS (Netherlands)

    Elsen, van den S.J.J.; Ave, M.; Schoenmakers, N.; Landeweert, R.; Bakker, J.; Helder, J.


    Potato cyst nematodes (PCN) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts which can contain 200-500 eggs protected by the hardened cuticle of a dead female, are either time and labor int

  9. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. (United States)

    Stockert, Juan C; Blázquez-Castro, Alfonso; Cañete, Magdalena; Horobin, Richard W; Villanueva, Angeles


    Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

  10. Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements. (United States)

    Forbes, Alaina M; Lin, Huimin; Meadows, Gary G; Meier, G Patrick


    Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

  11. A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions. (United States)

    Dehdashti, Seameen J; Abbott, Jennifer; Nguyen, Dac-Trung; McKew, John C; Williamson, Peter R; Zheng, Wei


    Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1% and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3'-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.

  12. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. (United States)

    Buttke, T M; McCubrey, J A; Owen, T C


    A new tetrazolium compound, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), has recently been described which in the presence of phenazine methosulfate (PMS) is reduced by living cells to yield a formazan product that can be assayed colorimetrically. An important advantage of MTS/PMS over other tetrazolium dyes (e.g., MTT) is the aqueous solubility of the reduced formazan product which eliminates the need for detergent solubilization or organic solvent extraction steps. Its advantages over XTT/PMS, another tetrazolium which yields a water-soluble formazan product, include the absorbance range of color produced (515-580 nm as opposed to 450 nm), the rapidity of color development, and the storage stability of the MTS/PMS reagent solution. In the present study, MTS/PMS was used to assay viability and proliferation of the IL-2-dependent HT-2 and CTLL-2 cell lines and the IL-3-dependent FDC-P1 and FL5.12 cell lines. With each cell line, the amount of formazan product was time-dependent and proportional to the number of viable cells. Furthermore, with both HT-2 and CTLL-2 cells it was found that cultures could be simultaneously labeled with MTS/PMS and [3H]thymidine, with relatively little effect of the dye on uptake of the latter. This feature was further capitalized upon in studies with FDC-P1 cells, in which the co-addition of MTS/PMS and [3H]thymidine was used to distinguish between cell viability and proliferation.

  13. Viability of developmental stages of Schistosoma mansoni quantified with xCELLigence worm real-time motility assay (xWORM). (United States)

    Rinaldi, Gabriel; Loukas, Alex; Brindley, Paul J; Irelan, Jeff T; Smout, Michael J


    Infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide. Where anthelmintic drugs are available, drug resistance is a major problem in livestock parasites, and a looming threat to public health. Monitoring the efficacy of these medicines and screening for new drugs has been hindered by the lack of objective, high-throughput approaches. Several cell monitoring technologies have been adapted for parasitic worms, including video-, fluorescence-, metabolism enzyme- and impedance-based tools that minimize the screening bottleneck. Using the xCELLigence impedance-based system we previously developed a motility-viability assay that is applicable for a range of helminth parasites. Here we have improved substantially the assay by using diverse frequency settings, and have named it the xCELLigence worm real-time motility assay (xWORM). By utilizing strictly standardized mean difference analysis we compared the xWORM output measured with 10, 25 and 50 kHz frequencies to quantify the motility of schistosome adults (human blood flukes) and hatching of schistosome eggs. Furthermore, we have described a novel application of xWORM to monitor movement of schistosome cercariae, the developmental stage that is infectious to humans. For all three stages, 25 kHz was either optimal or near-optimal for monitoring and quantifying schistosome motility. These improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large.

  14. Development and validation of a quick easily used biochemical assay for evaluating the viability of small immobile arthropods. (United States)

    Phillips, Craig B; Iline, Ilia I; Richards, Nicola K; Novoselov, Max; McNeill, Mark R


    Quickly, accurately, and easily assessing the efficacy of treatments to control sessile arthropods (e.g., scale insects) and stationary immature life stages (e.g., eggs and pupae) is problematic because it is difficult to tell whether treated organisms are alive or dead. Current approaches usually involve either maintaining organisms in the laboratory to observe them for development, gauging their response to physical stimulation, or assessing morphological characters such as turgidity and color. These can be slow, technically difficult, or subjective, and the validity of methods other than laboratory rearing has seldom been tested. Here, we describe development and validation of a quick easily used biochemical colorimetric assay for measuring the viability of arthropods that is sufficiently sensitive to test even very small organisms such as white fly eggs. The assay was adapted from a technique for staining the enzyme hexokinase to signal the presence of adenosine triphosphate in viable specimens by reducing a tetrazolium salt to formazan. Basic laboratory facilities and skills are required for production of the stain, but no specialist equipment, expertise, or facilities are needed for its use.

  15. Viability Reagent, PrestoBlue, in Comparison with Other Available Reagents, Utilized in Cytotoxicity and Antimicrobial Assays

    Directory of Open Access Journals (Sweden)

    Namrita Lall


    Full Text Available This study compared different commercially available viability reagents. The growth indicator reagents include p-iodonitrotetrazolium violet (INT, PrestoBlue, and Alamar Blue which were used for antimicrobial analysis against Streptococcus mutans, Prevotella intermedia, Propionibacterium acnes, and Mycobacterium tuberculosis. PrestoBlue and Alamar Blue are resazurin based reagents that resulted in a quick and easily distinguishable colour change that allowed for visual readings. INT and Sodium 3′-[1-(phenyl amino-carbonyl-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate (XTT are tetrazolium based reagents which are converted to a formazan dye in the presence of metabolically active mitochondria enzyme. For cell viability analysis, reagents XTT and PrestoBlue were compared. PrestoBlue was able to clearly indicate the minimum inhibitory concentration (MIC of various positive drug controls on various microbial strains. PrestoBlue was also a good indicator of the 50% inhibitory concentration (IC50 of positive drug controls on various cell lines.

  16. Optimizing cell viability in droplet-based cell deposition

    NARCIS (Netherlands)

    Hendriks, Jan; Willem Visser, Claas; Henke, Sieger; Leijten, Jeroen; Saris, Daniël B F; Sun, Chao; Lohse, Detlef; Karperien, Marcel


    Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here, w

  17. Fluorescent antibody-viability staining and beta-glucuronidase assay as rapid methods for monitoring Escherichia coli viability in coastal marine waters. (United States)

    Caruso, G; De Pasquale, F; Mancuso, M; Zampino, D; Crisafi, E


    A faecal pollution monitoring of coastal Messina waters was performed by comparing three (microscopic, enzyme, and culture) methods. Evidence of Escherichia coli cells (29.99 to 96.79% of the total enteropathogenic serotypes) retaining their viability into the marine environment was shown. beta-Glucuronidase activity rates suggested that living cells were also metabolically active. Heavily polluted sites were detected, where improperly treated urban wastes were discharged. Significant relationships between microscopic and enzymatic data proved both methods to be suitable alternatives to the culture method for E. coli detection, improving environmental quality assessment.

  18. Unsuitability of 2,3,5-Triphenyl-2H-Tetrazolium chloride(TTC) as a viability assay for plant cells in suspention

    Energy Technology Data Exchange (ETDEWEB)

    Kebler, M.; Furusaki, S. [Department of Chemistry and Biotechnology Univ, Tokyo (Japan). Graduate School of Engineering


    The well-known viability assay with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) is applied to plant cell suspensions. In this paper it is shown that parameters (pH, TTC concentration, incubation time) which are thought to b e only dependent on different cell lines are underlying at least two additional functions (age of the culture and shear stress). Each cell in a different state of activity requires a new set of the parameters mentioned above. Furthermore the time-dependent formazan production courses vary to such an extent that they cannot be used for viability determination. Therefore the usage of TTC as a viability test implies non negligible errors compared to the Evans` Blue staining method which does not involve cell metabolism. The values of the two different methods to determine the viability can differ by more than 50%. The data suggests abandoning the usage of TTC as a quantitative viability assay for plant cell suspensions. 22 refs., 7 figs., 1 tab.

  19. Assessment of cytotoxicity of carbon nanoparticles using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay. (United States)

    Schrand, Amanda M; Lin, Jonathan B; Hussain, Saber M


    Ever since the discovery of carbon nanotubes, there has been an increasing interest in technologies that rely upon these incredibly small particles for their unique properties. However, assessment of their biological consequences has been riddled with assay limitations. Here, we describe application of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay to study cytotoxicity of various carbon-based nanomaterials on cells and discuss some pitfalls of this method.

  20. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten


    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  1. Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells. (United States)

    Shenoy, Niraj; Stenson, Mary; Lawson, Joshua; Abeykoon, Jithma; Patnaik, Mrinal; Wu, Xiaosheng; Witzig, Thomas


    Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.18.

  2. Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro. (United States)

    Heredero-Bermejo, I; Criado-Fornelio, A; Soliveri, J; Díaz-Martín, J A; Matilla-Fuentes, J; Sánchez-Arias, J A; Copa-Patiño, J L; Pérez-Serrano, J


    A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism.

  3. PENGHAMBATAN CAJUPUTS CANDY TERHADAP VIABILITAS KHAMIR Candida albicans SECARA IN VITRO [Inhibition of Cajuputs Candy Toward the Viability of Candida albicans by using In Vitro Assay

    Directory of Open Access Journals (Sweden)

    C. Hanny Wijaya1*


    Full Text Available The utilization of cajuput essential oil as a flavor in candy may produce a physiological active added value. Some compounds of cajuput plant (Melaleuca cajuputi L have been reported for their anti-microbial activities. Candida albicans is a normal commensal organism in human mouth. However, it may become virulent and responsible for oral diseases known as oral candidiasis. This study aimed to determine the effect of cajuput and peppermint oil in cajuputs candy in inhibiting the C. albicans biofilms formation by using in vitro biofilm assay and viability assay. Furthermore, the influence of concentration of cajuput oil on the anti-microbial activities had been analyzed. All the tested concentration of cajuput oil in cajuputs candy was effective to inhibit the viability of C. albicans. The provision of flavor components of cajuput and peppermint oil could produce synergistic effects compared to a single flavor component. The addition of cajuput oil at 0.6% was able to inhibit the viability of C. albicans. The activities of the cajuput oil showed positive correlation to the concentration. The variable of plus and minus 0.1% addition of the cajuput oil concentration, however, produced no significant difference to inhibit the growth of C. albicans in biofilm. Sensory test, hedonic test, was conducted to evaluate the flavor, aroma, and overall attributes, resulting in no significant difference between 0.6 to 0.8% additions of cajuput oil upon the sensory acceptance.

  4. Experimental Calcium Silicate-Based Cement with and without Zirconium Oxide Modulates Fibroblasts Viability. (United States)

    Slompo, Camila; Peres-Buzalaf, Camila; Gasque, Kellen Cristina da Silva; Damante, Carla Andreotti; Ordinola-Zapata, Ronald; Duarte, Marco Antonio Hungaro; de Oliveira, Rodrigo Cardoso


    The aim of this study was to verify whether the use of zirconium oxide as a radiopacifier of an experimental calcium silicate-based cement (WPCZO) leads to cytotoxicity. Fibroblasts were treated with different concentrations (10 mg/mL, 1 mg/mL, and 0.1 mg/mL) of the cements diluted in Dulbecco's modified Eagle's medium (DMEM) for periods of 12, 24, and 48 h. Groups tested were white Portland cement (WPC), white Portland cement with zirconium oxide (WPCZO), and white mineral trioxide aggregate Angelus (MTA). Control group cells were not treated. The cytotoxicity was evaluated through mitochondrial-activity (MTT) and cell-density (crystal violet) assays. All cements showed low cytotoxicity. In general, at the concentration of 10 mg/mL there was an increase in viability of those groups treated with WPC and WPCZO when compared to the control group (pzirconium oxide as the radiopacifier showed low cytotoxicity as a promising material to be exploited for root-end filling.

  5. 改良Alamar Blue法检测结直肠微小组织块的活力%Viability of tissue fragments in colon and rectum detected using modified Alamar Blue assay

    Institute of Scientific and Technical Information of China (English)

    甘嘉亮; 高枫; 曹云飞; 廖存; 黄家豪; 唐双意


    BACKGROUND: Alamar Blue assay is one of the common methods to evaluate histic or cellular viability. It is widely used to detect cell viability at present. However, using Alamar Blue assay to detect the viability of tissue fragments has not been reported. OBJECTIVE: To detect the viability of the regular tissue fragments and cancer tissue fragments in the mucous membrane of colon and rectum using modified Alamar Blue assay. METHODS: Based on cell viability detect assay of Alamar Blue, the viability of the regular tissue fragments and cancer tissue fragments in the mucous membrane of colon and rectum was assessed using absorbance per unit mass. The detecting time was chosen at 0, 6, 12, 18, 24 , 30 and 36 hours according to the specification of the Alamar Blue assay and the pre-experiment results. Every specimen was divided into seven groups and the blank control hole was established. RESULTS AND CONCLUSIONS: Absorbance per unit mass reflected the change tendency of proliferation activity in tissue fragments as the culture time went on. Both regular tissue fragments and cancer tissue fragments in the mucous membrane of colon and rectum reached the best proliferative state at 12-24 hours after culture. It confirms that the modified Alamar Blue assay is a sensitive and effective way to evaluate the viability of colorectal tissue fragments.%背景:Alamar Blue法是测定组织细胞活性的方法之一,目前多数应用在细胞系活力测定方面,尚未见应用于微小组织块活力的评价.目的:采用改良Alamar Blue法评价结直肠正常黏膜组织块和癌组织块的活力.方法:以细胞活力测定的Alamar Blue法为基础,应用单位质量吸光度来评价结直肠正常黏膜组织块和癌组织块的活力.根据检测试剂盒说明书和预实验结果,实验选择0,6,12,18,24,30,36 h作为检测时间点,每一例标本分为7组,设空白对照孔.结果与结论:单位质量吸光度能够随培养时间的推移,很好地反映组织

  6. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

    Directory of Open Access Journals (Sweden)

    Emily Peak

    Full Text Available BACKGROUND: Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. METHODOLOGY/PRINCIPAL FINDINGS: We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase, praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed, relevant to industrial (384-well microtiter plate compatibility and academic (96-well microtiter plate compatibility settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative. CONCLUSIONS/SIGNIFICANCE: The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat

  7. Cell based assay for hypoglycemic drugs screening

    Institute of Scientific and Technical Information of China (English)

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)




    Lignin is a rich renewable source of aromatic compounds. As a potentialpetroleum feedstock replacement, lignin can reduce environmental impacts such ascarbon emission. Due to its complex chemical structure, lignin is currently underutilized.Exploiting lignin as a precursor for carbon fibre adds high economic value to lignin andencourages further development in lignin extraction technology. This report includes apreliminary cost analysis and identifies the key aspects of lignin-based carbon fi...

  9. Determination of pretransplantational cell viability by MTT colorimetric assay%噻唑蓝比色法测定待移植睾丸细胞的活性

    Institute of Scientific and Technical Information of China (English)

    金化民; 钱辉军; 程帆; 詹炳炎


    Objective To assess the role of MTT colorimetric assay in measuring mitochondrial succinate-dehydrogenase(SDH)and establish a new way to determine pretransplantational cell viability.Methods Human or rat testicular Leydig cell suspensions were incubated with MTT medium,isopropanol was used to dissolve MTT formazan.then the absorbance of supertants was measured at 563 nm wavelength.Cell culture and testosterone production were used to assess the reliability of MTT colorimetric assay with comparison with Trypan bluedye.Results 1 g/L MTT concentration,3 h incubation,and isopropanol as a solvent were the best optimum for the cell culture in vitro and testosterone determination showed that MTT colorimetric assay was more sensitive and accurate than Trypan bluedye.MTT colorimetric assay was employed to successfully measure cell viability for 10 cases of human testicular Leydig cell transplantation.Conclusion MTT colorimetric assay provided a reliable,objective,accurate method for determining the pretransplantational cell viability.%目的 探讨噻唑蓝(MTT)比色法测定细胞线粒体琥珀酸脱氢酶,建立细胞活性的比色测定法.方法 采用大鼠睾丸制备的Leydig细胞悬液,与MTT基质液共同温育后,以异丙醇溶解MTT formazan,上清液于563 nm测吸光度;用体外细胞培养和睾酮释放量评价方法的可靠性,与台盼蓝染色法对照.结果 基质浓度1 g/L,温育时间3小时,异丙醇作溶剂为反应最佳条件.细胞培养和睾酮测定表明MTT法较台盼蓝法灵敏、准确.应用MTT技术为10例人睾丸Leydig细胞移植术监测细胞活性获得成功.结论 MTT比色法为细胞移植术提供了一种监测细胞活性的客观、准确方法.

  10. Osteocyte-viability-based simulations of trabecular bone loss and recovery in disuse and reloading

    NARCIS (Netherlands)

    Wang, H.; Ji, B.; Liu, X.S.; van Oers, R.F.M.; Guo, X.E.; Huang, Y.; Hwang, K.C.


    Osteocyte apoptosis is known to trigger targeted bone resorption. In the present study, we developed an osteocyte-viability-based trabecular bone remodeling (OVBR) model. This novel remodeling model, combined with recent advanced simulation methods and analysis techniques, such as the element-by-ele

  11. Mass-based readout for agglutination assays (United States)

    Chunara, Rumi; Godin, Michel; Knudsen, Scott M.; Manalis, Scott R.


    We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63-630nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry.

  12. Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study

    Directory of Open Access Journals (Sweden)

    Li-li Cui


    Full Text Available Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL, storage solutions (complete growth medium, Dulbecco’s phosphate-buffered saline, and normal saline, storage time in suspension (0–9 h, and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%. A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

  13. A functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged BCG or M.Tb (BCGlux/M.Tb lux). (United States)

    Newton, Sandra; Martineau, Adrian; Kampmann, Beate


    Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been

  14. Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques. (United States)

    Gomez-Samblas, M; Vílchez, S; Racero, J C; Fuentes, M V; Osuna, A


    "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products.

  15. Rapid assessment of the viability of Mycobacterium avium subsp. paratuberculosis cells after heat treatment, using an optimized phage amplification assay. (United States)

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R


    Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10(6) to 10(7) CFU/ml) and dispensed in 100-microl aliquots in thin-walled 200-microl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63 degrees C for 3, 6, and 9 min; (ii) 68 degrees C for 20, 40, and 60 s; and (iii) 72 degrees C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r(2) = 0.943) and heated (r(2) = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D(68 degrees C), mean D(63 degrees C), and D(72 degrees C) for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9 degrees C. Complete inactivation of 10(6) to 10(7) CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log(10) reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.

  16. Standardisation of egg-viability assays for Fasciola hepatica and Calicophoron daubneyi: A tool for evaluating new technologies of parasite control. (United States)

    Chryssafidis, Andreas Lazaros; Fu, Yan; De Waal, Theo; Mulcahy, Grace


    Fasciola hepatica and Calicophoron daubneyi, liver and rumen flukes respectively, infect ruminants throughout Europe. There is considerable interest in the development of vaccines and in testing new potential anthelmintic agents against these species. One potential target of new control measures is the parasite egg, as interference at this stage of the life cycle could aid in blocking the transmission of infection, and some experimental vaccines have been shown to affect egg viability. In this study, we describe the standardisation of protocols to evaluate the viability of eggs of these two parasites. Eggs were recovered from adult parasites collected in a commercial abattoir, from naturally infected cattle. A protocol for in vitro development of F. hepatica eggs was optimised based on previously published methods, with variations in duration and temperature of incubation. A new protocol for measurement of rumen fluke egg development in vitro was designed, based on testing different temperatures and periods of incubation, with or without light exposure. The protocols described here may be used in the future for comparing experimental groups when new technologies for parasite control are tested. In addition, the methods described for C. daubneyi present new information on the biology of this parasite.

  17. The effect of phosphate based glasses on the formation and viability of oral bacterial biofilms (United States)

    Mulligan, April Miranda

    This study considered the antibacterial activity of a series of soluble phosphate-based glasses (based on the Na2O-CaO-P2O5 glass system) doped with increasing amounts of copper or silver against oral bacterial biofilms. Initially, a variety of phosphate-based glass compositions were produced. The dissolution rate of these glasses was determined, and the information obtained was used to decide which glass compositions would be investigated in future experiments for their antibacterial properties. Selected glass compositions were investigated for their antibacterial activity against Streptococcus sanguis biofilms and oral microcosm biofilms. These biofilms were produced on phosphate-based glass discs using a Constant Depth Film Fermenter (CDFF), which allows the conditions found in the oral cavity to be closely mimicked. Following disc removal from the CDFF, various analytical procedures were carried out. Under conditions designed to mimic the supragingival environment of the oral cavity, fewer viable cells of Streptococcus sanguis were detected on both copper and silver-containing glass discs than on control discs, during the initial stages of the experiments, the greatest reduction occurring on the silver-containing glasses. An increase in viable cell number was observed as the experiments continued. Under the same conditions, copper-containing glasses failed to reduce the viability of microcosm biofilms. Viable cell number was initially reduced on the silver-containing glasses, but by the end of the experiments the viability of microcosm biofilms was significantly similar to those observed on the controls. Attempts to determine the efficacy of silver-containing glasses at reducing the viability of microcosm biofilms, under conditions designed to mimic the subgingival environment of the oral cavity, were subsequently made. Viable cells were not detected on any type of disc, including the control discs. Various reasons for this were postulated. In conclusion, the

  18. Viability and Acidification by Promising Yeasts Intended as Potential Starter Cultures for Rice-based Beverages

    Directory of Open Access Journals (Sweden)

    Antonio Bevilacqua


    Full Text Available Over the last years, some innovative cereal-based beverages were designed using beneficial lactic acid bacteria; however, few data are available on the potential role of yeasts. The main topic of this research was to investigate the suitability of four promising yeast strains (Saccharomyces cerevisiae var. boulardii, Kluyveromyces lactis, Saccharomyces pastorianus and Kazachstania exigua as potential starter cultures for rice-based beverages. This aim was achieved through some intermediate scientific aims, i.e., by assessing cell viability and acidification in different cereal substrates (malt extract, soft wheat, rice and kamut flours; thereafter by studying acidification and persistence in an organic rice drink during a prolonged storage at 25 and 4°C. Rice flour provided appropriate growth for all the strains. K. exigua and S. pastorianus experienced a relatively fast acidification within 24 h. After 40 d the yeasts showed similar cell counts (ca. 7 log cfu/mL and acidification (experienced a relatively fast acidification within 24 h. After 40 d the yeasts showed similar cell counts (ca. 7 log cfu/mL and acidification (ΔpH of ca. 2.7 at 25°C and ca. 1.2-1.4 at 4°C in the organic rice drink. The evaluation of viability and acidification by promising candidates should be a simple procedure to screen yeast strains for potential use as starter cultures to design new rice-fermented functional beverages.

  19. Trehalose-Based Eye Drops Preserve Viability and Functionality of Cultured Human Corneal Epithelial Cells during Desiccation

    Directory of Open Access Journals (Sweden)

    Aneta Hill-Bator


    Full Text Available This paper presents the evaluation of cytoprotective ability of trehalose-based eye drops in comparison with commercially available preparations during the experimental desiccation of cultured human corneal epithelial cells. Cultured human corneal epithelial cells (hCEC underwent incubation with 7 different, commercially available medicaments used commonly in dry eye syndrome treatment, followed by desiccation trial performed on air under the flow hood for 5, 15, 30, and 45 minutes. Cell viability was quantified by live/dead fluorescent assay, while the presence of apoptotic cells was estimated by immunofluorescent staining for active caspase 3 protein. The preservation of membrane functions was evaluated using neutral red staining, while the preservation of proper morphology and phenotype was determined by fluorescent staining for actin filaments, nuclei, and p63 protein. The trehalose-based eye drops showed the highest efficiency in prevention of cell death from desiccation; moreover, this preparation preserved the normal cellular morphology, functions of cell membrane, and proliferative activity more effectively than other tested medicaments.

  20. Antibodies against mucin-based glycopeptides affect Trypanosoma cruzi cell invasion and tumor cell viability. (United States)

    Campo, Vanessa L; Riul, Thalita B; Carvalho, Ivone; Baruffi, Marcelo-Dias


    This study describes the synthesis of glycopeptides NHAc[βGal]-(Thr)2 -[αGalNAc]-(Thr)2 -[αGlcNAc]-(Thr)2 Gly-OVA (1-OVA) and NHAc[βGal-αGalNAc]-(Thr)3 -[αLacNAc]-(Thr)3 -Gly-OVA (2-OVA) as mimetics of both T. cruzi and tumor mucin glycoproteins. These glycopeptides were obtained by solid-phase synthesis, which involved the prior preparation of the protected glycosyl amino acids αGlcNAc-ThrOH (3), αGalNAc-ThrOH (4), βGal-ThrOH (5), αLacNAc-ThrOH (6), and βGal-αGalNAc-ThrOH (7) through glycosylation reactions. Immunizations of mice with glycopeptides 1-OVA and 2-OVA induced high antibody titers (1:16 000), as verified by ELISA tests, whereas flow cytometry assays showed the capacity of the obtained anti-glycopeptides 1-OVA and 2-OVA antibodies to recognize both T. cruzi and MCF-7 tumor cells. In addition, antisera induced by glycopeptides 1-OVA and 2-OVA were also able to inhibit T. cruzi fibroblast cell invasion (70 %) and to induce antibody-mediated cellular cytotoxicity (ADCC) against MCF-7 cells, with 50 % reduction of cell viability.

  1. An individual-based model for population viability analysis of humpback chub in Grand Canyon (United States)

    Pine, William Pine; Healy, Brian; Smith, Emily Omana; Trammell, Melissa; Speas, Dave; Valdez, Rich; Yard, Mike; Walters, Carl; Ahrens, Rob; Vanhaverbeke, Randy; Stone, Dennis; Wilson, Wade


    We developed an individual-based population viability analysis model (females only) for evaluating risk to populations from catastrophic events or conservation and research actions. This model tracks attributes (size, weight, viability, etc.) for individual fish through time and then compiles this information to assess the extinction risk of the population across large numbers of simulation trials. Using a case history for the Little Colorado River population of Humpback Chub Gila cypha in Grand Canyon, Arizona, we assessed extinction risk and resiliency to a catastrophic event for this population and then assessed a series of conservation actions related to removing specific numbers of Humpback Chub at different sizes for conservation purposes, such as translocating individuals to establish other spawning populations or hatchery refuge development. Our results suggested that the Little Colorado River population is generally resilient to a single catastrophic event and also to removals of larvae and juveniles for conservation purposes, including translocations to establish new populations. Our results also suggested that translocation success is dependent on similar survival rates in receiving and donor streams and low emigration rates from recipient streams. In addition, translocating either large numbers of larvae or small numbers of large juveniles has generally an equal likelihood of successful population establishment at similar extinction risk levels to the Little Colorado River donor population. Our model created a transparent platform to consider extinction risk to populations from catastrophe or conservation actions and should prove useful to managers assessing these risks for endangered species such as Humpback Chub.

  2. Viability Theory

    CERN Document Server

    Aubin, Jean-Pierre; Saint-Pierre, Patrick


    Viability theory designs and develops mathematical and algorithmic methods for investigating the adaptation to viability constraints of evolutions governed by complex systems under uncertainty that are found in many domains involving living beings, from biological evolution to economics, from environmental sciences to financial markets, from control theory and robotics to cognitive sciences. It involves interdisciplinary investigations spanning fields that have traditionally developed in isolation. The purpose of this book is to present an initiation to applications of viability theory, explai

  3. Computer-determined assay time based on preset precision

    Energy Technology Data Exchange (ETDEWEB)

    Foster, L.A.; Hagan, R.; Martin, E.R.; Wachter, J.R.; Bonner, C.A.; Malcom, J.E. [Los Alamos National Lab., NM (United States). Nuclear Materials Measurement and Accountability


    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.

  4. Cell-based Assays to Identify Inhibitors of Viral Disease (United States)

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong


    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  5. 川牛膝种子发芽试验与生活力测定方法的研究%Study on Method of Seed Germination Testing and Viability Assay of Cyathula officinalis Kuan.

    Institute of Scientific and Technical Information of China (English)

    刘千; 罗浩; 蔡文国; 吴卫


    针对目前尚无川牛膝种子发芽和生活力检测标准的现状,采用不同的发芽床、发芽温度和前处理方法进行发芽试验;采用红墨水法、BTB法和TTC法对川牛膝种子生活力进行测定.结果表明,褶裥纸、25℃和不作前处理为最适宜的发芽条件;第2天和第9天分别为初末次计数时间;浓度1%的TTC溶液染色3h为最佳生活力测定方法.%Considering the lack of seed germination testing and viability assay standards for Cyathula officinalis Kuan. ,this paper studied the effects of different germination beds, temperatures and pre-treatments on seed germination. Red ink staining, BTB staining and TTC staining were used for determining seed viability assay. The results showed that pleated paper,25 ℃ and no pre-treatment were the optimum conditions for seed germination. The 2 nd day and the 9 th day were the first and the last count time. The 1% TTC solution dyed 3 h was the best method for seed viability assay of Cyathula officinalis Kuan.

  6. A Cell-Based Assay to Assess Hemichannel Function (United States)

    Krishnan, Srinivasan; Fiori, Mariana C.; Cuello, Luis G.; Altenberg, Guillermo A.


    Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.

  7. Comparison of methodologies for enumerating and detecting the viability of Ascaris eggs in sewage sludge by standard incubation-microscopy, the BacLight Live/Dead viability assay and other vital dyes. (United States)

    Karkashan, Alaa; Khallaf, Basma; Morris, Jacqueline; Thurbon, Nerida; Rouch, Duncan; Smith, Stephen R; Deighton, Margaret


    The aim of this study was to evaluate the Live/Dead BacLight viability kit as a method for enumerating viable eggs of Ascaris suum in sewage sludge as a surrogate for the human roundworm. The number and viability status of eggs of A. suum were accurately measured directly in sewage sludge samples by the BacLight method, compared to the conventional incubation-microscopy procedure. BacLight stains were not toxic to A. suum eggs, in contrast to some conventional vital dyes which disrupted viable eggs. The method was effective for the direct examination of eggs in heavily contaminated samples or seeded sludge containing ∼200 eggs/g DS in sludge with 5% DS content. However, a recovery method would be necessary to examine samples with small numbers of eggs, for instance in sludge from regions where the prevalence of infection with Ascaris lumbricoides is low. The BacLight technique may therefore be an effective alternative to conventional incubation-microscopy for enumerating Ascaris eggs in contaminated field samples or to validate sludge treatment processes by examining decay rates of inoculated A. suum eggs in laboratory simulations. Most field samples would require recovery from an appropriate number of composite samples prior to vital staining.

  8. Traditional and Model Based Assay of Irregular Geometry Items

    Energy Technology Data Exchange (ETDEWEB)



    The Analytical Development Section (ADS) of SRNL was requested to perform a waste disposal assay of two heater boxes which had been used in the HB Line dissolvers. They had been sent to SRNL for study to make recommendations on how to prevent future failure of the units when they were replaced. The study having been completed, the units needed to be characterized prior to sending to Solid Waste for disposal. An assay station consisting of a turntable, HPGe detector, CANBERRA Inspector, transmission source and a portable computer was set up to do the required assays. The assays indicate the presence of U-235, Pu-239 and Cs-137. No measurable amounts of U-235 or Pu-239 were found. Therefore the Minimum Detectable Activities for U-235 and Pu-239 were calculated. For Heater Box 1, 0.23 grams of U-235 and 0.24 grams of Pu-239. For Heater Box 2, the results were 0.21 grams of U-235 and 0.21 grams of Pu-239. This paper describes and documents the assays employed to determine the amount of U, Pu and Cs contents of the heater boxes. The paper provides results of SNM assays using traditional calibration of the system and on one based on modeling. It also provides the scientific community with data that will assist the user in determining the method of choice for assaying items with irregular geometries.

  9. Comparison of microscopy and Alamar blue reduction in a larval based assay for schistosome drug screening.

    Directory of Open Access Journals (Sweden)

    Nuha R Mansour

    Full Text Available BACKGROUND: In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS to identify hits amongst large compound libraries. METHODOLOGY/PRINCIPAL FINDINGS: We have been carrying out low throughput (24-well plate in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB, a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes. CONCLUSION: We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome

  10. Liquid crystal based sensors monitoring lipase activity: a new rapid and sensitive method for cytotoxicity assays. (United States)

    Hussain, Zakir; Zafiu, Christian; Küpcü, Seta; Pivetta, Lucineia; Hollfelder, Nadine; Masutani, Akira; Kilickiran, Pinar; Sinner, Eva-Kathrin


    In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity.

  11. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas


    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

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

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

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

  13. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.

    Directory of Open Access Journals (Sweden)

    Piwen Wang

    Full Text Available BACKGROUND: The chemopreventive effect of green tea polyphenols, such as (--epigallocatechin-3-gallate (EGCG, has been well demonstrated in cell culture studies. However, a wide range of IC(50 concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature. METHODOLOGY/PRINCIPAL FINDINGS: We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP and deoxynucleic acid (DNA showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95. However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC(50 concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.

  14. A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

    Directory of Open Access Journals (Sweden)

    Prather Brittany


    Full Text Available Abstract Background Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS. These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes. Results A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE is described. This assay has been successfully applied to substrates as small as α-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of ± 0.2 of the actual relative mobilities. Conclusion The versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

  15. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

    Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon;


    Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...... of EPO in a high-throughput setting....

  16. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies. (United States)

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A


    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  17. Standardized Serum-Free Cryomedia Maintain Peripheral Blood Mononuclear Cell Viability, Recovery, and Antigen-Specific T-Cell Response Compared to Fetal Calf Serum-Based Medium (United States)

    Schulz, Julia C.; Kemp-Kamke, Beatrice; Zimmermann, Heiko; von Briesen, Hagen


    The ability to analyze cryopreserved peripheral blood mononuclear cells (PBMCs) from biobanks for antigen-specific T-cell immunity is necessary to evaluate responses to immune-based therapies. Comprehensive studies have demonstrated that the quality of frozen PBMCs is critical and the maintenance of cell viability and functionality by using appropriate cryopreservation techniques is a key to the successful outcome of assays using PBMCs. Different cryomedia additives affect cell viability. The most common additive is fetal calf serum (FCS), although it is widely known that each FCS lot has to be tested before usage to prevent nonspecific stimulation of T-cells. Also, shipping of samples containing FCS is critical because of many import restrictions. Often, dimethyl sulfoxide (DMSO) is added as a cryoprotectant. However, DMSO concentration has to be reduced significantly because of its toxic effect on cells at room temperature. Therefore, we have developed freezing approaches to minimize cytotoxicity of cryoprotectants and maintain T-cell functionality. We compared different additives to the widely used FCS and found bovine serum albumin fraction V to be an appropriate substitute for the potentially immune-modulating FCS. We also found that DMSO concentration can be reduced by the addition of hydroxyethyl starch. Using our serum-free cryomedia, the PBMC recovery was more than 83% and the PBMC viability was more than 98%. Also, the T-cell functionality measured by enzyme-linked immunospot (ELISpot) was optimal after cryopreservation with our new cryomedia. On the basis of our experimental results, we could finally design 2 different, fully working cryomedia that are standardized, serum free, and manufactured under GMP conditions. PMID:21977240

  18. Cell viability and PSA secretion assays in LNCaP cells: a tiered in vitro approach to screen chemicals with a prostate-mediated effect on male reproduction within the ReProTect project. (United States)

    Lorenzetti, Stefano; Marcoccia, Daniele; Narciso, Laura; Mantovani, Alberto


    Prostate function is critical for male fertility; nevertheless, prostate was so far overlooked in reproductive toxicity assays. Within the EU project ReProTect, the human prostate cell line LNCaP was utilized to identify molecules targeting prostate function by the integrated assessment of cell viability (MTS assay) and prostate-specific antigen (PSA) secretion as specific marker; a training set - five (anti)androgenic chemicals - and a ReProTect feasibility set - ten chemicals - were used. Several compounds reduced PSA only at cytotoxic concentrations. Androgens (DHT, MT) markedly increased PSA as did the herbicide glufosinate ammonium, not known as androgen agonist. Anti-androgens (2OH-flutamide, linuron, vinclozolin, di-n-butyl phthalate) also increased PSA, but the effect of magnitude was much lower than for androgens. The ER-binder bisphenol A reduced PSA, while increasing cell viability. At this stage, the approach can identify chemicals able to interfere with prostate function: further refinements may allow to include prostate effects in reproductive toxicity in vitro testing.

  19. Host-based Th2 cell therapy for prolongation of cardiac allograft viability.

    Directory of Open Access Journals (Sweden)

    Shoba Amarnath

    Full Text Available Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1 reduced the frequency of activated T cells in secondary lymphoid organs; (2 shifted post-transplant cytokines towards a Th2 phenotype; and (3 prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use "direct" host T cell therapy for prolongation of allograft viability as an alternative to "indirect" therapy mediated by donor T cell infusion.

  20. Defining viability in mammalian cell cultures


    Browne, Susan M.; Al-Rubeai, Mohamed


    Abstract A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure me...

  1. ApoHRP-based Assay to Measure Intracellular Regulatory Heme (United States)

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A.; Dhahbi, Joseph M.


    The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells. PMID:25525887

  2. Osmolarity of Coconut Water (Cocos nucifera Based Diluents and their Effect Over Viability of Frozen Boar Semen

    Directory of Open Access Journals (Sweden)

    Bottini Luzardo


    Full Text Available Problem statement: Boar sperm cells are sensitive to the freezing process, which compromises viability of frozen-thawed sperm. In a constant search for minimizing or suppressing sperm cell damage caused by the temperature and osmolarity changes during the freezing process, crioprotective and antioxidant substances have been added to the freezing media, such as coconut water, in order to increase the viability of frozen-thawed swine semen. The addition of any substance to the freezing diluent, directly affects osmolarity of the media, which can have positive or negative effects over the sperm cell. Approach: There are no published studies currently that indicate the effect of adding coconut water over the osmolarity of freezing media and their effect over viability of sperm cells, therefore, the objective of the present study was to evaluate the effect of coconut water based diluents osmolarity over the Motility (Mot, Acrosome Integrity (AI, Membrane Integrity (MI and Mitochondrial Activity (MA of thawed boar sperm cells. The treatments used were control T1 (LEY with bidistilled water + LEYGO with an osmolarity range of 296-368 mOsmol Kg-1, T2 (LEY and deionized coconut water + LEYGO between 381 and 480 mOsmol Kg-1 and T3 (LEY and in natura coconut water + LEYGO between 519 and 1041 mOsmol Kg-1. The Westendorf modified method was the freezing method used. The obtained data were statistically analyzed by GLM, using the SAS software (SAS, 2000. Results: A significant difference was observed on T2 compared to T1 regarding Mot 41.9 Vs 36.9% and MI 58.0 Vs 50.2. T3 had a detrimental effect on all studied variables. Conclusion: Under our study conditions, the osmolarity range of T2, due to the non ionic solutes content, contributed to improve the viability of frozen-thawed sperm cells.

  3. Miniaturized Aptamer-Based Assays for Protein Detection

    Directory of Open Access Journals (Sweden)

    Alessandro Bosco


    Full Text Available The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range assay conditions.

  4. A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay. (United States)

    Park, Jun Hee; Cho, Yea Seul; Kang, Sungmuk; Lee, Eun Jeong; Lee, Gwan-Ho; Hah, Sang Soo


    A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3',5'-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.

  5. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres. (United States)

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C


    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  6. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  7. Assessment of cell viability. (United States)

    Johnson, Simon; Nguyen, Vy; Coder, David


    Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation with amine-reactive dyes suitable for a range of excitation wavelengths. Membrane-impermeable dead cell and live cell dyes as well as dye-exclusion procedures for microscopy are also included.

  8. New optical sensing technique of tissue viability and blood flow based on nanophotonic iterative multi-plane reflectance measurements

    Directory of Open Access Journals (Sweden)

    Yariv I


    Full Text Available Inbar Yariv,1 Menashe Haddad,2,3 Hamootal Duadi,1 Menachem Motiei,1 Dror Fixler1 1Faculty of Engineering and the Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan, Israel; 2Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 3Mayanei Hayeshua Medical Center, Benei Brak, Israel Abstract: Physiological substances pose a challenge for researchers since their optical properties change constantly according to their physiological state. Examination of those substances noninvasively can be achieved by different optical methods with high sensitivity. Our research suggests the application of a novel noninvasive nanophotonics technique, ie, iterative multi-plane optical property extraction (IMOPE based on reflectance measurements, for tissue viability examination and gold nanorods (GNRs and blood flow detection. The IMOPE model combines an experimental setup designed for recording light intensity images with the multi-plane iterative Gerchberg-Saxton algorithm for reconstructing the reemitted light phase and calculating its standard deviation (STD. Changes in tissue composition affect its optical properties which results in changes in the light phase that can be measured by its STD. We have demonstrated this new concept of correlating the light phase STD and the optical properties of a substance, using transmission measurements only. This paper presents, for the first time, reflectance based IMOPE tissue viability examination, producing a decrease in the computed STD for older tissues, as well as investigating their organic material absorption capability. Finally, differentiation of the femoral vein from adjacent tissues using GNRs and the detection of their presence within blood circulation and tissues are also presented with high sensitivity (better than computed tomography to low quantities of GNRs (<3 mg. Keywords: Gerchberg-Saxton, optical properties, gold nanorods, blood vessel, tissue viability

  9. Cell based assays for anti-Plasmodium activity evaluation. (United States)

    Mokgethi-Morule, Thabang; N'Da, David D


    Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed.

  10. Use of fluorescent redox indicators to evaluate cell proliferation and viability

    DEFF Research Database (Denmark)

    Rasmussen, E.S.


    The performance of two cell viability test kits based on the use of redox indicators yielding fluorescent products, the AlamarBlue assay and a resazurin-based in vitro toxicology assay kit from Sigma, was compared in the present study. Cultures of human neonatal foreskin fibroblasts were exposed...... components were tentatively identified as resazurin and resorufin. The AlamarBlue assay has gained wide application as a cell viability indicator that allows continuous monitoring of cell proliferation or cytotoxicity in human and animal cells, bacteria, and fungi, but no studies with the deliberate use...

  11. ABAP: antibody-based assay for peptidylarginine deiminase activity. (United States)

    Zendman, Albert J W; Raijmakers, Reinout; Nijenhuis, Suzanne; Vossenaar, Erik R; Tillaart, Marloes van den; Chirivi, Renato G S; Raats, Jos M H; van Venrooij, Walther J; Drijfhout, Jan W; Pruijn, Ger J M


    Members of the family of peptidylarginine deiminases (PADs, EC catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.

  12. Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent. (United States)

    Kibbee, Richard J; Örmeci, Banu


    The detection and quantification of viable Escherichia coli cells in wastewater treatment plant effluent is very important as it is the main disinfection efficacy parameter for assessing its public health risk and environmental impact. The aim of this study was to develop a sensitive and false-positive free propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay to quantify the viable but non-culturable (VBNC) E. coli present in secondary wastewater effluent after chlorine disinfection. The qPCR target was the E. coli uidA gene, and native Taq was used to eliminate false positives caused by the presence of contaminant E. coli DNA in recombinant Taq polymerase reagents. Due to issues with qPCR inhibitors in wastewater, this study explored several pre-DNA extraction treatment methods for qPCR inhibitor removal. PMA-qPCR validation was done using salmon testes DNA (Sketa DNA) as an exogenous control added directly to the wastewater samples and amplified using a separate qPCR assay. After disinfection of secondary effluent with 2ppm chlorine at the plant, the mean Log10 CFU reduction in E. coli was 2.85 from a mean CFU of 3.48/10mL compared to 0.21 Log10 CCE mean reduction of the uidA gene from a mean CCE of 3.16/10mL. The VBNC cell concentrations were calculated as 2.32 Log10/10mL by subtracting the colony forming units (CFU) obtained from membrane filtration from the calculated CFU equivalent (CCE) values obtained from PMA-qPCR. These results demonstrate the effective use of a PMA-qPCR method for the quantification of the E. coli uidA gene and indicate there are high numbers (2.01×10(3)CCE/100mL) of VBNC E. coli cells leaving the wastewater treatment plant in the final effluent after chlorine treatment. VBNC bacterial cells are of concern as they have the potential to resuscitate and grow, regain virulence, affect natural microbiome in the discharge sites, and pass on antimicrobial resistant genes to other microorganisms.

  13. Viability and fertility of cooled equine semen diluted with skimmed milk or glycine egg yolk-based extenders

    Directory of Open Access Journals (Sweden)

    Guilherme Pugliesi


    Full Text Available Two semen extenders were compared for their ability to maintain viability of horse semen during 24 hours of cold preservation, and for the pregnancy rate after artificial insemination. In the experiment 1, five ejaculates from three stallions were split-diluted in either a skimmed milk-based extender (Kenney extender or a glycine egg yolk-based extender (Foote extender and cooled at 6-8 ºC for 24 hours. Semen samples stored in Kenney extender for 24 hours had higher motility and spermatic vigor compared with those stored in Foote extender. However, samples stored in Foote extender had higher number of reactive sperm by hypoosmotic test and greater viability by epifluorescence test compared with those in Kenney extender. In the experiment 2, 17 and 23 ejaculates from two stallions were split-diluted with Kenney extender and Foote extender. The sperm concentration in each extender was adjusted to 500 million viable sperms per insemination dose. Semen was cooled to 6-8 ºC and stored for 24 hours. Seventy-four cycles of crossbred mares were inseminated with either semen diluted in Kenney extender or semen diluted in Foote extender. The pregnancy rate was higher from semen diluted in Kenney extender than that from semen in Foote extender (0.553 vs. 0.306. The Kenney extender is effective in preserving the motility, vigor and fertility of stallion semen after 24 hours of cold storage, whereas the Foote extender is not acceptable.

  14. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.


    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  15. Effects of two plant growth regulators, indole-3-acetic acid and β-naphthoxyacetic acid, on genotoxicity in Drosophila SMART assay and on proliferation and viability of HEK293 cells from the perspective of carcinogenesis. (United States)

    Karadeniz, Asuman; Kaya, Bülent; Savaş, Burhan; Topcuoğlu, Ş Fatih


    In this study, the mutagenic and recombinogenic effects of indole-3-acetic acid (IAA), a plant growth regulator naturally synthesized in plants but produced synthetically, and β-naphthoxyacetic acid (BNOA), a synthetic plant growth regulator widely used in agricultural regions, were investigated using the somatic mutation and recombination test (SMART) in Drosophila wings. The effect of the same plant growth regulators against the proliferation and viability of a human immortalized embryonic kidney HEK293 cells which is at the early stage of carcinogenesis were also examined with MTT and trypan-blue exclusion assays. For the SMART assay, two different crosses were used: a standard and a high-bioactivation (HB) cross, involving the flare-3 and the multiple wing hairs markers. The HB cross involved flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits the more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. The results show that IAA and BNOA are not mutagenic or recombinogenic in the wing cells of Drosophila. Furthermore, neither plant growth regulator affected the proliferation rate of HEK293 cells; however, both of them induced cell death at high concentrations.

  16. Automated microfluidic screening assay platform based on DropLab. (United States)

    Du, Wen-Bin; Sun, Meng; Gu, Shu-Qing; Zhu, Ying; Fang, Qun


    This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.

  17. A functional assay-based strategy for nanomaterial risk forecasting

    Energy Technology Data Exchange (ETDEWEB)

    Hendren, Christine Ogilvie, E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Lowry, Gregory V., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Carnegie Mellon University, 119 Porter Hall, Pittsburgh, PA 15213 (United States); Unrine, Jason M., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Plant and Soil Sciences, University of Kentucky, Agricultural Science Center, Lexington, KY 40546 (United States); Wiesner, Mark R., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Duke University, 121 Hudson Hall PO Box 90287, Durham, NC 27708 (United States)


    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical–chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical–chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. - Highlights: • Approaches to predict risk directly from nanomaterial (NM) properties are problematic. • We propose

  18. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting. (United States)

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E


    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

  19. The economic viability of value-based food chain for dairy farms in mountain regions: an econometric analysis approach

    Directory of Open Access Journals (Sweden)

    Jernej Prišenk


    Full Text Available The attention of this paper is drawn to analyze the economic potential of involvement of farmers into the small-medium sized value-based food chain (VBFC. The survey represents a solid dana basis from which econometric modelling approach was further developed. Empirical results reveal the positive economic viability on a general level; this means more stable purchase price of raw milk for dairy farms, which are the part of value-based food chain. Results point at inelastic demand for milk and milk related products. Furthermore, there are some accompanying and underlying indirect social benefits, such as production of high-quality food products, more stable and constant demand for raw milk, steady payments and better social situation. The last one is especially important for the farms operating in less-favored mountain areas where the survey was actually conducted.

  20. Dakin Solution Alters Macrophage Viability and Function (United States)


    crobial for wound care. DS has been shown to be toxic to host cells, but effects on immune cells are not well documented. Materials and methods: DS at 0.5...characterize the impact of DS on macrophage viability and function in vitro. 2. Materials and methods 2.1. Cell lines and reagents Murine macrophages...strainer to separate conidia from mycelium , and stored in DMEM at 4C. 2.3. Cellular viability assays Effect of DS on cellular viability was

  1. Preparation and characterization of an anti-inflammatory agent based on a zinc-layered hydroxide-salicylate nanohybrid and its effect on viability of Vero-3 cells

    Directory of Open Access Journals (Sweden)

    Ramli M


    Full Text Available Munirah Ramli,1,2 Mohd Zobir Hussein,1,3 Khatijah Yusoff41Advanced Materials and Nanotechnology Laboratory, Institute of Advanced Technology, Universiti Putra Malaysia, Serdang, Selangor, 2Department of Industrial Biotechnology, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, Skudai, Johor, 3Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, 4Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, MalaysiaAbstract: A new organic-inorganic nanohybrid based on zinc-layered hydroxide intercalated with an anti-inflammatory agent was synthesized through direct reaction of salicylic acid at various concentrations with commercially available zinc oxide. The basal spacing of the pure phase nanohybrid was 15.73 Å, with the salicylate anions arranged in a monolayer form and an angle of 57 degrees between the zinc-layered hydroxide interlayers. Fourier transform infrared study further confirmed intercalation of salicylate into the interlayers of zinc-layered hydroxide. The loading of salicylate in the nanohybrid was estimated to be around 29.66%, and the nanohybrid exhibited the properties of a mesoporous-type material, with greatly enhanced thermal stability of the salicylate compared with its free counterpart. In vitro cytotoxicity assay revealed that free salicylic acid, pure zinc oxide, and the nanohybrid have a mild effect on viability of African green monkey kidney (Vero-3 cells.Keywords: anti-inflammatory, salicylic acid, zinc-layered hydroxide, zinc oxide, nanohybrid, cytotoxicity

  2. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies. (United States)

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A


    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  3. Development of a Drosophila cell-based error correction assay

    Directory of Open Access Journals (Sweden)

    Jeffrey D. Salemi


    Full Text Available Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT interactions before anaphase onset results in chromosomal instability (CIN, which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5. Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due to the activity of kinesin-14 (Ncd when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC. Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and

  4. High content cell-based assay for the inflammatory pathway (United States)

    Mukherjee, Abhishek; Song, Joon Myong


    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  5. Development of a Drosophila cell-based error correction assay. (United States)

    Salemi, Jeffrey D; McGilvray, Philip T; Maresca, Thomas J


    Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

  6. Viability of brown trout embryos positively linked to melanin-based but negatively to carotenoid-based colours of their fathers. (United States)

    Wedekind, Claus; Jacob, Alain; Evanno, Guillaume; Nusslé, Sébastien; Müller, Rudolf


    'Good-genes' models of sexual selection predict significant additive genetic variation for fitness-correlated traits within populations to be revealed by phenotypic traits. To test this prediction, we sampled brown trout (Salmo trutta) from their natural spawning place, analysed their carotenoid-based red and melanin-based dark skin colours and tested whether these colours can be used to predict offspring viability. We produced half-sib families by in vitro fertilization, reared the resulting embryos under standardized conditions, released the hatchlings into a streamlet and identified the surviving juveniles 20 months later with microsatellite markers. Embryo viability was revealed by the sires' dark pigmentation: darker males sired more viable offspring. However, the sires' red coloration correlated negatively with embryo survival. Our study demonstrates that genetic variation for fitness-correlated traits is revealed by male colour traits in our study population, but contrary to predictions from other studies, intense red colours do not signal good genes.

  7. The relationship between sperm viability and DNA fragmentation rates


    Mary K. Samplaski; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D.; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A


    Background In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of > 30% indicating subfertility. We postulated that in some men, the sperm viability assay coul...

  8. Exposure-based validation list for developmental toxicity screening assays

    NARCIS (Netherlands)

    Daston, George P.; Beyer, Bruce K.; Carney, Edward W.; Chapin, Robert E.; Friedman, Jan M.; Piersma, Aldert H.; Rogers, John M.; Scialli, Anthony R.


    Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as "positive" or "negative" in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a

  9. 基于 WST-8细胞活性实验的表面增强拉曼光谱研究%WST-8 Assay for Living Cell Viability Detection by Surface-enhanced Raman Spectroscopy†

    Institute of Scientific and Technical Information of China (English)

    杨津; 刘卓; 王晓慧; 隋会敏; 毛竹; 赵冰


    采用常规的用于测定活细胞个数的试剂2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐(WST-8四唑盐)建立了一种快速测定细胞活性的表面增强拉曼散射光谱(SERS)新方法.考察了pH 值及还原剂烟酰胺腺嘌噙二核苷酸(NADH)用量对 WST-8甲瓒信号的影响.结果表明,与传统的 WST-8比色法相比, SERS 法具有更高的检测灵敏度和选择性,对 WST-8甲瓒的检测限可达到10μmol/ L.%Surface-enhanced Raman scattering(SERS) technique was used to develop and optimize a novel and quantitative 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt(WST-8) assay for living cell viability. The influences of pH and dosage of reductant nicoti-namide adenine dinucleotide(NADH) on WST-8 formazan signal were examined. SERS signals of formazan with high resolution and great diversity between WST-8 and formazan were observed in neutral conditions. Meanwhile, the signals of formazan(560, 1351, 1598, 1616 cm-1 ) could be found clearly when the concen-tration of NADH was four times that of WST-8. Compared with the traditional method, this assay appears higher sensitivity and selectivity for WST-8 formazan. Moreover, the detection limit of formazan is as low as 10μmol/ L.

  10. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment. (United States)

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T


    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

  11. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Lotte Hatting Pugholm


    Full Text Available Extracellular vesicles (EVs are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA. This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

  12. GTP-specific fab fragment-based GTPase activity assay. (United States)

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri


    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  13. Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Seema, E-mail: [Department of Applied Mechanics (Biotechnology), Motilal Nehru National Institute of Technology, Allahabad 211004 (India); Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Tripathi, Vinay [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Singh, Harpal [Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Shrivastav, Tulsidas G. [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India)


    A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ng mL{sup -1} with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.

  14. Nanobeads-based assays. The case of gluten detection (United States)

    Venditti, Iole; Fratoddi, Ilaria; Vittoria Russo, Maria; Bellucci, Stefano; Crescenzo, Roberta; Iozzino, Luisa; Staiano, Maria; Aurilia, Vincenzo; Varriale, Antonio; Rossi, Mosè; D'Auria, Sabato


    In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

  15. A label-free, fluorescence based assay for microarray (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  16. Strategic framework for socioeconomic viability of community-based early warning system

    NARCIS (Netherlands)

    Homberg, M.J.C. van den; Posthumus, A.L.


    Christian Aid, Cordaid, PVGS and Practical Action established a community-based early warning system for cross-border floods between India and Nepal in 45 Indian villages. The project will scale to 95 villages early 2016. The number of stakeholders and organizational levels of this system with four

  17. Viability of mycelial growth of Coprinus Comatus in culture medium based on organic residues


    de Almeida, Lais Benes; Sales-Campos,Ceci; Melo de Carvalho, Cristiane Suely; de Almeida Minhoni, Marli Teixeira; Nogueira de Andrade, Meire Cristina


    The objective of this work was to evaluate the mycelial growth of the Coprinus comatus strain CCO 01/01 in culture based on organic residues of Saccharum officinarum (sugarcane bagasse), Citrus sinensis (orange bagasse), Ananas comosus (pineapple residues) and Musa sp. (banana leaf), supplemented with wheat bran in the proportions of 0, 10 and 20%, kept at 27 degrees C. The mycelial growth of C. comatus was evaluated daily by measurement of the diameter of the colony during seven days of incu...

  18. Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants (United States)

    Abdal-hay, Abdalla; Dewidar, Montasser; Lim, Jae Kyoo


    The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might widen the use of Mg based implants.

  19. Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants

    Energy Technology Data Exchange (ETDEWEB)

    Abdal-hay, Abdalla [Departmentt of Bionano System Engineering, College of Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Mechanical Design Engineering, Advanced wind power system research institute, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Dewidar, Montasser [Department of Materials and Mechanical Design, Faculty of Energy Engineering, South Valley University, Aswan (Egypt); Lim, Jae Kyoo, E-mail: [Department of Mechanical Design Engineering, Advanced wind power system research institute, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)


    Highlights: Black-Right-Pointing-Pointer The corrosion behavior of magnesium for orthopedic applications is extremely poor. Black-Right-Pointing-Pointer The solvent (DCM, THF and DMF) had a strong effect on the coatings performance. Black-Right-Pointing-Pointer Mg bar alloy coated with PVAc/DCM layers provided an excellent bonding strength. Black-Right-Pointing-Pointer Treated samples indicated significant damping for the degradation rate. Black-Right-Pointing-Pointer Cytocompatibility on MC3T3 cells of the PVAc/DCM samples revealed a good behavior. - Abstract: The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might

  20. The application of flow cytometry and fluorescent probe technology for detection and assessment of viability of plant pathogenic bacteria

    NARCIS (Netherlands)

    Chitarra, L.G.; Bulk, van den R.W.


    Conventional methods to detect and assess the viability of plant pathogenic bacteria are usually based on plating assays or serological techniques. Plating assays provide information about the number of viable cells, expressed as colony-forming units, but are time-consuming and laborious. Serologica

  1. Expanding the available assays: adapting and validating In-Cell Westerns in microfluidic devices for cell-based assays. (United States)

    Paguirigan, Amy L; Puccinelli, John P; Su, Xiaojing; Beebe, David J


    Microfluidic methods for cellular studies can significantly reduce costs due to reduced reagent and biological specimen requirements compared with many traditional culture techniques. However, current types of readouts are limited and this lack of suitable readouts for microfluidic cultures has significantly hindered the application of microfluidics for cell-based assays. The In-Cell Western (ICW) technique uses quantitative immunocytochemistry and a laser scanner to provide an in situ measure of protein quantities in cells grown in microfluidic channels of arbitrary geometries. The use of ICWs in microfluidic channels was validated by a detailed comparison with current macroscale methods and shown to have excellent correlation. Transforming growth factor-β-induced epithelial-to-mesenchymal transition of an epithelial cell line was used as an example for further validation of the technique as a readout for soluble-factor-based assays performed in high-throughput microfluidic channels. The use of passive pumping for sample delivery and laser scanning for analysis opens the door to high-throughput quantitative microfluidic cell-based assays that integrate seamlessly with existing high-throughput infrastructure.

  2. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays. (United States)

    Henriksen, Ulla; Fog, Jacob; Loechel, Frosty; Praestegaard, Morten


    Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.

  3. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique


    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  4. Different cytotoxicity responses to antimicrobial nanosilver coatings when comparing extract-based and direct-contact assays. (United States)

    Sussman, Eric M; Casey, Brendan J; Dutta, Debargh; Dair, Benita J


    This study was performed to understand how the choice of cytotoxicity assay format affects the observed biocompatibility of nanosilver (nAg). nAg coatings are physical coatings containing silver (Ag) that have feature sizes of 100 nm or less, often in the form of nanoparticles or grains. They are used on medical devices to prevent infection, but in spite of this intended benefit, observations of potential cytotoxicity from nAg have been reported in numerous published studies. For medical device regulation, cytotoxicity testing is part of a biocompatibility evaluation, in which specific test methods are chosen based on the technological characteristics and intended use of a device. For this study, nAg-coated tissue culture polystyrene surfaces were prepared using magnetron sputter coating, resulting in nAg films of 0.2 to 311 µg cm(-2) Ag. These coatings exhibited nanometer-scale morphologies and demonstrated a > 4log10 reduction in Escherichia coli viability. It was observed that extracts of nAg caused no cytotoxicity to L929 mouse fibroblasts, but cells cultured directly on nAg coatings (direct-contact assay format) showed a dose-dependent reduction in viability by up to 100% (P < 0.001). Results using inductively coupled plasma mass spectrometry to measure Ag release suggested that extracts of nAg are not toxic because the dissolved Ag in those samples becomes less cytotoxic over time, probably owing to the reaction with cell culture media and serum (six-fold cytotoxicity reductions observed over a 24-h period). These findings highlight the potential value of direct-contact cytotoxicity testing for nAg in predicting biological interactions with cells or tissue in vivo.

  5. p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures. (United States)

    Di Paolo, Carolina; Müller, Yvonne; Thalmann, Beat; Hollert, Henner; Seiler, Thomas-Benjamin


    The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

  6. Pseudotype-based neutralization assays for influenza: a systematic analysis

    Directory of Open Access Journals (Sweden)

    George William Carnell


    Full Text Available The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI-competent antibodies that target the globular head of HA thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D towards the production of a universal vaccine has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1 to H16. The accurate and sensitive measurement of antibody responses elicited by these next-generation influenza vaccines is however hampered by the lack of sensitivity of the traditional influenza serological assays hemagglutinin inhibition (HI, single radial hemolysis (SRH and microneutralization (MN. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly-neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies.

  7. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay. (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G


    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

  8. Cell Viability Assessment: Toward Content-Rich Platforms (United States)

    Ramirez, Christina Nicole; Antczak, Christophe; Djaballah, Hakim


    Importance of the field Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening (HTS) toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete endpoint measurements such as membrane permeability. Areas covered in this review/What the reader will gain This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function. We describe each approach, and using case studies, we emphasize their limitations. Take home message Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously, on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately. PMID:22823019

  9. Receptor-based screening assays for the detection of antibiotics residues - A review. (United States)

    Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui


    Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics.

  10. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays (United States)

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


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

  11. A protein-based oxygen biosensor for high-throughput monitoring of cell growth and cell viability

    NARCIS (Netherlands)

    Strianese, M.; Zauner, G.; Tepper, A.W.J.W.; Bubacco, L.; Breukink, E.J.; Aartsma, J.; Canters, G.W.; Tabares, L.C.


    Fluorescently labeled hemocyanin has been previously proposed as an oxygen sensor. In this study, we explored the efficacy of this biosensor for monitoring the biological oxygen consumption of bacteria and its use in testing bacterial cell growth and viability of Escherichia coli, Pseudomonas aerugi

  12. Cell-based assays in GPCR drug discovery. (United States)

    Siehler, Sandra


    G protein-coupled receptors (GPCRs) transmit extracellular signals into the intracellular space, and play key roles in the physiological regulation of virtually every cell and tissue. Characteristic for the GPCR superfamily of cell surface receptors are their seven transmembrane-spanning alpha-helices, an extracellular N terminus and intracellular C-terminal tail. Besides transmission of extracellular signals, their activity is modulated by cellular signals in an auto- or transregulatory fashion. The molecular complexity of GPCRs and their regulated signaling networks triggered the interest in academic research groups to explore them further, and their drugability and role in pathophysiology triggers pharmaceutical research towards small molecular weight ligands and therapeutic antibodies. About 30% of marketed drugs target GPCRs, which underlines the importance of this target class. This review describes current and emerging cellular assays for the ligand discovery of GPCRs.

  13. Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay). (United States)

    Schmid, Claudia; Arndt, Christian; Reifferscheid, Georg


    The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

  14. Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

    NARCIS (Netherlands)

    Chitarra, L.G.


    Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on serological techni

  15. Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

    NARCIS (Netherlands)

    Tanneberger, K.; Rico Rico, A.; Kramer, N.I.; Busser, F.J.M.; Hermens, J.L.M.; Schirmer, K.


    Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly underst

  16. Development of a lipase-based optical assay for detection of DNA

    DEFF Research Database (Denmark)

    Pinijsuwan, Suttiporn; Shipovskov, Stepan; Surareungchai, Werasak


    A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate...

  17. Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.

    Directory of Open Access Journals (Sweden)

    Taylor R Paskin

    Full Text Available Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli, planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green, as well as ultraviolet (UV and infrared (IR which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV causing the most intense photophobic responses while longer wavelengths produce no effect (red or an apparent attraction (IR. In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment.

  18. Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification. (United States)

    Derda, Ratmir; Lockett, Matthew R; Tang, Sindy K Y; Fuller, Renee C; Maxwell, E Jane; Breiten, Benjamin; Cuddemi, Christine A; Ozdogan, Aysegul; Whitesides, George M


    This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

  19. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus. (United States)

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng


    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  20. Fluorescence Hybridization Assay Based On Chitosan-Linked Softarrays (United States)


    was incubated in the wells to reduce the Schiff base resulting from the reaction of aldehyde and amine groups. After this reaction, the yellowish...color representative of a Schiff base disappeared and the background fluorescence signal dropped to the initial ~8 to 12 fluorescence intensity (FI

  1. Influence of carvacrol and 1,8-cineole on cell viability, membrane integrity, and morphology of Aeromonas hydrophila cultivated in a vegetable-based broth. (United States)

    de Sousa, Jossana Pereira; de Oliveira, Kataryne Árabe Rimá; de Figueiredo, Regina Celia Bressan Queiroz; de Souza, Evandro Leite


    This study investigated the effects of carvacrol (CAR) and 1,8-cineole (CIN) alone (at the MIC) or in combination at subinhibitory amounts (both at 1/8 MIC) on the cell viability, membrane permeability, and morphology of Aeromonas hydrophila INCQS 7966 (A. hydrophila) cultivated in a vegetable-based broth. CAR and CIN alone or in combination severely affected the viability of the bacteria and caused dramatic changes in the cell membrane permeability, leading to cell death, as observed by confocal laser microscopy. Scanning and transmission electron microscopy images of bacterial cells exposed to CAR or CIN or the mixture of both compounds revealed severe changes in cell wall structure, rupture of the plasma membrane, shrinking of cells, condensation of cytoplasmic content, leakage of intracellular material, and cell collapse. These findings suggest that CAR and CIN alone or in combination at subinhibitory amounts could be applied to inhibit the growth of A. hydrophila in foods, particularly as sanitizing agents in vegetables.

  2. The Impact of a Values-Based Supply Chain (VBSC on Farm-Level Viability, Sustainability and Resilience: Case Study Evidence

    Directory of Open Access Journals (Sweden)

    Teresa Hooks


    Full Text Available The ‘Agriculture of the Middle’ (AotM development paradigm emphasises that in order to survive, family farms must transition from a supply chain approach to a values-based supply chain (VBSC approach, involving amendments to both product type and actor dynamics within the chain. This paper presents a qualitative case study of a beef co-operative integrated to a VBSC. We use an analytical framework of viability, sustainability and resilience to analyse impacts at farm-level. Our analysis highlights a number of positive effects on farm-level viability, sustainability and resilience. These benefits stemmed largely from improvements to market orientation, price stability, and members’ capacities in responding to problems. However, the autonomy of the co-operative was challenged by VBSC chain members, which impacted negatively on the stability of the co-operative.

  3. Degradation characteristics, cell viability and host tissue responses of PDLLA-based scaffold with PRGD and β-TCP nanoparticles incorporation. (United States)

    Yi, Jiling; Xiong, Feng; Li, Binbin; Chen, Heping; Yin, Yixia; Dai, Honglian; Li, Shipu


    This study is aimed to evaluate the degradation characteristics, cell viability and host tissue responses of PDLLA/PRGD/β-TCP (PRT) composite nerve scaffold, which was fabricated by poly(d, l-lactic acid) (PDLLA), RGD peptide(Gly-Arg-Gly-Asp-Tyr, GRGDY, abbreviated as RGD) modified poly-{(lactic acid)-co-[(glycolic acid)-alt-(l-lysine)]}(PRGD) and β-tricalcium phosphate (β-TCP). The scaffolds' in vitro degradation behaviors were investigated in detail by analysing changes in weight loss, pH and morphology. Then, the 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2 -H-tetrazolium bromide (MTT) assay and cell live/dead assay were carried out to assess their cell viability. Moreover, in vivo degradation patterns and host inflammation responses were monitored by subcutaneous implantation of PRT scaffold in rats. Our data showed that, among the tested scaffolds, the PRT scaffold had the best buffering capacity (pH = 6.1-6.3) and fastest degradation rate (12.4%, 8 weeks) during in vitro study, which was contributed by the incorporation of β-TCP nanoparticles. After in vitro and in vivo degradation, the high porosity structure of PRT could be observed using scanning electron microscopy. Meanwhile, the PRT scaffold could significantly promote cell survival. In the PRT scaffold implantation region, less inflammatory cells (especially for neutrophil and lymphocyte) could be detected. These results indicated that the PRT composite scaffold had a good biodegradable property; it could improve cells survival and reduced the adverse host tissue inflammation responses.

  4. Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays (United States)

    de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra


    Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

  5. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo


    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  6. Microchip-based ultrafast serodiagnostic assay for tuberculosis (United States)

    Mani, Vigneshwaran; Paleja, Bhairav; Larbi, Karima; Kumar, Pavanish; Tay, Jo Ann; Siew, Jie Yee; Inci, Fatih; Wang, ShuQi; Chee, Cynthia; Wang, Yee Tang; Demirci, Utkan; De Libero, Gennaro; Singhal, Amit


    Access to point-of-care (POC), rapid, inexpensive, sensitive, and instrument-free tests for the diagnosis of tuberculosis (TB) remains a major challenge. Here, we report a simple and low-cost microchip-based TB ELISA (MTBE) platform for the detection of anti-mycobacterial IgG in plasma samples in less than 15 minutes. The MTBE employs a flow-less, magnet-actuated, bead-based ELISA for simultaneous detection of IgG responses against multiple mycobacterial antigens. Anti-trehalose 6,6′-dimycolate (TDM) IgG responses were the strongest predictor for differentiating active tuberculosis (ATB) from healthy controls (HC) and latent tuberculosis infections (LTBI). The TDM-based MTBE demonstrated superior sensitivity compared to sputum microscopy (72% vs. 56%) with 80% and 63% positivity among smear-positive and smear-negative confirmed ATB samples, respectively. Receiver operating characteristic analysis indicated good accuracy for differentiating ATB from HC (AUC = 0.77). Thus, TDM-based MTBE can be potentially used as a screening device for rapid diagnosis of active TB at the POC. PMID:27775039

  7. A cost effective base-matching assay with low backgrounds.


    SU, X.; Mushinsky, G; Comeau, A.M.


    Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.

  8. Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method

    DEFF Research Database (Denmark)

    Zhang, Changyi; Guo, Li; Deng, Ling;


    Organisms belonging to the Crenarchaeota lineage contain three PCNA subunits (proliferating cell nuclear antigen) while those in Euryarchaeota have only one as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandic...... genes are absolutely required for host cell viability. Because the only prerequisite for this assay is to generate a MID transformant, this approach can be applied generally to any microorganisms proficient in homologous recombination....

  9. Discovery of novel BRD4 inhibitors by high-throughput screening, crystallography, and cell-based assays. (United States)

    Sun, Zhongya; Zhang, Hao; Chen, Zhifeng; Xie, Yiqian; Jiang, Hao; Chen, Limin; Ding, Hong; Zhang, Yuanyuan; Jiang, Hualiang; Zheng, Mingyue; Luo, Cheng


    As an epigenetic reader, BRD4 regulates the transcription of important downstream genes that are essential for the survival of tumor cells. Small molecular inhibitors targeting the first bromodomain of BRD4 (BRD4-BD1) have showed promising potentials in the therapies of BRD4-related cancers. Through AlphaScreen-based high-throughput screening assay, a novel small molecular inhibitor was identified, and named DCBD-005, which inhibited the binding between BRD4-BD1 and acetylated lysines with an IC50 value of 0.81±0.03μM. The compound DCBD-005 effectively inhibited the viability, caused cell cycle arrest, and induced apoptosis in human leukemia MV4-11 cells. Moreover, the crystal structure of compound DCBD-005 with the BRD4-BD1 was determined at 1.72Å resolution, which revealed the binding mechanism of the leading compound, and also provided solid basis for further structure-based optimization. These results indicated that this novel BRD4-BD1 inhibitor DCBD-005 is promising to be developed into a drug candidate in the treatment of BRD4-related diseases.

  10. Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost. (United States)

    Berry, Scott M; Pezzi, Hannah M; Williams, Eram D; Loeb, Jennifer M; Guckenberger, David J; Lavanway, Alex J; Puchalski, Alice A; Kityo, Cissy M; Mugyenyi, Peter N; Graziano, Franklin M; Beebe, David J


    Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R2 = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shukor, Yunus [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)]. E-mail:; Baharom, Nor Azlan [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Rahman, Fadhil Abd. [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Mohd. Puad [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Shamaan, Nor Aripin [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Syed, Mohd. Arif [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)


    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{sub 50} (concentration of toxicant giving 50% inhibition) of Hg{sup 2+}, Ag{sup 2+}, Pb{sup 2}, Zn{sup 2+} is 0.39, 0.40, 2.16, 2.11 mg l{sup -1}, respectively. For Cu{sup 2+} and Cd{sup 2+} the LOQ (limits of quantitation) is 0.004 and 0.1 mg l{sup -1}, respectively. The IC{sub 50} and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox{sup 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.

  12. Using Exclusion-Based Sample Preparation (ESP to Reduce Viral Load Assay Cost.

    Directory of Open Access Journals (Sweden)

    Scott M Berry

    Full Text Available Viral load (VL measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD, accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1 and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP.71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL and high accuracy (average difference between methods of 0.08 log, R2 = 0.97. Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.

  13. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki


    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  14. Digital magnetic tagging for multiplexed suspension-based biochemical assays (United States)

    Mitrelias, T.; Trypiniotis, T.; Palfreyman, J. J.; Hong, B.; Vyas, K.; Hayward, T. J.; Llandro, J.; Kopper, K. P.; Bland, J. A. C.; Robertson, P. A.; Barnes, C. H. W.


    Microarrays and suspension (or bead)-based technologies have attracted significant interest for their broad applications in high throughput molecular biology. However, the throughput of microarrays will always be limited by the array density and the slow diffusion of molecules to their binding sites. Suspension-based technologies, in which all the reactions take place directly on the surface of microcarriers functionalized with molecular probes, could offer true multiplexing due to the possibility of extending their detection capability by a straightforward expansion of the size of the chemical library of probes. To fully exploit their potential, the microcarriers must be tagged, but the number of distinct codes available from spectrometric/graphical/physical encoding methods is currently fairly limited. A digital magnetic tagging method based on magnetic microtags, which have been anisotropy engineered to provide stable magnetization directions which correspond to digital codes, is reported. The tags can be suspended in solution and functionalized with a variety of biological molecular probes. Magnetic tagging offers several benefits compared to the traditional optical encoding techniques currently employed. It offers minimal background signals, potential for a large number of distinct codes, miniaturization of devices, and the ability to write a code in situ. Experimental data showing the reading of individual magnetic microbars from samples comprising 50×20 μm2 Ni elements, as well as micromagnetic simulations that show the feasibility of stray field detection, are presented. The stray fields of the magnetic microbars spanning a range of 60 mOe were detected by a microfabricated fluxgate sensor scanned in a raster fashion over the sample that was placed about 70 μm away. Free floating tags have also been fabricated for use in microfluidic systems. A magnetic lab-on-a-chip device could be used for tagging biomolecular probes for applications in genome

  15. The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity. (United States)

    Gillissen, M A; Yasuda, E; de Jong, G; Levie, S E; Go, D; Spits, H; van Helden, P M; Hazenberg, M D


    Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

  16. Smartphone based visual and quantitative assays on upconversional paper sensor. (United States)

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


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

  17. Spectrophotometric total reducing sugars assay based on cupric reduction. (United States)

    Başkan, Kevser Sözgen; Tütem, Esma; Akyüz, Esin; Özen, Seda; Apak, Reşat


    As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method.

  18. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays. (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M


    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  19. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason


    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  20. Nanoparticle-based assays in automated flow systems: A review

    Energy Technology Data Exchange (ETDEWEB)

    Passos, Marieta L.C. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Pinto, Paula C.A.G., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Santos, João L.M., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Araujo, André R.T.S. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, Av. Dr. Francisco de Sá Carneiro, n° 50, 6300-559 Guarda (Portugal)


    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section.

  1. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins (United States)


    streptavidin-phycoerythrin (PE) similar to sandwich enzyme-linked immunosorbent assays (ELISAs). The 3 fluorescent markers (2 beads plus PE) allow for...least expensive platform. It uses a magnetic plate to create a monolayer of beads that can be imaged with a light-emitting-diode-based imager capable... Magnetic Luminex Screening Assay Rat Premixed Multi-Analyte Kit, a kit was purchased that included all of the 17 analytes included in company’s catalog

  2. A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology. (United States)

    Abderrahim, Mohamed; Arribas, Silvia M; Condezo-Hoyos, Luis


    Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H2O2) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL(-1)), HRP (1UmL(-1)) and H2O2 (250µmolL(-1)) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL(-1)) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H2O2 as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL(-1) (R(2)=0.993) and very sensitive with limits of detection (LOD) of 0.005UmL(-1) and quantitation (LOQ) of 0.01UmL(-1). PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples.

  3. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned. (United States)

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile


    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  4. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay. (United States)

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K


    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  5. A versatile microparticle-based immunoaggregation assay for macromolecular biomarker detection and quantification.

    Directory of Open Access Journals (Sweden)

    Haiyan Wu

    Full Text Available The rapid, sensitive and low-cost detection of macromolecular biomarkers is critical in clinical diagnostics, environmental monitoring, research, etc. Conventional assay methods usually require bulky, expensive and designated instruments and relative long assay time. For hospitals and laboratories that lack immediate access to analytical instruments, fast and low-cost assay methods for the detection of macromolecular biomarkers are urgently needed. In this work, we developed a versatile microparticle (MP-based immunoaggregation method for the detection and quantification of macromolecular biomarkers. Antibodies (Abs were firstly conjugated to MP through streptavidin-biotin interaction; the addition of macromolecular biomarkers caused the aggregation of Ab-MPs, which were subsequently detected by an optical microscope or optical particle sizer. The invisible nanometer-scale macromolecular biomarkers caused detectable change of micrometer-scale particle size distributions. Goat anti-rabbit immunoglobulin and human ferritin were used as model biomarkers to demonstrate MP-based immunoaggregation assay in PBS and 10% FBS to mimic real biomarker assay in the complex medium. It was found that both the number ratio and the volume ratio of Ab-MP aggregates caused by biomarker to all particles were directly correlated to the biomarker concentration. In addition, we found that the detection range could be tuned by adjusting the Ab-MP concentration. We envision that this novel MP-based immunoaggregation assay can be combined with multiple detection methods to detect and quantify macromolecular biomarkers at the nanogram per milliliter level.

  6. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate. (United States)

    Bi, Xiaodong; Liu, Zhen


    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  7. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan


    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  8. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne


    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  9. HIV-1 Reverse Transcriptase based assay to determine cellular dNTP concentrations (United States)

    Hollenbaugh, Joseph A.; Kim, Baek


    Summary Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis are tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection. PMID:26714705

  10. Long term response of a Concanavalin-A based fluorescence glucose sensing assay (United States)

    Locke, Andrea K.; Cummins, Brian M.; Abraham, Alexander A.; Coté, Gerard L.


    Competitive binding assays comprised of the protein Concanavalin A (ConA) have shown potential for use in continuous glucose monitoring devices. However, its time-dependent, thermal instability can impact the lifetime of these ConA based assays. In an attempt to design sensors with longer in vivo lifetimes, different groups have immobilized the protein to various surfaces. For example, Ballerstadt et al. have shown that immobilizing ConA onto the interior of a micro-dialysis membrane and allowing dextran to be freely suspended within solution allowed for successful in vivo glucose sensing up to 16 days. This work explores the glucose response of an assay comprised of modified ConA and a single fluorescently labeled competing ligand in free solution to increase the in vivo sensing lifetime without immobilization,. The behavior of this assay in the presence of varying glucose concentrations is monitored via fluorescence anisotropy over a 30 day period.

  11. Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

    Directory of Open Access Journals (Sweden)

    Frick A


    were generated using GraphPad Prism 6. Results: Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion: Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies. Keywords: immunomodulation, cytotoxicity, chemotherapy, precision medicine

  12. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. (Univ. of Pittsburgh School of Medicine, PA (USA))


    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

  13. Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay. (United States)

    Dossou, K S S; Devkota, K P; Kavanagh, P V; Beutler, J A; Egan, J M; Moaddel, R


    Cannabinoid (CB) receptors are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed antiproliferative activity against breast cancer and prostate cancer in animal models. To study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [(3)H]Win55-212-2 for CB1 and [(3)H]CP55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630, and AM6545. The data obtained were consistent with previously reported values, thereby confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors. In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.

  14. Processing of nanolitre liquid plugs for microfluidic cell-based assays

    Directory of Open Access Journals (Sweden)

    Junji Fukuda, Shintaro Takahashi, Tatsuya Osaki, Naoto Mochizuki and Hiroaki Suzuki


    Full Text Available Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

  15. Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit. (United States)

    Mather, Stuart T; Wright, Edward; Scott, Simon D; Temperton, Nigel J


    Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

  16. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay. (United States)

    Sonavane, Manoj; Creusot, Nicolas; Maillot-Maréchal, Emmanuelle; Péry, Alexandre; Brion, François; Aїt-Aïssa, Selim


    Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish.

  17. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening. (United States)

    Caballero, Iván; Lafuente, María José; Gamo, Francisco-Javier; Cid, Concepción


    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

  18. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. (United States)

    Christopher-Hennings, Jane; Araujo, Karla P C; Souza, Carlos J H; Fang, Ying; Lawson, Steven; Nelson, Eric A; Clement, Travis; Dunn, Michael; Lunney, Joan K


    Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

  19. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides (United States)

    Rogers, Kim R.; Wang, Yi; Mulchandani, Ashok; Mulchandani, P.; Chen, Wilfred


    We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphate pesticides. This assay is based on a substrate-dependant change in pH near the active site of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC) which is covalently immobilized to the enzyme. This method employs FITC-labeled enzyme adsorbed to polymethylmethacrylate beads. Analytes were measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 (mu) M to 400 (mu) M for paraoxon with a detection limit of 8 (mu) M. This assay compared favorably to an HPLC method for monitoring the concentration of coumaphos in bioremediation filtrate samples.

  20. Cultivar origin and admixture detection in Turkish olive oils by SNP-based CAPS assays. (United States)

    Uncu, Ali Tevfik; Frary, Anne; Doganlar, Sami


    The aim of this study was to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils. To reach this aim, we sequenced short fragments from five olive genes for SNP (single nucleotide polymorphism) identification and developed CAPS (cleaved amplified polymorphic DNA) assays for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. We also tested the efficiency and limit of our approach for detecting olive oil admixtures. As a result of the analysis, we were able to detect admixing down to a limit of 20%. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.

  1. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity. (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I


    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  2. A robotics-based automated assay for inorganic and organic phosphates. (United States)

    Cogan, E B; Birrell, G B; Griffith, O H


    Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.

  3. Conference report: the 5th cell-based assay and bioanalytical method development conference. (United States)

    Ma, Mark


    Approximately 80 participants met at the Marriot Hotel, San Francisco, CA, USA, between the 4th and 6th October 2010 to share novel techniques and discuss the emerging approaches in the evolving field of cell-based assay and bioanalytical method development. This report highlights the discussion and summary of the meeting.

  4. An assay for secologanin in plant tissues based on enzymatic conversion into strictosidine

    DEFF Research Database (Denmark)

    Hallard, Didier; van der Heijden, Robert; Contin, Adriana;


    The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding...

  5. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    NARCIS (Netherlands)

    Nicolas, J.A.Y.


    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby rep

  6. Uncertainty budget for final assay of a pharmaceutical product based on RP-HPLC

    DEFF Research Database (Denmark)

    Heydorn, Kaj; Anglov, Thomas; Byrialsen, Kirsten


    Compliance with specified limits for the content of active substance in a pharmaceutical drug requires knowledge of the uncertainty of the final assay. The uncertainty of measurement is based on the ISO recommendation as expressed in the Guide to the Expression of Uncertainty in Measurement (GUM...

  7. Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs. (United States)

    Ren, Xi; Tapias, Luis F; Jank, Bernhard J; Mathisen, Douglas J; Lanuti, Michael; Ott, Harald C


    Decellularized organ scaffolds allow whole organ regeneration and study of cell behavior in three-dimensional culture conditions. Cell viability within the bio-engineered organ constructs is an essential parameter reflecting the performance of participating cells during long-term ex vivo culture, and is a prerequisite for further functional performance. Resazurin-based redox metabolic assays have been used to monitor cell viability in both two- and three-dimensional cell cultures. Here we developed a method for monitoring cell viability and proliferation in bio-engineered organ constructs using a resazurin perfusion assay. This method allows non-invasive, repetitive and rapid estimation of viable cell numbers during long-term ex vivo culture. As a proof-of-principle, we assessed the performance of two different endothelial sources and the impact of different perfusion programs on endothelial viability after re-endothelialization of decellularized lung scaffolds. The resazurin-based perfusion assay revealed changes in endothelial viability and proliferation during long-term ex vivo culture, which was consistent with histological assessment at different time points. Finally, we showed that this method could be used for assessment of proliferation and cytotoxicity after pharmacological treatment on a three-dimensional non-small cell lung cancer culture model.

  8. Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India.

    Directory of Open Access Journals (Sweden)

    Shanmugam Vanithamani

    Full Text Available Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS was evaluated by enzyme linked immunosorbent assay (ELISA, dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA. Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%, Autumnalis (11.7%, Ballum (25.8%, Grippotyphosa (12.5%, Pomona (10% and were used as antigens in the diagnostics to detect IgM antibodies in patients' sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05.The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.

  9. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo


    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  10. Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers. (United States)

    Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru


    The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  11. Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

    Directory of Open Access Journals (Sweden)

    Phairote Teeranaipong


    Full Text Available Introduction: A dual split reporter protein system (DSP, recombining Renilla luciferase (RL and green fluorescent protein (GFP split into two different constructs (DSP1–7 and DSP8–11, was adapted to create a novel rapid phenotypic tropism assay (PTA for HIV-1 infection (DSP-Pheno. Methods: DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4 or CD4/CCR5 (N4R5, respectively. An expression vector with DSP8–11 (pRE11 was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results: The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP. The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions: A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and

  12. Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay. (United States)

    Wasserstrom, Adam; Frumkin, Dan; Davidson, Ariane; Shpitzen, Moshe; Herman, Yael; Gafny, Ron


    Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This

  13. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风


    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  14. [Changes in serum vitamin D assay usage and the need for evidence-based recommendations]. (United States)

    Pilon, Antoine; Lim, Soo-Kyung; Guéchot, Jérôme


    Result of renewed interest due to the large amount of literature that reported numerous epidemiological data demonstrating the high prevalence of vitamin D deficiency, the number of prescriptions of serum vitamin D assays has grown exponentially in recent years with a cost for health insurance that increased almost fivefold in four years. The quantitative and qualitative analysis of assays carried out from 2007 to 2011 in a French university adult short-stay hospital shows changes in practices not only quantitatively but also qualitatively resulting in an overtime increase in the frequency of prescriptions in patients younger, less vitamin D deficient and more frequently male. In the absence of French guidelines, this development cannot be qualified as deviant but justifies the urgent need to establish evidence-based recommendations for good prescriptions and adequate assays of blood vitamin D.

  15. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. (United States)

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger


    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  16. Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

    Institute of Scientific and Technical Information of China (English)

    陈宝玉; 郭勤; 郭智; 王希成


    This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid-reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N-phospho-L-arginine (PArg) formed in the forward catalysis reaction.The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized.For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L)-1 · cm-1 for the phosphate.Standard curves for phosphate show a good linearity of 0.999.Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies.This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

  17. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification. (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng


    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  18. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays. (United States)

    Langer, Gernot


    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  19. Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics. (United States)

    Tao, Ye; Rotem, Assaf; Zhang, Huidan; Chang, Connie B; Basu, Anindita; Kolawole, Abimbola O; Koehler, Stephan A; Ren, Yukun; Lin, Jeffrey S; Pipas, James M; Feldman, Andrew B; Wobus, Christiane E; Weitz, David A


    A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

  20. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay. (United States)

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz


    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  1. Quantification of microglial phagocytosis by a flow cytometer-based assay. (United States)

    Pul, Refik; Chittappen, Kandiyil Prajeeth; Stangel, Martin


    Microglia represent the largest population of phagocytes in the CNS and have a principal role in immune defense and inflammatory responses in the CNS. Their phagocytic activity can be studied by a variety of techniques, including a flow cytometry-based approach utilizing polystyrene latex beads. The flow cytometry-based microglial phagocytosis assay, which is presented here, offers the advantage of rapid and reliable analysis of thousands of cells in a quantitative fashion.

  2. Viability, invariance and applications

    CERN Document Server

    Carja, Ovidiu; Vrabie, Ioan I


    The book is an almost self-contained presentation of the most important concepts and results in viability and invariance. The viability of a set K with respect to a given function (or multi-function) F, defined on it, describes the property that, for each initial data in K, the differential equation (or inclusion) driven by that function or multi-function) to have at least one solution. The invariance of a set K with respect to a function (or multi-function) F, defined on a larger set D, is that property which says that each solution of the differential equation (or inclusion) driven by F and issuing in K remains in K, at least for a short time.The book includes the most important necessary and sufficient conditions for viability starting with Nagumo's Viability Theorem for ordinary differential equations with continuous right-hand sides and continuing with the corresponding extensions either to differential inclusions or to semilinear or even fully nonlinear evolution equations, systems and inclusions. In th...

  3. Terminology for pregnancy loss prior to viability

    DEFF Research Database (Denmark)

    Kolte, A M; Bernardi, L A; Christiansen, O B


    Pregnancy loss prior to viability is common and research in the field is extensive. Unfortunately, terminology in the literature is inconsistent. The lack of consensus regarding nomenclature and classification of pregnancy loss prior to viability makes it difficult to compare study results from...... different centres. In our opinion, terminology and definitions should be based on clinical findings, and when possible, transvaginal ultrasound. With this Early Pregnancy Consensus Statement, it is our goal to provide clear and consistent terminology for pregnancy loss prior to viability....

  4. Sperm viability staining in ecology and evolution: potential pitfalls

    DEFF Research Database (Denmark)

    Holman, Luke


    The causes and consequences of variation in sperm quality, survival and ageing are active areas of research in ecology and evolution. In order to address these topics, many recent studies have measured sperm viability using fluorescent staining. Although sperm viability staining has produced...... a number of interesting results, it has some potential pitfalls that have rarely been discussed. In the present paper, I review the major findings of ecology and evolution studies employing sperm viability staining and outline the method's principle limitations. The key problem is that the viability assay...... may itself kill sperm, which is likely to confound many common experimental designs in addition to producing artificially low estimates of sperm viability. I further suggest that sperm number should be routinely measured in sperm viability studies, as it may be an important but overlooked source...

  5. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Directory of Open Access Journals (Sweden)

    Iole Macchia


    Full Text Available The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs and tumor-associated antigens (TAAs and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM- based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

  6. Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions. (United States)

    Tan, Yen Nee; Su, Xiaodi; Liu, Edison T; Thomsen, Jane S


    Gold nanoparticles (AuNPs) are widely used as colorimetric probes for biosensing, relying on their unique particle size-dependent and/or interparticle distance-dependent extinction spectrum and solution color. Herein, we describe an AuNP-based colorimetric assay to detect binding interactions between nuclear hormone receptors and their corresponding DNA-binding elements, particularly the human estrogen receptors (ERalpha and ERbeta) and their cognate estrogen response elements (EREs). We found that the protein-DNA (ER-ERE) complexes can stabilize citrate anion-capped AuNPs against salt-induced aggregation to a larger extent than the protein (ER) or the DNA (ERE) alone, due to their unique molecular size and charge properties that provide a strong electrosteric protection. Moreover, our results show that the extent of stabilization is sequence-dependent and can distinguish a single base variation in the ERE associated with minor changes in protein-DNA binding affinity. With this assay, many important parameters of protein-DNA binding events (e.g., sequence selectivity, distinct DNA binding properties of protein subtypes, binding stoichiometry, and sequence-independent transient binding) can be determined instantly without using labels, tedious sample preparations, and sophisticated instrumentation. These benefits, in particular the high-throughput potential, could enable this assay to become the assay of choice to complement conventional techniques for large scale characterization of protein-DNA interactions, a key aspect in biological research.

  7. Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F. (United States)

    Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole


    Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.

  8. Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation. (United States)

    Wu, Shangquan; Liu, Xiaoli; Zhou, Xiarong; Liang, Xin M; Gao, Dayong; Liu, Hong; Zhao, Gang; Zhang, Qingchuan; Wu, Xiaoping


    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.

  9. Determination of protease subsite preference on SPOT peptide array by fluorescence quenching-based assay. (United States)

    Kim, Do-Hyun; Shin, Dong-Sik; Lee, Yoon-Sik


    A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc-amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl-X-X-P(2)-Arg-X-X-X-Lys(FITC) (X: equimolar mixture of 19 amino acids, P(2): one of 19 amino acids) was synthesized on a chitosan-grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching-based assay. The P(2) subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P(2) subsite showed the highest specificity for thrombin based on both the fluorescence quenching-based assay and the solution phase assay. From these results, we confirmed that our mixture-based peptide SPOT array format on the chitosan-grafted glass chips could be used to determine protease subsite preference.

  10. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

    Directory of Open Access Journals (Sweden)

    Van Neste Leander


    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  11. Cell biological analyses of anther morphogenesis and pollen viability in Arabidopsis and rice. (United States)

    Chang, Fang; Zhang, Zaibao; Jin, Yue; Ma, Hong


    Major advances have been made in recent years in our understanding of anther development through a combination of genetic studies, cell biological technologies, biochemical analysis, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize the widely used protocols for pollen viability staining; the investigation of anther morphogenesis by light microscopy of semi-thin sections; TUNEL assay for programmed tapetum cell death; and laser microdissection procedures to obtain specialized cells or cell layers for carrying out transcriptomics.

  12. A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans. (United States)

    Lai, Yiling; Xiang, Meichun; Liu, Shuchun; Li, Erwei; Che, Yongsheng; Liu, Xingzhong


    Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

  13. Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis. (United States)

    Rascoe, Lisa N; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W; Handali, Sukwan


    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.

  14. Strategies to develop strain-specific PCR based assays for probiotics. (United States)

    Treven, P


    Since health benefits conferred by probiotics are strain-specific, identification to the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotic in a product or in a gastrointestinal tract. Compared to standard plate counts, the reduced duration of the assays and higher specificity makes PCR-based methods (standard PCR and quantitative PCR) very appropriate for detection or quantification of probiotics. Development of strain-specific assay consists of 4 main stages: (1) strain-specific marker identification; (2) construction of potential strain-specific primers; (3) validation on DNA from pure cultures of target and related strains; and (4) validation on spiked samples. The most important and also the most challenging step is the identification of strain-specific sequences, which can be subsequently targeted by specific primers or probes. Such regions can be identified on sequences derived from 16S-23S internally transcribed spacers, randomly amplified polymorphic DNA, representational difference analysis and suppression subtractive hybridisation. Already known phenotypic or genotypic characteristics of the target strain can also be used to develop the strain-specific assay. However, the initial stage of strain-specific assay development can be replaced by comparative genomics analysis of target genome with related genomes in public databases. Advances in whole genome sequencing (WGS) have resulted in a cost reduction for bacterial genome sequencing and consequently have made this approach available to most laboratories. In the present paper I reviewed the available literature on PCR and qPCR assays developed for detection of a specific probiotic strain and discussed future WGS and comparative genomics-based approaches.

  15. A simple technique for reducing edge effect in cell-based assays. (United States)

    Lundholt, Betina Kerstin; Scudder, Kurt M; Pagliaro, Len


    Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.

  16. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay. (United States)

    Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H; Xia, Menghang


    In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

  17. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors. (United States)

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B


    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  18. A highly sensitive and selective diagnostic assay based on virus nanoparticles (United States)

    Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon


    Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

  19. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA. (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman


    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

  20. GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

    Directory of Open Access Journals (Sweden)

    Beatriz A Santillan

    Full Text Available Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

  1. Assessing the applicability of FISH-based prematurely condensed dicentric chromosome assay in triage biodosimetry. (United States)

    Suto, Yumiko; Gotoh, Takaya; Noda, Takashi; Akiyama, Miho; Owaki, Makiko; Darroudi, Firouz; Hirai, Momoki


    The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

  2. Fluorescence-based assay as a new screening tool for toxic chemicals (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey


    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

  3. Novel Cell-Based Assays for Detecting Low Levels of Active Ricin Following Decontamination (United States)


    of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which is converted to a purple formazan dye by cellular reductive power...general decay in cell viability. Loss in cell viability is detected as loss in absorbance of formazan at 550–620 nm, resulting from the reduction of MTT

  4. Electrochemical Genotoxicity Assay Based on a SOS/umu Test Using Hydrodynamic Voltammetry in a Droplet


    Kazuharu Sugawara; Masami Fukushima; Shigeru Taguchi; Noriko Hata; Kazuto Sazawa; Yasuaki Nanayama; Hideki Kuramitz


    The SOS/umu genotoxicity assay evaluates the primary DNA damage caused by chemicals from the β-galactosidase activity of S. typhimurium. One of the weaknesses of the common umu test system based on spectrophotometric detection is that it is unable to measure samples containing a high concentration of colored dissolved organic matters, sediment, and suspended solids. However, umu tests with electrochemical detection techniques prove to be a better strategy because it causes less inter...

  5. Performance of a MALDI-TOF MS-based imipenem hydrolysis assay incorporating zinc sulfate. (United States)

    Knox, James; Palombo, Enzo


    A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.

  6. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes


    Alvaro Díaz-Badillo; María de Lourdes Muñoz; Gerardo Perez-Ramirez; Victor Altuzar; Juan Burgueño; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Alejandro Cisneros; Joel Navarrete-Espinosa; Feliciano Sanchez-Sinencio


    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybrid...

  7. The use of nanocrystal quantum dot as fluorophore reporters in molecular beacon-based assays (United States)

    Adegoke, Oluwasesan; Park, Enoch Y.


    The utilization of molecular beacon (MB) biosensor probes to detect nucleic acid targets has received enormous interest within the scientific community. This interest has been stimulated by the operational qualities of MB-based probes with respect to their unique sensitivity and specificity. The design of MB biosensors entails not only optimizing the sequence of the loop to hybridize with the nucleic acid target or optimization of the length of the stem to tune the sensitivity but also the selection of the appropriate fluorophore reporter to generate the signal transduction read-out upon hybridization of the probe with the target sequence. Traditional organic fluorescent dyes are mostly used for signal reporting in MB assays but their optical properties in comparison to semiconductor fluorescent quantum dot (Qdot) nanocrystals are at a disadvantage. This review highlights the progress made in exploiting Qdot as fluorophore reporters in MB-based assays with the aim of instigating further development in the field of Qdot-MB technology. The development reported to date indicates that unparalleled fluorescence signal reporting in MB-based assays can be achieved using well-constructed Qdot fluorophores.

  8. A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms. (United States)

    Kim, Young Hwa; Kim, Eung Soo; Ko, Byong Seob; Oh, Seung-Eun; Ryuk, Jin-Ah; Chae, Seong Wook; Lee, Hye Won; Choi, Go Ya; Seo, Doo Won; Lee, Mi Young


    This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

  9. A novel mass spectrometry-based assay for GSK-3β activity

    Directory of Open Access Journals (Sweden)

    Gan Bing Siang


    Full Text Available Abstract Background As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of γ-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3β activity. Results Synthetic peptide substrates designed with a GSK-3β phosphorylation site were assayed with both recombinant enzyme and GSK-3β immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da. was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS in a GSK-3β target peptide (2B-Sp. Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3β used in this assay, this shift was also inhibited by lithium chloride (LiCl, in a dose-dependent manner. Conclusion We present here a novel method to sensitively measure peptide phosphorylation by GSK-3β that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3β in vivo, and to screen enzyme activity in relation to a variety of GSK-3β related disorders.

  10. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.


    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  11. Comparative cytotoxic and genotoxic potential of 13 drinking water disinfection by-products using a microplate-based cytotoxicity assay and a developed SOS/umu assay. (United States)

    Zhang, Shao-Hui; Miao, Dong-Yue; Tan, Li; Liu, Ai-Lin; Lu, Wen-Qing


    The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively.

  12. A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging. (United States)

    Tseng, Hubert; Gage, Jacob A; Shen, Tsaiwei; Haisler, William L; Neeley, Shane K; Shiao, Sue; Chen, Jianbo; Desai, Pujan K; Liao, Angela; Hebel, Chris; Raphael, Robert M; Becker, Jeanne L; Souza, Glauco R


    An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5'-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (-control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z' = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

  13. A Cell-Based Pharmacokinetics Assay for Evaluating Tubulin-Binding Drugs (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W.; Ying, Wenbin


    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels. PMID:24688312

  14. A cell-based pharmacokinetics assay for evaluating tubulin-binding drugs. (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W; Ying, Wenbin


    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.

  15. Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

    Directory of Open Access Journals (Sweden)

    Edilaine Mauricia Gelinski Grabicoski


    Full Text Available Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max. Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction primer set and seed soaking (without DNA extraction for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence and one naturally infected seed in a 300-seed sample (0.33 % incidence. The PCR-based assay was rapid (< 9 h, did not require DNA extraction and was very sensitive.

  16. Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine. (United States)

    Sechi, Deidre; Greer, Brady; Johnson, Jesse; Hashemi, Nastaran


    The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

  17. An enzyme thermistor-based assay for total and free cholesterol. (United States)

    Raghavan, V; Ramanathan, K; Sundaram, P V; Danielsson, B


    A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit.

  18. Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food. (United States)

    Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K


    The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

  19. Financial viability and conservation role of betel leaf based agroforestry: an indigenous hill farming system of Khasia community in Bangladesh

    Institute of Scientific and Technical Information of China (English)

    Mizanur Rahman; Mohammad Mahfuzur Rahman; Mahmuda Islam


    A study was conducted to investigate the cultural and financial management techniques of betel leaf based agroforestry system practiced in or near homegardens of Khasia community in Jaintapur Upazila in the district of Sylhet, Bangladesh. The Khasia is an educated community where 100% of Khasia people were literate, a stunning fact for this ethnic community in Bangladesh. The average family size in the study area was 7.68, with a ration of male and females of 141:100. The homegardens of the Khasia are rich in species composition, which 15 timber species, 22 horticultural species, six medicinal species, 13 annual crops including leafy vegetables, seven species of spices and five species of bamboo were identified along with betel leaf. The Khasia is an economically prosperous community with the minimum family incomes of Tk 4000 per month (Tk. 70=1 US Dollar). Betel leaf based agroforestry is very common being a prevalent source of income. About 95.45% of the households are involved in betel leaf husbandry. The mean annual income from one hectare of betel leaf plantation was estimated to be Tk. 80979. This practice was proven to be a profitable business where the benefit cost ratio was calculated to be 4.47. Moreover, the species composition in the betel leaf plantation area (the forest area once utilized by Khasia for shifting cultivation) was found to be very promising to play the significant role in conservation of biological diversity making the practice a sustainable agroforestry system.

  20. SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

    Directory of Open Access Journals (Sweden)

    Wai-Hoe Lau


    Full Text Available Abstract Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P

  1. An extended set of yeast-based functional assays accurately identifies human disease mutations (United States)

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.


    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  2. Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis. (United States)

    Yen, Ching-Yu; Wu, Yu-Wei; Hsiung, Chao-Nan; Yeh, Min-I; Lin, Yi-Ming; Lee, Sheng-Yang


    Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

  3. A rapid, micro-scale preliminary screening method for active components in Galangal with protective effect against hydrogen peroxide induced cell apoptosis through "thin layer chromatography" and "tetrazolium-based colorimetric assay" array correspondence. (United States)

    Cheng, Yuan; Li, Yuanting; Li, Jin; Deng, Yifeng


    A new method has been established for rapid preliminary screening active ingredients in natural products through thin layer chromatography (TLC) array responding with tetrazolium-based colorimetric assay (MTT assay) along with post LC-MS in micro-scale. The extract of the natural product was first separated by TLC. The separated spots obtained from TLC were visualized in situ with vanillin-ethanolic sulfuric acid agent to define the array correspondence between TLC spots and 384-cell culture plate for MTT cell viability assay. The TLC spots from the replicate TLC plates were then eluted and transferred into the wells of 384-cell culture plates according to the array respondence. The TLC spots with significant antioxidant activities were further screened by MTT assay, and subsequently traced and identified by LC-MS based on the TLC-MTT assay array correspondence. This new method was successfully applied to screen active ingredients in a Chinese medicine known as Galangal. Two major inhibitors for the decline of PC12 cell survival (Galangin, m/z 269.1, and 5-hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone, m/z 327.2) were effectively screened and identified by this method.

  4. Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies. (United States)

    Belouski, Shelley S; Born, Danika; Jacques, Susan; Harder, Brandon; Reynhardt, Kai; Kaliyaperumal, Arunan; Gupta, Shalini


    We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

  5. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR. (United States)

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R


    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  6. Effects of diluents on cell culture viability measured by automated cell counter (United States)

    Chen, Aaron; Leith, Matthew; Tu, Roger; Tahim, Gurpreet; Sudra, Anish; Bhargava, Swapnil


    Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions. PMID:28264018

  7. A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

    Directory of Open Access Journals (Sweden)

    Anli Zhang


    Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

  8. Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

    Directory of Open Access Journals (Sweden)

    Abdel-Rahman Hamdy M


    Full Text Available Abstract The formation of a colored charge-transfer (CT complex between atorvastatin calcium (ATR-Ca as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995 was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

  9. Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells. (United States)

    Kim, Hak Jun; Shim, Hye Eun; Lee, Jun Hyuck; Kang, Yong-Cheol; Hur, Young Baek


    Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

  10. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes. (United States)

    Ding, Xiaokang; Yang, Kun-Lin


    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  11. A novel in vitro image-based assay identifies new drug leads for giardiasis. (United States)

    Hart, Christopher J S; Munro, Taylah; Andrews, Katherine T; Ryan, John H; Riches, Andrew G; Skinner-Adams, Tina S


    Giardia duodenalis is an intestinal parasite that causes giardiasis, a widespread human gastrointestinal disease. Treatment of giardiasis relies on a small arsenal of compounds that can suffer from limitations including side-effects, variable treatment efficacy and parasite drug resistance. Thus new anti-Giardia drug leads are required. The search for new compounds with anti-Giardia activity currently depends on assays that can be labour-intensive, expensive and restricted to measuring activity at a single time-point. Here we describe a new in vitro assay to assess anti-Giardia activity. This image-based assay utilizes the Perkin-Elmer Operetta(®) and permits automated assessment of parasite growth at multiple time points without cell-staining. Using this new approach, we assessed the "Malaria Box" compound set for anti-Giardia activity. Three compounds with sub-μM activity (IC50 0.6-0.9 μM) were identified as potential starting points for giardiasis drug discovery.

  12. A simple, versatile and sensitive cell-based assay for prions from various species.

    Directory of Open Access Journals (Sweden)

    Zaira E Arellano-Anaya

    Full Text Available Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

  13. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants. (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan


    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  14. EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis. (United States)

    Bandeira-Melo, Christianne; Paiva, Ligia Almeida; Amorim, Natália R T; Weller, Peter F; Bozza, Patricia T


    Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.

  15. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes. (United States)

    Warrilow, David; Northill, Judith A; Pyke, Alyssa; Smith, Greg A


    Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.

  16. An Acetylcholinesterase-Based Chronoamperometric Biosensor for Fast and Reliable Assay of Nerve Agents

    Directory of Open Access Journals (Sweden)

    Rene Kizek


    Full Text Available The enzyme acetylcholinesterase (AChE is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer, it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol /L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples.

  17. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay. (United States)

    Folman, C C; von dem Borne, A E; Rensink, I H; Gerritsen, W; van der Schoot, C E; de Haas, M; Aarden, L


    In this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs). The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 +/- 0.8 A.U./ml, mean +/- sd) was lower than the concentration found in controls (11 +/- 8 A.U./ml, mean +/- sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma. We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels. In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

  18. Applications of monolithic solid-phase extraction in chromatography-based clinical chemistry assays. (United States)

    Bunch, Dustin R; Wang, Sihe


    Complex matrices, for example urine, serum, plasma, and whole blood, which are common in clinical chemistry testing, contain many non-analyte compounds that can interfere with either detection or in-source ionization in chromatography-based assays. To overcome this problem, analytes are extracted by protein precipitation, solid-phase extraction (SPE), and liquid-liquid extraction. With correct chemistry and well controlled material SPE may furnish clean specimens with consistent performance. Traditionally, SPE has been performed with particle-based adsorbents, but monolithic SPE is attracting increasing interest of clinical laboratories. Monoliths, solid pieces of stationary phase, have bimodal structures consisting of macropores, which enable passage of solvent, and mesopores, in which analytes are separated. This structure results in low back-pressure with separation capabilities similar to those of particle-based adsorbents. Monoliths also enable increased sample throughput, reduced solvent use, varied support formats, and/or automation. However, many of these monoliths are not commercially available. In this review, application of monoliths to purification of samples from humans before chromatography-based assays will be critically reviewed.

  19. Naked-eye quantitative aptamer-based assay on paper device. (United States)

    Zhang, Yun; Gao, Dong; Fan, Jinlong; Nie, Jinfang; Le, Shangwang; Zhu, Wenyuan; Yang, Jiani; Li, Jianping


    This work initially describes the design of low-cost, naked-eye quantitative aptamer-based assays by using microfluidic paper-based analytical device (μPAD). Two new detection motifs are proposed for quantitative μPAD measurement without using external electronic readers, which depend on the length of colored region in a strip-like μPAD and the number of colorless detection microzones in a multi-zone μPAD. The length measuring method is based on selective color change of paper from colorless to blue-black via formation of iodine-starch complex. The counting method is conducted on the basis of oxidation-reduction reaction between hydrogen peroxide and potassium permanganate. Their utility is well demonstrated with sensitive, specific detection of adenosine as a model analyte with the naked eye in buffer samples and undiluted human serum. These equipment-free quantitative methods proposed thus hold great potential for the development of more aptamer-based assays that are simple, cost-efficient, portable, and user-friendly for various point-of-care applications particularly in resource-constrained environments.

  20. Establishing lunar resource viability (United States)

    Carpenter, J.; Fisackerly, R.; Houdou, B.


    Recent research has highlighted the potential of lunar resources as an important element of space exploration but their viability has not been demonstrated. Establishing whether or not they can be considered in future plans is a multidisciplinary effort, requiring scientific expertise and delivering scientific results. To this end various space agencies and private entities are looking to lunar resources, extracted and processed in situ, as a potentially game changing element in future space architectures, with the potential to increase scale and reduce cost. However, before any decisions can be made on the inclusion of resources in exploration roadmaps or future scenarios some big questions need to be answered about the viability of different resource deposits and the processes for extraction and utilisation. The missions and measurements that will be required to answer these questions, and which are being prepared by agencies and others, can only be performed through the engagement and support of the science community. In answering questions about resources, data and knowledge will be generated that is of fundamental scientific importance. In supporting resource prospecting missions the science community will de facto generate new scientific knowledge. Science enables exploration and exploration enables science.

  1. Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

    Institute of Scientific and Technical Information of China (English)

    Pei-Yuan Li; Xiao-Jun Zhou; Lan Yao; Xin-Hua Fang; Jiang-Nan Ren; Jia-Wu Song


    AIM:To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rsS099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 103-104 white cells/lμL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray

  2. Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

    Directory of Open Access Journals (Sweden)

    Claudia Cozma

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS, resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance.We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS. For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry. 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h. With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays.The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

  3. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  4. Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.

    NARCIS (Netherlands)

    Quint, K.D.; Porras, C.; Safaeian, M.; Gonzalez, P.; Hildesheim, A.; Quint, W.G.V.; Doorn, L.J. van; Silva, S.; Melchers, W.J.G.; Schiffman, M.; Rodriguez, A.C.; Wacholder, S.; Freer, E.; Cortes, B.; Herrero, R.


    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a

  5. A facile low-cost enzymatic paper-based assay for the determination of urine creatinine. (United States)

    Talalak, Kwanrutai; Noiphung, Julaluk; Songjaroen, Temsiri; Chailapakul, Orawon; Laiwattanapaisal, Wanida


    Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries.

  6. Restored viability and function of dental pulp cells on poly-methylmethacrylate (PMMA)-based dental resin supplemented with N-acetyl cysteine (NAC). (United States)

    Kojima, N; Yamada, M; Paranjpe, A; Tsukimura, N; Kubo, K; Jewett, A; Ogawa, T


    This study examines cytotoxicity of poly-methylmethacrylate (PMMA)-based dental temporary filling resin to dental pulp cells, and the potential amelioration of the toxicity with an anti-oxidant amino-acid, N-acetyl cysteine (NAC). Dental pulp cells extracted from rat maxillary incisors were cultured on the resin material with or without NAC incorporation, or on the polystyrene. The cultures were supplied with osteoblastic media, containing dexamethasone. Forty five percent of cells on the PMMA dental resin were necrotic at 24h after seeding. However, this percentage was reduced to 27% by incorporating NAC in the resin, which was the level equivalent to that in the culture on polystyrene. The culture on the untreated resin was found to be negative for alkaline phosphate (ALP) activity at days 5 and 10 or von Kossa mineralized nodule formation at day 20. In contrast, some areas of the cultures on NAC-incorporated resin substrates were ALP and von Kossa positive. Collagen I and dentin sialoprotein genes were barely expressed in day 7 culture on the untreated resin. However, those genes were expressed in the culture on the resin with NAC. These results suggest that the decreased cell viability and the nearly completely suppressed odontoblast-like cell phenotype of dental pulp cells cultured on PMMA dental resin can be salvaged to a biologically significant degree by the incorporation of NAC in the resin.

  7. Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability (United States)

    Magrì, Andrea; Belfiore, Ramona; Reina, Simona; Tomasello, Marianna Flora; Di Rosa, Maria Carmela; Guarino, Francesca; Leggio, Loredana; De Pinto, Vito; Messina, Angela


    Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies. PMID:27721436

  8. Advances and Challenges in Viability Detection of Foodborne Pathogens. (United States)

    Zeng, Dexin; Chen, Zi; Jiang, Yuan; Xue, Feng; Li, Baoguang


    Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually

  9. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins. (United States)

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


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

  10. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay (United States)

    Zhou, Huijuan; Wu, Baoyan


    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  11. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform. (United States)

    Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B


    An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

  12. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis. (United States)

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman


    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  13. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements. (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David


    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

  14. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Institute of Scientific and Technical Information of China (English)

    Vikrant Sudan; A K Tewari; R Singh; Harkirat Singh


    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  15. A practical method for extending the biuret assay to protein determination of corn-based products. (United States)

    Liu, Zelong; Pan, Junhui


    A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.

  16. Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay. (United States)

    Khan, Sadia Afrin; DeGrasse, Jeffrey A; Yakes, Betsy Jean; Croley, Timothy R


    Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV-Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.

  17. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design (United States)

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.


    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  18. Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.

    Directory of Open Access Journals (Sweden)

    Jasmine I Daksis

    Full Text Available Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP, without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

  19. Verification of cell viability in bioengineered tissues and organs before clinical transplantation. (United States)

    Jungebluth, Philipp; Haag, Johannes C; Lim, Mei L; Lemon, Greg; Sjöqvist, Sebastian; Gustafsson, Ylva; Ajalloueian, Fatemeh; Gilevich, Irina; Simonson, Oscar E; Grinnemo, Karl H; Corbascio, Matthias; Baiguera, Silvia; Del Gaudio, Costantino; Strömblad, Staffan; Macchiarini, Paolo


    The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety.

  20. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)


    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  1. A parallel and quantitative cell migration assay using a novel multi-well-based device. (United States)

    Quan, Qianghua; Zhang, Shuwen; Wang, Xudong; Ouyang, Qi; Wang, Yugang; Yang, Gen; Luo, Chunxiong


    Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-β(TGF-β) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-β + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

  2. A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape. (United States)

    Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale


    Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.

  3. Gold nanoparticle aggregation-based colorimetric assay for β-casein detection in bovine milk samples. (United States)

    Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Song, F; Lu, S Y; Ren, H L; Hu, P; Liu, Z S; Zhang, J H


    Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-consuming sample preparation and two analyses to determine the difference between non-protein nitrogen content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparticles (AuNPs), we described a colorimetric method for β-casein (β-CN) detection in bovine milk samples. The linear dynamic range and the LOD were 0.08-250 μg mL(-1), and 0.03 μg mL(-1) respectively. In addition, the real content of β-CN in bovine milk was measured by using the developed assay. The results are closely correlated with those from Kjeldahl method. The advantages of β-CN triggered AuNP aggregation-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as no need of complicated sample preparation, which make it for on-site detection of β-CN in bovine milk samples.

  4. Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses. (United States)

    Sudarshana, M R; Reddy, D V


    A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.

  5. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells. (United States)

    Wang, Xiao; Peden, Keith; Murata, Haruhiko


    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  6. Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides. (United States)

    Long, Qian; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo


    This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays.

  7. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye. (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta


    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

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

    Directory of Open Access Journals (Sweden)

    Zhang PF


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

  9. A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

    NARCIS (Netherlands)

    van Ameijde, Jeroen; Overvoorde, John; Knapp, Stefan; den Hertog, Jeroen; Ruijtenbeek, Rob; Liskamp, Rob M J


    A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production

  10. Microplate assay for screening the antibacterial activity of Schiff bases derived from substituted benzopyran-4-one. (United States)

    Amin, Rehab M; Abdel-Kader, Nora S; El-Ansary, Aida L


    Schiff bases (SB(1)-SB(3)) were synthesized from the condensation of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one with 2-aminopyridine (SB(1)), p-phenylenediamine (SB(2)) and o-phenylenediamine (SB(3)), while Schiff bases (SB(4)-SB(6)) were synthesized by condensation of 5,7-dihydroxy-6-formyl-2-methylbenzopyran-4-one with 2-aminopyridine (SB(4)), p-phenylenediamine (SB(5)) and o-phenylenediamine (SB(6)). Schiff bases were characterized using elemental analysis, IR, UV-Vis, (1)H NMR, (13)C NMR and mass spectroscopy. These compounds were screened for antibacterial activities by micro-plate assay technique. Escherichia coli and Staphylococcus capitis were exposed to different concentrations of the Schiff bases. Results showed that the antibacterial effect of these Schiff bases on Gram-negative bacteria were higher than that on Gram-positive bacteria moreover, the Schiff bases containing substituent OCH(3) on position five have higher antibacterial activity than that containing hydroxy group on the same position.

  11. Ozone Depletion Caused by Rocket Engine Emissions: A Fundamental Limit on the Scale and Viability of Space-Based Geoengineering Schemes (United States)

    Ross, M. N.; Toohey, D.


    Emissions from solid and liquid propellant rocket engines reduce global stratospheric ozone levels. Currently ~ one kiloton of payloads are launched into earth orbit annually by the global space industry. Stratospheric ozone depletion from present day launches is a small fraction of the ~ 4% globally averaged ozone loss caused by halogen gases. Thus rocket engine emissions are currently considered a minor, if poorly understood, contributor to ozone depletion. Proposed space-based geoengineering projects designed to mitigate climate change would require order of magnitude increases in the amount of material launched into earth orbit. The increased launches would result in comparable increases in the global ozone depletion caused by rocket emissions. We estimate global ozone loss caused by three space-based geoengineering proposals to mitigate climate change: (1) mirrors, (2) sunshade, and (3) space-based solar power (SSP). The SSP concept does not directly engineer climate, but is touted as a mitigation strategy in that SSP would reduce CO2 emissions. We show that launching the mirrors or sunshade would cause global ozone loss between 2% and 20%. Ozone loss associated with an economically viable SSP system would be at least 0.4% and possibly as large as 3%. It is not clear which, if any, of these levels of ozone loss would be acceptable under the Montreal Protocol. The large uncertainties are mainly caused by a lack of data or validated models regarding liquid propellant rocket engine emissions. Our results offer four main conclusions. (1) The viability of space-based geoengineering schemes could well be undermined by the relatively large ozone depletion that would be caused by the required rocket launches. (2) Analysis of space- based geoengineering schemes should include the difficult tradeoff between the gain of long-term (~ decades) climate control and the loss of short-term (~ years) deep ozone loss. (3) The trade can be properly evaluated only if our

  12. Viability of Hybrid Systems A Controllability Operator Approach

    CERN Document Server

    Labinaz, G


    The problem of viability of hybrid systems is considered in this work. A model for a hybrid system is developed including a means of including three forms of uncertainty: transition dynamics, structural uncertainty, and parametric uncertainty. A computational basis for viability of hybrid systems is developed and applied to three control law classes. An approach is developed for robust viability based on two extensions of the controllability operator. The three-tank example is examined for both the viability problem and robust viability problem. The theory is applied through simulation to an active magnetic bearing system and to a batch polymerization process showing that viability can be satisfied in practice. The problem of viable attainability is examined based on the controllability operator approach introduced by Nerode and colleagues. Lastly, properties of the controllability operator are presented.

  13. A fourier transform infrared spectroscopy (FTIR) based assay for Candida parapsilosis ATCC 7330 mediated oxidation of aryl alcohols. (United States)

    Sudhakara, Sneha; Chadha, Anju


    We present an FTIR based assay to monitor the whole cell mediated oxidation of aryl alcohols by measuring the characteristic IR absorption of the hydroxyl group [OH] of the substrate and the carbonyl group [CO] of the corresponding oxidized product. This method expedites the analysis of whole cell mediated catalysis which is usually done by GC and/or HPLC. The FTIR assay had linearity with R(2)≥0.980 and sensitivity up to 10μM. The accuracy and precision of FTIR assay was found ≥81% and ≥94%, respectively. This assay was validated by GC which exhibited ≥82% accuracy and ≥79% precision. The time of analysis taken by this assay was 2-3min per sample in comparison with 20-40min by GC.

  14. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen


    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  15. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna


    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  16. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  17. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

    Directory of Open Access Journals (Sweden)

    Chan Koon H


    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  18. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

    Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

  19. SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

    Directory of Open Access Journals (Sweden)

    Wing-Seng Leong


    Full Text Available Abstract Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05 at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.

  20. Recommendations for the development and validation of flow cytometry-based receptor occupancy assays. (United States)

    Green, Cherie L; Stewart, Jennifer J; Högerkorp, Carl-Magnus; Lackey, Alan; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Ferbas, John; Moulard, Maxime; Czechowska, Kamila; Mc Closkey, Thomas W; van der Strate, Barry W A; Wilkins, Danice E C; Lanham, David; Wyant, Timothy; Litwin, Virginia


    Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

  1. Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers

    Institute of Scientific and Technical Information of China (English)

    Kai SUN; Qian WANG; Xiao-hui HUANG; Mao-chuan ZHEN; Wen LI; Long-juan ZHANG


    Aim: The multiplexed, microsphere-based flow cytometric assay (MFCA) for mul- tiple human tumor markers was established for the early screening and detection of suspected cancer patients. Methods: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were iden- tified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. Results: The identifications revealed that the coupling proce- dures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality con- trol of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. Conclusion: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

  2. Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device. (United States)

    Neves, Marta M P S; Bobes-Limenes, Pablo; Pérez-Junquera, Alejandro; González-García, María Begoña; Hernández-Santos, David; Fanjul-Bolado, Pablo


    Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (μSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the μSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol-H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.

  3. Novel arginine deiminase-based method to assay L-arginine in beverages. (United States)

    Stasyuk, N Ye; Gayda, G Z; Fayura, L R; Boretskyy, Y R; Gonchar, M V; Sibirny, A A


    A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 μM to 24 μM with the detection limit of 0.25 μM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 μM to 50 μM with the detection limit of 0.55 μM. The new method was tested on real samples of wines and juices. A high correlation (R=0.978) was shown for the results obtained with the proposed and the reference enzymatic method.

  4. A High-resolution Typing Assay for Uropathogenic Escherichia coli Based on Fimbrial Diversity. (United States)

    Ren, Yi; Palusiak, Agata; Wang, Wei; Wang, Yi; Li, Xiao; Wei, Huiting; Kong, Qingke; Rozalski, Antoni; Yao, Zhi; Wang, Quan


    Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, causing cystitis, pyelonephritis, and renal failure. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTIs. Accurate and rapid discrimination of UPEC lineages is useful for epidemiological surveillance. Fimbriae are necessary for the adherence of UPEC strains to host uroepithelia, and seem to be abundant and diverse in UPEC strains. By analyzing all the possible fimbrial operons in UPEC strains, we found that closely related strains had similar types of chaperone-usher fimbriae, and the diversity of fimbrial genes was higher than that of multilocus sequence typing (MLST) genes. A typing assay based on the polymorphism of four gene sequences (three fimbrial genes and one housekeeping gene) and the diversity of fimbriae present was developed. By comparison with the MLST, whole-genome sequence (WGS) and fumC/fimH typing methods, this was shown to be accurate and have high resolution, and it was also relatively inexpensive and easy to perform. The assay can supply more discriminatory information for UPEC lineages, and have the potential to be applied in epidemiological surveillance of UPEC isolates.

  5. Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors (United States)

    Andresen, Liis; Varik, Vallo; Tozawa, Yuzuru; Jimmy, Steffi; Lindberg, Stina; Tenson, Tanel; Hauryliuk, Vasili


    The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors – a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 – showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries. PMID:27775002

  6. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase. (United States)

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga


    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  7. Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system. (United States)

    StGelais, Corine; Tuthill, Tobias J; Clarke, Dean S; Rowlands, David J; Harris, Mark; Griffin, Stephen


    Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.

  8. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals. (United States)

    Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P


    A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.

  9. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay. (United States)

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan


    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  10. Detection of Cronobacter sakazakii in powdered infant formula using an immunoliposome-based immunomagnetic concentration and separation assay (United States)

    Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Jung Hyun; Cho, Hyunjeong; Lee, Eun Ju; Kim, Myunghee


    This study aimed to optimize the applicability of an immunoliposome-based immunomagnetic concentration and separation assay to facilitate rapid detection of Cronobacter sakazakii in powdered infant formula (PIF). To determine the detection limit, specificity, and pre-enrichment incubation time (0, 4, 6, and 8 h), assay tests were performed with different cell numbers of C. sakazakii (2 × 100 and 2 × 101 CFU/ml) inoculated in 10 g of PIF. The assay was able to detect as few as 2 cells of C. sakazakii/10 g of PIF sample after 6 h of pre-enrichment incubation with an assay time of 2 h 30 min. The assay was assessed for cross-reactivity with other bacterial strains and exhibited strong specificity to C. sakazakii. Moreover, the assay method was applied to the detection of C. sakazakii in PIF without pre-enrichment steps, and the results were compared with INC-ELISA and RT-PCR. The developed method was able to detect C. sakazakii in spiked PIF without pre-enrichment, whereas INC-ELISA failed to detect C. sakazakii. In addition, when compared with the results obtained with RT-PCR, our developed assay required lesser detection time. The developed assay was also not susceptible to any effect of the food matrix or background contaminant microflora. PMID:27721500

  11. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  12. Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay. (United States)

    Rücker, Hannelore; Amslinger, Sabine


    The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

  13. Application of bioluminescence ATP measurement for evaluation of fungal viability of foxing spots on old documents. (United States)

    Rakotonirainy, Malalanirina Sylvia; Dubar, Pauline


    An adenosine triphosphate (ATP) bioluminescence-based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish-red stains, damaging the aesthetics of documents and their long-term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold-like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm(2), suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm(2). ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots.

  14. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies

    DEFF Research Database (Denmark)

    Van Elssen, Catharina H M J; Clausen, Henrik; Germeraad, Wilfred T V;


    to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary......-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique...... in vaccination studies as well as for functional assays....

  15. The regulation of circulating ghrelin - with recent updates from cell-based assays. (United States)

    Iwakura, Hiroshi; Kangawa, Kenji; Nakao, Kazuwa


    Ghrelin is a stomach-derived orexigenic hormone with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of appetite control in body energy homeostasis. Earlier studies revealed that circulating ghrelin levels are under the control of both acute and chronic energy status: at the acute scale, ghrelin levels are increased by fasting and decreased by feeding, whereas at the chronic scale, they are high in obese subjects and low in lean subjects. Subsequent studies revealed that nutrients, hormones, or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin secretion can assess whether and how individual factors affect ghrelin secretion from cells. In this review, on the basis of numerous human, animal, and cell-based studies, we summarize current knowledge on the regulation of circulating ghrelin levels and enumerate the factors that influence ghrelin levels.

  16. A carbon nanotubes based ATP apta-sensing platform and its application in cellular assay. (United States)

    Zhang, Libing; Wei, Hui; Li, Jing; Li, Tao; Li, Dan; Li, Yunhui; Wang, Erkang


    In this paper, a sensitive and selective fluorescent aptasensor for adenosine triphosphate (ATP) detection is constructed, based on the noncovalent assembly of dye-labeled ATP aptamer and single-walled carbon nanotubes (SWNTs). In the absence of ATP, the dye tethered to the ATP aptamer is close to SWNTs, which can effectively quench fluorescence of the dye. Upon adding ATP, the fluorophore keeps away from the quencher, since ATP specifically binds to the aptamer and competes with carbon nanotubes, resulting in an increase in the fluorescence intensity. This enables ATP to be detected down to 4.5nM. To the best of our knowledge, this is the most sensitive fluorescent ATP aptasensor. In addition, prominent fluorescence signals were obtained in cellular ATP assays, thus the aptasensor could be used to detect ATP in real samples.

  17. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo


    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  18. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano


    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  19. Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses. (United States)

    Sánchez-Matamoros, A; Nieto-Pelegrín, E; Beck, C; Rivera-Arroyo, B; Lecollinet, S; Sailleau, C; Zientara, S; Sánchez-Vizcaíno, J M


    African horse sickness (AHS) is considered a fatal re-emergent vector-borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non-structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV-free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex-based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex-based assay for detecting anti-NS3 antibodies showed good positive correlation with results from an in-house enzyme-linked immunosorbent assay (ELISA). Thus, the Luminex-based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2 h, and it may prove adaptable for the development of robust, multiplex serological assays.

  20. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences


    Ågren, Joakim; Raditijo A Hamidjaja; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; van Rotterdam, Bart; Derzelle, Sylviane


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. ...

  1. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples. (United States)

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T


    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  2. Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    Full Text Available BACKGROUND: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of +/-25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4-8 determinations from each amplification reaction. FINDINGS: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. CONCLUSIONS: Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the

  3. Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays

    Directory of Open Access Journals (Sweden)

    Ahlinder Jon


    Full Text Available Abstract Background Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.

  4. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU


    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  5. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Institute of Scientific and Technical Information of China (English)

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU


    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  6. A digital microfluidic method for multiplexed cell-based apoptosis assays. (United States)

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R


    Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.

  7. Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation. (United States)

    Mota, Caroline M; Ferreira, Marcela D; Costa, Lourenço F; Barros, Patrício S C; Silva, Murilo V; Santiago, Fernanda M; Mineo, José R; Mineo, Tiago W P


    Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.

  8. Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

    Directory of Open Access Journals (Sweden)

    Haifeng Geng

    Full Text Available Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs, inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA activity found in patients with metachromatic leukodystrophy (MLD, a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS, detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an

  9. Fault Detection and Isolation using Viability Theory and Interval Observers (United States)

    Ghaniee Zarch, Majid; Puig, Vicenç; Poshtan, Javad


    This paper proposes the use of interval observers and viability theory in fault detection and isolation (FDI). Viability theory develops mathematical and algorithmic methods for investigating the adaptation to viability constraints of evolutions governed by complex systems under uncertainty. These methods can be used for checking the consistency between observed and predicted behavior by using simple sets that approximate the exact set of possible behavior (in the parameter or state space). In this paper, fault detection is based on checking for an inconsistency between the measured and predicted behaviors using viability theory concepts and sets. Finally, an example is provided in order to show the usefulness of the proposed approach.

  10. Detection for Viability of Wheat Based on Ultra -Weak Delayed Bioluminescence%基于超弱延迟发光的小麦生活力检测方法研究

    Institute of Scientific and Technical Information of China (English)

    吴才章; 牛群峰; 张兰静


    The variation of the ultra -weak delayed bioluminescence under different viability and different test conditions was investigated with zhengmai 9023 as an experimental object.The different viability wheat sample could be obtained by rapid artificial aging,and the characteristics of the delayed bioluminescence from different viability and the influence of temperature on the delayed bioluminescence were studied.The results showed that obvious differences were found in the delayed bioluminescence ability for different viability with the same seeds.There was a negative correlation between the delayed bioluminescence and its viability.In other words,its viability was strengthened as the delayed bi-oluminescence was weakened.The influence of temperature on the delayed bioluminescence was obvious.As the temper-ature rose,the intensity of ultra -weak photon emission decreased gradually.The research demonstrated that it was feasi-ble to detect wheat viability based on the ultra -weak delayed bioluminescence under the strict control of test conditions.%以郑麦9023为试验对象,研究了不同生活力和不同测试条件下小麦样品超弱延迟发光的变化情况。通过人工快速陈化的方法获得不同生活力的小麦样品,对这些小麦样品的延迟发光特性进行系统研究,在此基础上以温度为例研究了测试条件对小麦延迟发光的影响。结果表明,同一品种不同生活力小麦样品的延迟发光能力差异明显,小麦的延迟发光强度与其生活力显著负相关,生活力越强,其延迟发光能力越弱;小麦的延迟发光能力受其自身温度的影响显著,随着温度的升高,其延迟发光能力减弱。本研究发现,利用超弱延迟发光进行小麦生活力检测是可行的,但需要对测试条件进行严格控制。

  11. Cell-based galactosemia diagnosis system based on a galactose assay using a bioluminescent Escherichia coli array. (United States)

    Woo, Min-Ah; Kim, Moon Il; Cho, Daeyeon; Park, Hyun Gyu


    A new cell-based galactose assay system, which is comprised of two bioluminescent Escherichia coli strains immobilized within an agarose gel arrayed on a well plate, has been developed. For this purpose, a galT knockout strain [galT(-) cell] of E. coli was genetically constructed so that cell growth is not promoted by galactose but rather by glucose present in a sample. Another E. coli W strain (normal cell), which grows normally in the presence of either glucose or galactose, was employed. A luminescent reporter gene, which produces luminescence as cells grow, was inserted into both of the E. coli strains, so that cell growth could be monitored in a facile manner. The two strains were separately grown for 4 h on gel arrays to which test samples were individually supplied. The relative luminescence unit (RLU) values caused by cell growth were determined for each array, one of which is resulted by glucose only and the other of which is resulted by both glucose and galactose present in the sample. By employing this protocol, galactose concentrations present in the test sample are reflected in the differences between the RLU values for each array. The practical utility of the new assay system was demonstrated by its use in determining galactose levels in clinical blood spot specimens coming from newborn babies. Because it can be employed to diagnosis of galactosemia in newborn babies in a more rapid, convenient, and cost-effective manner, this cell-based solid-phase galactose assay system should become a powerful alternative to conventional methods, which require labor-intensive and time-consuming procedures and/or complicated and expensive equipment.

  12. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay. (United States)

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung


    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm(2). A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times.

  13. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.


    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  14. Sperm morphometry: a tool for detecting biophysical changes associated with viability in cryopreserved bovine spermatozoa. (United States)

    García-Herreros, M; Leal, C L V


    The aim of this study was to determine whether computerised sperm head morphometric analysis can be used as a diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa. Ejaculates from five bulls (4 ejaculates/bull) were pooled and processed for computerised morphometric analysis, and SYBR-14 green/ethidium homodimer-1 fluorescence-based live/dead viability assay was used simultaneously to confirm the viability index of frozen-thawed spermatozoa. Sperm samples were assigned to three experimental groups. The first group was enriched in live spermatozoa (after a double Percoll selection), the second group was enriched in dead spermatozoa (after a refreeze-thaw procedure), and the last group was a 50 : 50 pool of live/dead spermatozoa (from first and second group samples). There were significant differences (P sperm morphometric dimensional parameters among the three groups analysed, being the lowest overall sperm head dimension found in the second (dead spermatozoa) group. In conclusion, sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa.

  15. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources. (United States)

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar


    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.

  16. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  17. CCL18 in a multiplex urine-based assay for the detection of bladder cancer.

    Directory of Open Access Journals (Sweden)

    Virginia Urquidi

    Full Text Available The early detection of bladder cancer (BCa is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA; C-C motif chemokine 18 (CCL18, Plasminogen Activator Inhibitor 1 (PAI-1 and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak© and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674. Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001 and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033 as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to 0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.

  18. Prediction of Non-Genotoxic Carcinogenicity Based on Genetic Profiles of Short Term Exposure Assays (United States)

    Pérez, Luis Orlando; González-José, Rolando; García, Pilar Peral


    Non-genotoxic carcinogens are substances that induce tumorigenesis by non-mutagenic mechanisms and long term rodent bioassays are required to identify them. Recent studies have shown that transcription profiling can be applied to develop early identifiers for long term phenotypes. In this study, we used rat liver expression profiles from the NTP (National Toxicology Program, Research Triangle Park, USA) DrugMatrix Database to construct a gene classifier that can distinguish between non-genotoxic carcinogens and other chemicals. The model was based on short term exposure assays (3 days) and the training was limited to oxidative stressors, peroxisome proliferators and hormone modulators. Validation of the predictor was performed on independent toxicogenomic data (TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System, Osaka, Japan). To build our model we performed Random Forests together with a recursive elimination algorithm (VarSelRF). Gene set enrichment analysis was employed for functional interpretation. A total of 770 microarrays comprising 96 different compounds were analyzed and a predictor of 54 genes was built. Prediction accuracy was 0.85 in the training set, 0.87 in the test set and increased with increasing concentration in the validation set: 0.6 at low dose, 0.7 at medium doses and 0.81 at high doses. Pathway analysis revealed gene prominence of cellular respiration, energy production and lipoprotein metabolism. The biggest target of toxicogenomics is accurately predict the toxicity of unknown drugs. In this analysis, we presented a classifier that can predict non-genotoxic carcinogenicity by using short term exposure assays. In this approach, dose level is critical when evaluating chemicals at early time points. PMID:27818731

  19. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers. (United States)

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick


    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  20. Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay. (United States)

    Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro


    Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP ). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

  1. Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua. (United States)

    Chikeka, Ijeuru; Matute, Armando J; Dumler, J Stephen; Woods, Christopher W; Mayorga, Orlando; Reller, Megan E


    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.

  2. Establishing Guidelines to Retain Viability of Probiotics during Spray Drying

    NARCIS (Netherlands)

    Perdana, J.A.; Fox, M.B.; Boom, R.M.; Schutyser, M.A.I.


    We present the application of a model-based approach to map processing conditions suitable to spray dry probiotics with minimal viability loss. The approach combines the drying history and bacterial inactivation kinetics to predict the retention of viability after drying. The approach was used to sy

  3. Establishing guidelines to retain viability of probiotics during spray drying

    NARCIS (Netherlands)

    Perdana, J.A.; Fox, M.B.; Boom, R.M.; Schutyser, M.A.I.


    We present a model-based approach to map processing conditions suitable to spray dry probiotics with minimal viability loss. The approach combines the drying history and bacterial inactivation kinetics to predict the retention of viability after drying. The approach was used to systematically assess

  4. Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation. (United States)

    Choulier, Laurence; Nominé, Yves; Zeder-Lutz, Gabrielle; Charbonnier, Sebastian; Didier, Bruno; Jung, Marie-Louise; Altschuh, Danièle


    We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

  5. A simple dot-blot-Sirius red-based assay for collagen quantification. (United States)

    Rodríguez-Rodríguez, Pilar; Arribas, Silvia M; de Pablo, Angel Luis López; González, M Carmen; Abderrahim, Fatima; Condezo-Hoyos, Luis


    The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

  6. Analytical assays based on chromogenic and fluorogenic chemosensors for the detection of cyanide

    Directory of Open Access Journals (Sweden)

    Vanderléia Gava Marini


    Full Text Available Cyanide (CN– is an anion well–known for its toxicity, being a chemical agent often related to cases of homicide and suicide. Despite being responsible for the toxicity of many animals and plants, it is used in several industrial activities, with innumerous implications in terms of the environment. Due to its high toxicity, the maximum level of CN– concentration allowed by the World Health Organization in potable water is 1.7 µmol/L. This low concentration limit requires methods of visual detection and quantitative determination which are ever more sensitive, simple, reliable, and economical. Advancements in the field of chromogenic and fluorogenic chemosensors for anionic analytes have led to the development of several methodologies for the detection of CN–. Therefore, this review aims to present the main strategies that have been used in the study of quantitative and naked–eye detection of CN– by means of chromogenic and fluorogenic chemosensors. Aspects related to CN–, such as its reactivity, toxicity, applications, and implications in different domains of knowledge, are presented. Recent work involving the development of chemosensors for CN– based on acid–base reactions, chemodosimeters, chromoreactands, and competition assays is also described. In addition, recent studies that make use of nanotechnology to develop strategies for the detection of CN– are also discussed, as well as the prospects envisioned in this field.

  7. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening. (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun


    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions.

  8. A high-throughput pipeline for designing microarray-based pathogen diagnostic assays

    Directory of Open Access Journals (Sweden)

    Reifman Jaques


    Full Text Available Abstract Background We present a methodology for high-throughput design of oligonucleotide fingerprints for microarray-based pathogen diagnostic assays. The oligonucleotide fingerprints, or DNA microarray probes, are designed for identifying target organisms in environmental or clinical samples. The design process is implemented in a high-performance computing software pipeline that incorporates major algorithmic improvements over a previous version to both reduce computation time and improve specificity assessment. Results The algorithmic improvements result in significant reduction in runtimes, with the updated pipeline being nearly up to five-times faster than the previous version. The improvements in specificity assessment, based on multiple specificity criteria, result in robust and consistent evaluation of cross-hybridization with nontarget sequences. In addition, the multiple criteria provide finer control on the number of resulting fingerprints, which helps in obtaining a larger number of fingerprints with high specificity. Simulation tests for Francisella tularensis and Yersinia pestis, using a well-established hybridization model to estimate cross-hybridization with nontarget sequences, show that the improved specificity criteria yield a larger number of fingerprints as compared to using a single specificity criterion. Conclusion The faster runtimes, achieved as the result of algorithmic improvements, are critical for extending the pipeline to process multiple target genomes. The larger numbers of identified fingerprints, obtained by considering broader specificity criteria, are essential for designing probes for hard-to-distinguish target sequences.

  9. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays. (United States)

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark


    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

  10. Microtiter plate based colorimetric assay for characterization of dehalogenation activity of GAC/Fe0 composite

    DEFF Research Database (Denmark)

    Hwang, Yuhoon; Salatas, Apostolos; Mines, Paul D.


    of nZVI and its composite with granular activated carbon (GAC). The assay focused on analysis of reaction products rather than its mother compounds, which gives more accurate quantification of reductive activity. The colorimetric assays were developed to quantify three reaction products, ammonia...

  11. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat


    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  12. Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation. (United States)

    Shapiro-Ilan, David I; Hazir, Selcuk; Lete, Luis


    Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone

  13. Membrane-based assay for iodide ions based on anti-leaching of gold nanoparticles. (United States)

    Shen, Yu-Wei; Hsu, Pang-Hung; Unnikrishnan, Binesh; Li, Yu-Jia; Huang, Chih-Ching


    We report a label-free colorimetric strategy for the highly selective and sensitive detection of iodide (I(-)) ions in human urine sample, seawater and edible salt. A poly(N-vinyl-2-pyrrolidone)-stabilized Au nanoparticle (34.2-nm) was prepared to detect I(-) ions using silver (Ag(+)) and cyanide (CN(-)) ions as leaching agents in a glycine-NaOH (pH 9.0) solution. For the visual detection of the I(-) ions by naked eye, and for long time stability of the probe, Au nanoparticles (NPs) decorated mixed cellulose ester membrane (MCEM) was prepared (Au NPs/MCEM). The Au NPs-based probe (CN(-)/Ag(+)-Au NPs/MCEM) operates on the principle that Ag(+) ions form a monolyar silver atoms/ions by aurophilic/argentophilic interactions on the Au NPs and it accelerates the leaching rate of Au atoms in presence of CN(-) ions. However, when I(-) is introduced into this system, it inhibits the leaching of Au atoms because of the strong interactions between Ag/Au ions and I(-) ions. Inductively coupled plasma mass spectrometry, surface-assisted laser desorption/ionization time-of-flight mass spectrometry were used to characterize the surface properties of the Au NPs in the presence of Ag(+) and I(-). Under optimal solution conditions, the CN(-)/Ag(+)-Au NPs/MCEM probe enabled the detection of I(-) by the naked eye at nanomolar concentrations with high selectivity (at least 1000-fold over other anions). In addition, this cost-effective probe allowed the determination of I(-) ions in complex samples, such as urine, seawater, and edible salt samples.

  14. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

    DEFF Research Database (Denmark)

    Ørsted, Michael; Roslev, Peter


    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl o...... that the fluorescence based hydrolytic enzyme activity assay (FLEA assay) can be used as an index of D. magna stress. Combining enzyme activity with fluorescence measurements may be applied as a simple and quantitative supplement for toxicity testing with D. magna....

  15. A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins in human peripheral blood. (United States)

    Sun, Peifang; Beckett, Charmagne; Danko, Janine; Burgess, Timothy; Liang, Zhaodong; Kochel, Tadeusz; Porter, Kevin


    Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53-513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0-45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4(+) and CD8(+) T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

  16. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum. (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai


    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

  17. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis (United States)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.


    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  18. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics (United States)

    Joubert, Marisa K.; Deshpande, Meghana; Yang, Jane; Reynolds, Helen; Bryson, Christine; Fogg, Mark; Baker, Matthew P.; Herskovitz, Jonathan; Goletz, Theresa J.; Zhou, Lei; Moxness, Michael; Flynn, Gregory C.; Narhi, Linda O.; Jawa, Vibha


    An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. PMID:27494246

  19. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  20. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity. (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin


    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  1. "Molecular beacon"-based fluorescent assay for selective detection of glutathione and cysteine. (United States)

    Xu, Hui; Hepel, Maria


    We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.

  2. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening. (United States)

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua


    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  3. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay. (United States)

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues


    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  4. Functional screening with a live cell imaging-based random cell migration assay. (United States)

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob


    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  5. A novel flow-based procedure for automation of respirometric assays in soils. (United States)

    Silva, Claudineia R; Oliveira, Eliezer; Zagatto, Elias A G; Henriquez, Camelia


    A flow-based strategy involving a gas-diffusion sampling probe was proposed for evaluating the respiration rate in soils. The amount of CO2 collected after a pre-defined time interval was proportional to the free CO2 released by the soil ecosystem. The 500-mL incubation flasks typically used for soil respirometric assays were adapted and a special cover was designed for connecting a tubular gas diffusion membrane, a fan, and a septum for adding the CO2(g) standards required for calibration. The method relied on the pH-dependent absorbance variations resulting from the CO2 collection. A 1.3mmolL(-1) bromothymol blue solution (pH 7.0) acted as both acceptor and carrier streams. In order to widen the dynamical working range to 0.003-0.2mmol CO2, two analytical curves were obtained, each related to a different time interval for the CO2 collection. Kinetic curves related to CO2 release by the soil samples were straightforwardly attained. Repeatability and detection limit were estimated as 2.0% and 0.001mmol CO2 (n=10), and accuracy was assessed in relation to a recommended titrimetric procedure.

  6. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications. (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo


    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C3N4) and transition metal dichalcogenides (e.g. MoS2, MnO2, and WS2). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring.

  7. An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays. (United States)

    Qureshi, Anjum; Roci, Irena; Gurbuz, Yasar; Niazi, Javed H


    An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.

  8. Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

    Directory of Open Access Journals (Sweden)

    Milla Luis A


    Full Text Available Abstract Background The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1 and novel Hh-regulated genes in zebrafish embryos. Results The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. Conclusion A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in

  9. Flavocytochrome b2-Based Enzymatic Method of L-Lactate Assay in Food Products

    Directory of Open Access Journals (Sweden)

    Oleh Smutok


    Full Text Available L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b2 (EC; FC b2 isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ=525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate, simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

  10. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M


    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  11. Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system. (United States)

    Karadeniz, Hakan; Erdem, Arzum; Kuralay, Filiz; Jelen, Frantisek


    An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N=3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.

  12. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon (United States)

    Goodridge, Lawrence (Inventor)


    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  13. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays


    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Marcotte, Edward M.


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential ap...

  14. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays


    Iole Macchia; Francesca Urbani; Enrico Proietti


    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometr...

  15. Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator. (United States)

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


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

  16. The xCELLigence system for real-time and label-free monitoring of cell viability. (United States)

    Ke, Ning; Wang, Xiaobo; Xu, Xiao; Abassi, Yama A


    We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.

  17. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts

    Directory of Open Access Journals (Sweden)

    Luc Séro


    Full Text Available Advanced glycation end-products (AGEs are involved in the pathogenesis of numerous diseases. Among them, cellular accumulation of AGEs contributes to vascular complications in diabetes. Besides using drugs to lower blood sugar, a balanced diet and the intake of herbal products potentially limiting AGE formation could be considered beneficial for patients’ health. The current paper presents a simple and cheap high-throughput screening (HTS assay based on AGE fluorescence and suitable for plant extract screening. We have already implemented an HTS assay based on vesperlysines-like fluorescing AGEs quickly (24 h formed from BSA and ribose under physiological conditions. However, interference was noted when fluorescent compounds and/or complex mixtures were tested. To overcome these problems and apply this HTS assay to plant extracts, we developed a technique for systematic quantification of both vesperlysines (λexc 370 nm; λem 440 nm and pentosidine-like (λexc 335 nm; λem 385 nm AGEs. In a batch of medicinal and food plant extracts, hits were selected as soon as fluorescence decreased under a fixed threshold for at least one wavelength. Hits revealed during this study appeared to contain well-known and powerful anti-AGE substances, thus demonstrating the suitability of this assay for screening crude extracts (0.1 mg/mL. Finally, quercetin was found to be a more powerful reference compound than aminoguanidine in such assay.

  18. Antitumour efficacy of Piper tuberculatum and piplartine based on the hollow fiber assay. (United States)

    Bezerra, Daniel P; Ferreira, Paulo Michel P; Machado, Camila Maria L; de Aquino, Nayara C; Silveira, Edilberto R; Chammas, Roger; Pessoa, Claudia


    Piper tuberculatum, popularly known in Brazil as "jaborandi falso" and "pimenta darta", is widely used in folk medicine for the treatment of several diseases. In this study, the in vivo hollow fiber assay was used to investigate the antitumour efficacy of the crude extract and piplartine obtained from P. tuberculatum roots. Human glioblastoma (SF-295) and colon carcinoma (HCT-8) cell lines were used. In vitro cytotoxicity was assayed by the MTT assay. In the hollow fiber assay, nude mice implanted with tumour cells in hollow fibers were treated for four consecutive days via the intraperitoneal route, and tumour cell populations were assessed by the MTT assay. Both the crude extract and piplartine displayed cytotoxicity. In the hollow fiber assay, tumour growth inhibition rates were 24.6-54.8 % for the crude extract and 33.7-62.2 % for piplartine. No signal of toxicity was noticed. In conclusion, the crude extract and piplartine obtained from P. tuberculatum roots displayed in vitro and in vivo anticancer efficacy.

  19. Bioluminescence-Sensing Assay for Microbial Growth Recognition


    Heba Ramadan Eed; Nora S. Abdel-Kader; Mahmoud Helmy El Tahan; Tianhong Dai; Rehab Amin


    The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP) bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A biolumine...

  20. Medical Devices; Immunology and Microbiology Devices; Classification of Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay. Final order. (United States)


    The Food and Drug Administration (FDA) is classifying a gastrointestinal microorganism multiplex nucleic acid-based assay into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

  1. Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016). (United States)

    Berns, Eric J; Cabezas, Maria D; Mrksich, Milan


    On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

  2. Optimal Concentrations and Synergistic Effects of Some Herbal Extracts on Viability of Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)



    Full Text Available Background Many studies examine the antibacterial effects of medicinal plants; however, little research is done to evaluate their effects on different cell types, especially dermal fibroblasts. Objectives The current study aimed to study the effect of different concentrations of Aloe Vera, henna, chamomile, myrtle, mint, licorice, cinnamon, ginger and cedar extracts and their synergistic effects on the viability of dermal fibroblasts. Methods To evaluate the performance of herbal extracts on dermal fibroblasts, in the first experiment different concentrations (6.25, 12.5, 25, 50, 100, 250, 500 and 1000 µg/mL of the extracts were evaluated by the MTT cell proliferation assay. In the second experiment, the minimum effective concentrations of the plant extracts in triple combination were evaluated in the cells under study. Results The minimum effective concentrations of henna, chamomile, myrtle, mint, cinnamon, ginger and cedar were 12.5, 6.25, 6.25, 6.25, 6.25, 12.5 and 12.5µg/mL, respectively. Results showed that, by comparing the minimum effective concentration of herbal extracts, the viability of dermal fibroblasts significantly increased by cedar extract (P < 0.05. Combination of Aloe Vera, licorice and mint extracts significantly increased the viability of dermal fibroblasts (P < 0.05. Conclusions Based on the results of the current study, it was concluded that Aloe vera, licorice and mint extracts had synergistic effects on the viability of dermal fibroblasts; in addition, the combination of Aloe vera and licorice with either henna or myrtle, and Aloe vera and mint with either cedar or ginger resulted in synergistic effects on viability of dermal fibroblasts. The third category of triple combinations of herbal extracts with synergistic effects on the cells under study was the combination of Aloe Vera and mint with either chamomile or cinnamon and also Aloe vera and licorice with either myrtle or cedar.

  3. Bioluminescence-Sensing Assay for Microbial Growth Recognition

    Directory of Open Access Journals (Sweden)

    Heba Ramadan Eed


    Full Text Available The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A bioluminescent recombinant E. coli strain was used with luciferase extracted from transformed bacteria. Results showed that there is a direct correlation between the bioluminescence intensity of the ATP bioluminescence-sensing assay and the microbial viability. Bacterial counts from food samples were detected using the developed sensing assay and validated by the traditional plate-counting method. Compared with the plate-counting method, ATP bioluminescence-sensing assay is a more rapid and efficient approach for detecting microbial viability.

  4. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment (United States)

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


    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. 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:; Loo, Say Chye Joachim, E-mail: [Nanyang Technological University, School of Materials Science and Engineering (Singapore)


    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.

  6. Evaluation of a gp63-PCR based assay as a molecular diagnosis tool in canine leishmaniasis in Tunisia.

    Directory of Open Access Journals (Sweden)

    Souheila Guerbouj

    Full Text Available A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania (L. parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture or serological (IFAT techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18 and control dogs (N = 45 originating from non-endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8-95.4% or serology IFAT technique (47.4%, 95% CI: 23.5-71.3%. However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

  7. Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia (United States)

    Guerbouj, Souheila; Djilani, Fattouma; Bettaieb, Jihene; Lambson, Bronwen; Diouani, Mohamed Fethi; Ben Salah, Afif; Ben Ismail, Riadh; Guizani, Ikram


    A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia. PMID:25153833

  8. Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

    Energy Technology Data Exchange (ETDEWEB)

    Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.; Kuhn, Eric; Liu, Tao; Massoni, Sam A.; Thomas, Stefani N.; Townsend, Reid; Zimmerman, Lisa J.; Boja, Emily; Chen, Jing; Crimmins, Daniel L.; Davies, Sherri; Gao, Yuqian; Hiltke, Tara R.; Ketchum, Karen; Kinsinger, Christopher; Mesri, Mehdi; Meyer, Matthew R.; Qian, Weijun; Schoenherr, Regine M.; Scott, Mitchell; Shi, Tujin; Whiteley, Gordon; Wrobel, John; Wu, Chaochao; Ackermann, Bradley L.; Aebersold, Ruedi; Barnidge, David R.; Bunk, David M.; Clarke, Nigel; Fishman, Jordan B.; Grant, Russ P.; Kusebauch, Ulrike; Kushnir, Mark M.; Lowenthal, Mark S.; Moritz, Robert; Neubert, Hendrik; Patterson, Scott D.; Rockwood, Alan L.; Rogers, John; Singh, Ravinder J.; Van Eyk, Jennifer; Wong, Steven H.; Zhang, Shucha; Chan, Daniel W.; Chen, Xian; Ellis, Matthew J.; Liebler, Daniel; Rodland, Karin D.; Rodriguez, Henry; Smith, Richard D.; Zhang, Zhen; Zhang, Hui; Paulovich, Amanda G.


    The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

  9. Making the cut: Innovative methods for optimizing perfusion-based migration assays. (United States)

    Holt, Andrew W; Howard, William E; Ables, Elizabeth T; George, Stephanie M; Kukoly, Cindy A; Rabidou, Jake E; Francisco, Jake T; Chukwu, Angel N; Tulis, David A


    Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell

  10. Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora. (United States)

    Cheng, Zhongzhe; Huang, Meilin; Chen, Guiying; Yang, Guangjie; Zhou, Xin; Chen, Chang; Zhang, Yang; Xu, Yong; Feng, Yulin; Zhang, Lin; Jiang, Hongliang


    Many plant-derived glycosides are used as medications. It is common that these glycosides show poor intestinal absorption but their metabolites generated by intestinal microflora demonstrate strong bioactivity. Hence, it is crucial to develop a method for the identification and characterization of the metabolites, and consequently reveal the pathway in which the glycosides are processed in gut. In this study, cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) were developed for rapid discovery and evaluation of the metabolites of a glycoside compound, capilliposide C (LC-C). 92.7% of LC-C was biotransformed by rat intestinal microflora after 36-h incubation at 37°C. Human cancer cell lines HepG2, PC-3 and A549 was treated with metabolites pool, respectively, which was followed by cell viability assays and characterization of metabolites using UHPLC-QTOF-MS/MS. As a result, significant cytotoxicity was observed for the metabolites pool, from which six metabolites were identified. Based on the metabolites identified, deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-C in rat intestinal microflora. In addition, M4, an esterolysis product of LC-C, was obtained and evaluated for its bioactivity in vitro. As a result, M4 exhibited a reduction in cell viability in HepG2 with an IC50 value of 17.46±1.55μg/mL.

  11. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine


    on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S......Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely......-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal...

  13. A fluorescence turn on assay for alkaline phosphatase based on the Cu(2+) catalyzed Fenton-like reaction. (United States)

    Zhang, Qingfeng; Zhang, Cuiyun; Shahzad, Sohail Anjum; Yu, Cong


    A fluorescence turn-on assay was established for ALP (alkaline phosphatase) based on Cu(2+) catalyzed Fenton-like reaction and Graphene Oxide (GO). GO was utilized to quench the fluorescence of fluorescein (FAM) labeled single strand DNA (F-DNA). ALP can remove the phosphate group in sodium ascorbyl phosphate (SAP), and convert it into reducing ascorbate. Highly reactive hydroxyl radicals (·OH) were generated in the presence of ascorbate and Cu(2+) through the Fenton-like reaction. The reactive radicals generated in situ caused the cleavage of F-DNA into small fragments. When GO was added, the fluorescence emission of the sample without ALP was quenched and fluorescence emission recovered in the presence of ALP. The intensity of the recovered fluorescence was directly related to the concentration of ALP in the assay solution, and a sensitive and selective facile ALP assay is therefore established.

  14. Toward the authentication of wines of Nemea denomination of origin through cleaved amplified polymorphic sequence (CAPS)-based assay. (United States)

    Spaniolas, Stelios; Tsachaki, Maroussa; Bennett, Malcolm J; Tucker, Gregory A


    In the present study, we developed a cleaved amplified polymorphic sequence (CAPS)-based assay as a first attempt to detect fraud in grapevine musts with a long-term objective to establish an analytical methodology to authenticate wines of Nemea denomination of origin (Agiorgitiko). The analytical assay makes use of a single nucleotide polymorphism that discriminates Agiorgitiko and Cabernet Sauvignon varieties. The latter grape variety is one of the major adulterants for Nemea wines. Agiorgitiko grapevine must was spiked with Cabernet Sauvignon in several ratios (v/v) from 50 down to 10%, and the subsequent mixes were subjected to alcoholic microfermentation. DNA was extracted from all mixture samples up to the end of the fermentation process and was subjected to the CAPS assay. Both standard agarose gel and lab-on-a-chip capillary electrophoresis illustrated the ability of the method to detect the presence of Cabernet Sauvignon down to 10% throughout the whole fermentation process.

  15. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus? (United States)

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen


    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

  16. Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates. (United States)

    Mirazizi, Fatemeh; Bahrami, Azita; Haghbeen, Kamahldin; Shahbani Zahiri, Hossein; Bakavoli, Mehdi; Legge, Raymond L


    Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.

  17. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity. (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John


    Dipeptidyl peptidase 1 (DPP1) (EC; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  18. Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

    Directory of Open Access Journals (Sweden)

    Yu Jingjing


    Full Text Available Abstract Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP and methylated DNA (MeDIP represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation

  19. Dose-response curve of a microfluidic magnetic bead-based surface coverage sandwich assay. (United States)

    Cornaglia, Matteo; Trouillon, Raphaël; Tekin, H Cumhur; Lehnert, Thomas; Gijs, Martin A M


    Magnetic micro- and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fgmL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8μm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0μm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves.

  20. Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products. (United States)

    Randlev, Britta; Huang, Li-chun; Watatsu, Mitsuko; Marcus, Matthew; Lin, Andy; Shih, Shian-Jiun


    GVAX immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

  1. Effects of Ciprofloxacin-Containing Antimicrobial Scaffolds on Dental Pulp Stem Cell Viability — In Vitro Studies (United States)

    Kamocki, Krzysztof; Nör, Jacques E.; Bottino, Marco C.


    Objective A combination of antibiotics, including but not limited to metronidazole (MET) and ciprofloxacin (CIP), has been indicated to eradicate bacteria in necrotic immature permanent teeth prior to regenerative procedures. It has been shown clinically that antibiotic pastes may lead to substantial stem cell death. The aim of this study was to synthesize scaffolds containing various concentrations of CIP to enhance cell viability while preserving antimicrobial properties. Design Polydioxanone (PDS)-based electrospun scaffolds were processed with decreasing CIP concentrations (25 – 1 wt.%) and morphologically evaluated using scanning electron microscopy (SEM). Cytotoxicity assays were performed to determine whether the amount of CIP released from the scaffolds would lead to human dental pulp stem cell (hDPSC) toxicity. Similarly, WST-1 assays were performed to evaluate the impact of CIP release on hDPSC proliferation. Pure PDS scaffolds and saturated double antibiotic solution MET/CIP (DAP) served as both positive and negative controls, respectively. Antibacterial efficacy against E. faecalis (Ef) was tested. Results A significant decrease in hDPSC’ viability at concentrations 5–25 wt.% was observed. However, concentrations below 5 wt.% did not impair cell viability. Data from the WST-1 assays indicated no detrimental impact on cell proliferation for scaffolds containing 2.5 wt.% CIP or less. Significant antimicrobial properties were seen for CIP-scaffolds at lower concentrations (i.e., 1 and 2.5 wt.%). Conclusion The obtained data demonstrated that a reduced concentration of CIP incorporated into PDS-based scaffolds maintains its antimicrobial properties while enhancing viability and proliferation of dental pulp stem cells. PMID:26042622

  2. LFR Demonstrator Materials Viability

    Energy Technology Data Exchange (ETDEWEB)

    Caro, M


    Interest in fast reactor development has increased with the Department of Energy's introduction of the Global Nuclear Energy Partnership (GNEP) [1]. The GNEP program plans development of a sodium cooled Advanced Burner Reactor (ABR) that can be used to reduce the amount spent LWR fuel in storage and the number of high level waste sites needed for expansion of nuclear power throughout the world over the 21st century. In addition, the program proposes to make nuclear power more available while reducing the proliferation concerns by revising policies and technology for control of weapons useable materials. This would be accomplished with establishment of new institutional arrangements based on selective siting of reprocessing, enrichment and waste disposal facilities. The program would also implement development of small reactors suitable for use in developing countries or remote regions with small power grids. Over the past several years, under the Department of Energy (DOE) NERI and GEN IV programs research has been conducted on small lead cooled reactors. The Small Secure Transportable Autonomous Reactor (SSTAR) [2] is the most recent version of this type of reactor and research is continuing on it in the GEN IV program in parallel with GNEP. SSTAR is a small (10MWe-100MWe) reactor that is fueled once for life. It complements the GNEP program very well in that it serves one of the world markets not currently addressed by large reactors and its development requirements are similar to those for the ABRs. In particular, the fuel and structural materials for these fast spectrum reactors share common thermal and neutron environments. The coolants, sodium in ABR and lead or lead-bismuth eutectic (LBE) in SSTAR, are the major developmental difference. This report discusses the status of structural materials for fast reactor core and primary system components and selected aspects of their development.

  3. Fast and automated DNA assays on a compact disc (CD)-based microfluidic platform (United States)

    Jia, Guangyao

    Nucleic acid-based molecular diagnostics offers enormous potential for the rapid and accurate diagnosis of infectious diseases. However, most of the existing commercial tests are time-consuming and technically complicated, and are thus incompatible with the need for rapid identification of infectious agents. We have successfully developed a CD-based microfluidic platform for fast and automated DNA array hybridization and a low cost, disposable plastic microfluidic platform for polymerase chain reaction (PCR). These platforms have proved to be a promising approach to meet the requirements in terms of detection speed and operational convenience in diagnosis of infectious diseases. In the CD-based microfluidic platform for DNA hybridization, convection is introduced to the system to enhance mass transport so as to accelerate the hybridization rate since DNA hybridization is a diffusion limited reaction. Centrifugal force is utilized for sample propulsion and surface force is used for liquid gating. Standard microscope glass slides are used as the substrates for capture probes owing to their compatibility with commercially available instrumentation (e.g. laser scanners) for detection. Microfabricated polydimethylsiloxane (PDMS) structures are used to accomplish the fluidic functions required by the protocols for DNA hybridization. The assembly of the PDMS structure and the glass slide forms a flow-through hybridization unit that can be accommodated onto the CD platform for reagent manipulation. The above scheme has been validated with oligonucleotides as the targets using commercially available enzyme-labeled fluorescence (ELF 97) for detection of the hybridization events, and tested with amplicons of genomic staphylococcus DNA labeled with Cy dye. In both experiments, significantly higher fluorescence intensities were observed in the flow-through hybridization unit compared to the passive assays. The CD fluidic scheme was also adapted to the immobilization of

  4. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals


    ALEPEE Nathalie; VIEGAS BARROSO JOAO FILIPE; De Smedt, Ann; Wever, Bart De; Hibatallah, Jalila; Klaric, Martina; MEWES Karsten R.; Millet, Marion; Pfannenbecker, Uwe; Tailhardat, Magalie; TEMPLIER Marie; McNamee, Pauline


    A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLCsp...

  5. Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

    Directory of Open Access Journals (Sweden)

    Kramer Marianne F


    Full Text Available Abstract An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.

  6. Sensitivity and Selectivity on Aptamer-Based Assay: The Determination of Tetracycline Residue in Bovine Milk

    Directory of Open Access Journals (Sweden)

    Sohee Jeong


    Full Text Available A competitive enzyme-linked aptamer assay (ELAA to detect tetracycline in milk was performed by using two different aptamers individually; one is 76 mer-DNA aptamer and the other is 57 mer-RNA aptamer. The best optimum condition was obtained without monovalent ion, Na+ and also by adding no Mg2+ ion in the assay buffer, along with RT incubation. The optimized ELAA showed a good sensitivity (LOD of 2.10 × 10−8 M with a wide dynamic range (3.16 × 10−8 M ~ 3.16 × 10−4 M. In addition, the average R.S.D. across all data points of the curve was less than 2.5% with good recoveries (~101.8% from the milk media. Thus, this method provides a good tool to monitor tetracycline in milk from MRLs’ point of view. However, this ELAA method was not superior to the ELISA method in terms of specificity. This paper describes that it does not always give better sensitivity and specificity in assays even though aptamers have several advantages over antibodies and have been known to be good binders for binding assays.

  7. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae (United States)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  8. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.


    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two...

  9. Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-e; WANG Cheng-ke; LIU Dian-jun; WANG Zhen-xin


    The interactions of antibiotic with living cells were studied by iectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.

  10. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate. (United States)

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred


    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

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


    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.

  12. Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. (United States)

    Ai, L; Dong, S J; Zhang, W Y; Elsheikha, H M; Mahmmod, Y S; Lin, R Q; Yuan, Z G; Shi, Y L; Huang, W Y; Zhu, X Q


    Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.

  13. 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.

  14. Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes. (United States)

    Montón, Helena; Medina-Sánchez, Mariana; Soler, Joan Antoni; Chałupniak, Andrzej; Nogués, Carme; Merkoçi, Arben


    Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

  15. Evaluation of a new serological test for syphilis based on chemiluminescence assay in a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Aseem K Tiwari


    Full Text Available Context: Syphilis is a transfusion transmissible infections and it is mandatory to do serological test for syphilis (STS on all donor blood samples. STS is usually based on detection of antibodies against the cardiolipin-lecithin antigen or against the Treponema-specific antigen. STS with good sensitivity and specificity helps enhance blood safety and consolidation of STS along with other transfusion transmittable infections such as human immunodeficiency virus, hepatitis-C virus, and hepatitis-B virus helps in reducing the errors and enhances efficiency. Aims: This study was designed to evaluate the performance of newly introduced VITROS ® syphilis Treponema pallidum agglutination (TPA assay based on enhanced chemiluminescence principle for its analytical performance for use as a STS on donor blood samples at a tertiary care health center in National Capital Region, India. Materials and Methods: A total of 108 random blood units collected from the donors (both voluntary and replacement donors and 28 known syphilis sero-reactive samples stored at −20°C, were used to evaluate the performance of VITROS ® syphilis TPA assay based on enhanced chemiluminescence assay on VITROS ® ECiQ immunodiagnostics system along with its analytical performance in terms of its sensitivity, precision, cross-reactivity and interference studies. Results: VITROS ® syphilis TPA showed 100% sensitivity and specificity with precision (20 days study of <10% co-efficient of variation. There was no cross-reactivity with other viral and auto-immune antibodies. No interference was observed from endogenous interfering substances like free hemoglobin or fats. Conclusions: Performance of the VITROS ® syphilis TPA assay meets the requirements for its use as STS in blood bank, thus allowing consolidation with other transfusion transmittable infections screening assay on chemiluminescence platform, which is highly valuable for optimizing workflow and efficiency.

  16. Genetic diversity in Silene sennenii Pau (Caryophyllaceae assayed through DNA-based techniques

    Directory of Open Access Journals (Sweden)

    Blanché, C.


    Full Text Available Silene sennenii is a narrow endemic species from the NE of the Iberian Peninsula. It is considered as EN (“Endangered” according to the IUCN criteria and is under legal protection in Catalonia. In the present work we report an assay using three different approaches for surveying the genetic diversity in this rare, endangered campion: analysis of chloroplast haplotypes, AFLPs and transferability of microsatellites previously developed for Silene latifolia. None of the nine chloroplast regions sequenced showed any variability. Five out of the 21 microsatellite loci tested were polymorphic although more loci are required in order to perform a robust population genetics study. Regarding the AFLP analysis, five out of the 26 pairs of primers tested exhibited moderate levels of variability and therefore they could be useful for further investigating the genetic structure of S. sennenii. Although preliminary, our results based on three different genetic markers are in agreement with the low values of genetic variation already reported for this species employing allozymes.Silene sennenii es una especie endémica, circunscrita a un área extremadamente reducida al NE de la Península Ibérica. Está catalogada como EN («En Peligro» según criterios UICN y se encuentra legalmente protegida en Cataluña. En el presente trabajo se expone el ensayo de tres aproximaciones diferentes al estudio de su diversidad genética: análisis de haplotipos cloroplásticos, AFLPs y transferibilidad de microsatélites diseñados previamente para Silene latifolia. Ninguna de las nueve regiones cloroplásticas secuenciadas ha presentado variabilidad. Se han obtenido cinco loci microsatélites polimórficos de los 21 ensayados, cantidad insuficiente para llevar a cabo un estudio robusto sobre genética poblacional. En cuanto a AFLPs, cinco combinaciones de cebadores de las 26 probadas han mostrado niveles moderados de variabilidad siendo así útiles para posteriores

  17. High throughput microwell spectrophotometric assay for olmesartan medoxomil in tablets based on its charge-transfer reaction with DDQ

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A.


    Full Text Available The study describes the development and validation of a new microwell-based spectrophotometric assay for determination of olmesartan medoxomil (OLM in tablets. The formation of a colored charge-transfer (CT complex between OLM as an n-electron donor and 2,3-dichloro- -5,6-dicyano-1,4-benzoquinone (DDQ as a p-electron acceptor was investigated, and employed as the basis for the development of the new assay. The proposed assay was conducted in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm with a microplate reader. Optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, a linear relationship with a good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 2-200 μg per well. The limits of detection and quantitation were 0.53 and 1.61 μg per well, respectively. No interference was observed from the excipients present in OLM tablets or from hydrochlorothiazide and amlodipine besylate that were co-formulated with OLM in some of its formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has a great practical value in the routine analysis of OLM in quality control laboratories, since it has a high throughput property and consumes low volumes of organic solvent. It thus offers a reduction in the exposure of analysts to the toxic effects of organic solvents, as well as a reduction in the cost of analysis.

  18. 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. (United States)

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


    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.

  19. The viability of perilabyrinthine osteocytes

    DEFF Research Database (Denmark)

    Bloch, Sune Land; Kristensen, Søren Lund; Sørensen, Mads Sølvsten


    Bone remodeling is highly inhibited around the inner ear space, most likely by the anti-resorptive action of the inner ear cytokine osteoprotegerin (OPG) entering perilabyrinthine bone through the lacuno-canalicular porosity (LCP). This extracellular signaling pathway depends on the viability of ...

  20. Scintigraphic Assessment of Myocardial Viability

    Energy Technology Data Exchange (ETDEWEB)

    Bom, Hee Seung [Chonnam University Hospital, Kwangju (Korea, Republic of)


    The identification of viable myocardium in patients with coronary artery disease and left ventricular dysfunction is an issue of increasing clinical relevance in the current era of myocardial revascularization. There are at least two forms of reversible myocardial dysfunction. Early reperfusion does not always lead to immediate functional improvement; rather, the return of contractility in tissue salvaged by reperfusion is delayed for hours, days or even weeks, a phenomenon that has been termed {sup s}tunned myocardium{sup .} Some patients with coronary artery disease show myocardial dysfunction ar rest which are associated with reduced perfusion, and which disappear after revascularization; this phenomenon has been termed {sup h}ibernating myocardium{sup .} Recently, cardiac imaging techniques that evaluate myocardial viability on the basis of perfusion contraction mismatch and inotropic reserve have gained substantial popularity and clinical success. This review focus on the application of {sup 201}T1 and {sup 99m}Tc-MIBI to address myocardial viability in patients with hibernating and stunned myocardium. It is clear that 4-hour redistribution images of {sup 201}T1 underestimate ischemia and overestimate scar. Delayed imaging and reinjection imaging have been developed for the assessment of viability. Among many protocols suggested, stress-redistribution-reinjection imaging gained most popularity. Although {sup 99m}Tc- MIBI could identify myocardial viability, {sup 201}T1 reinjection technique was regarded as superior to it. In conclusion, {sup 201}T1 stress, 4-hr rest redistribution, and reinjection imaging technique may be the most preferable method for evaluation of myocardial viability.

  1. Fluorescence microscopy methods for determining the viability of bacteria in association with mammalian cells. (United States)

    Johnson, M Brittany; Criss, Alison K


    Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

  2. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  3. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Xiao-yi ZHANG; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG


    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  4. A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry. (United States)

    de Boer, Arjen R; Bruyneel, Ben; Krabbe, Johannes G; Lingeman, Henk; Niessen, Wilfried M A; Irth, Hubertus


    The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.

  5. Development of fully automated determination of marker-specific immunoglobulin G (IgG) avidity based on the avidity competition assay format: application for Abbott Architect cytomegalovirus and Toxo IgG Avidity assays. (United States)

    Curdt, Ingo; Praast, Gerald; Sickinger, Eva; Schultess, Jan; Herold, Iris; Braun, Hans Bertram; Bernhardt, Stephanie; Maine, Gregory T; Smith, Darwin D; Hsu, Stephen; Christ, Heike M; Pucci, Dominick; Hausmann, Michael; Herzogenrath, Jörg


    Determination of the avidity of immunoglobulin G (IgG) directed against a specific marker has become an established diagnostic tool for identifying or excluding acute infections with pathogens. A novel assay format termed AVIcomp (avidity competition based on mass action) circumventing the conventional chaotropic format has been developed for determination of the avidity of marker-specific IgG in patient specimens. Its applications for cytomegalovirus (CMV) and Toxoplasma gondii are presented. Specific high-avidity IgG from the patient specimen is selectively blocked using a soluble antigen in a sample pretreatment reagent, and the amount of remaining specific low-avidity IgG is determined relative to that in an untreated control. The comparison of the conventional chaotropic format, represented by the Radim CMV IgG Avidity assay, and the newly developed AVIcomp method, as exemplified by the Architect CMV IgG Avidity assay, on blood drawn within 4 months after seroconversion revealed a sensitivity of 100% (97.3% by an alternative calculation) for the AVIcomp format versus 87.5% (75.7% by an alternative calculation) for the chaotropic avidity assay. The specificity on 312 CMV IgG reactive and CMV IgM nonreactive specimens from pregnant women was 100% for the AVIcomp assay and 99.7% for the conventional avidity assay. The Architect Toxo IgG Avidity assay showed an agreement of 97.2% with the bioMérieux Vidas Toxo IgG Avidity Assay employing chaotropic reagents. These performance data suggest that the AVIcomp format shows superior sensitivity and equivalent specificity for the determination of IgG avidity to assays based on the chaotropic method and that the AVIcomp format may also be applicable to other disease states.

  6. Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors. (United States)

    Doughan, Samer; Noor, M Omair; Han, Yi; Krull, Ulrich J


    Quantum dots (QDs) and upconverting nanoparticles (UCNPs) are luminescent nanoparticles (NPs) commonly used in bioassays and biosensors as resonance energy transfer (RET) donors. The narrow and tunable emissions of both QDs and UCNPs make them versatile RET donors that can be paired with a wide range of acceptors. Ratiometric signal processing that compares donor and acceptor emission in RET-based transduction offers improved precision, as it accounts for fluctuations in the absolute photoluminescence (PL) intensities of the donor and acceptor that can result from experimental and instrumental variations. Immobilizing NPs on a solid support avoids problems such as those that can arise with their aggregation in solution, and allows for facile layer-by-layer assembly of the interfacial chemistry. Paper is an attractive solid support for the development of point-of-care diagnostic assays given its ubiquity, low-cost, and intrinsic fluid transport by capillary action. Integration of nanomaterials with paper-based analytical devices (PADs) provides avenues to augment the analytical performance of PADs, given the unique optoelectronic properties of nanomaterials. Herein, we describe methodology for the development of PADs using QDs and UCNPs as RET donors for optical transduction of nucleic acid hybridization. Immobilization of green-emitting QDs (gQDs) on imidazole functionalized cellulose paper is described for use as RET donors with Cy3 molecular dye as acceptors for the detection of SMN1 gene fragment. We also describe the covalent immobilization of blue-emitting UCNPs on aldehyde modified cellulose paper for use as RET donors with orange-emitting QDs (oQDs) as acceptors for the detection of HPRT1 gene fragment. The data described herein is acquired using an epifluorescence microscope, and can also be collected using technology such as a typical electronic camera.

  7. Multidimensional GPCR profiling and screening using impedance-based label-free and real-time assay. (United States)

    Ke, Ning; Nguyen, Khanh; Irelan, Jeffery; Abassi, Yama A


    GPCRs constitute one of the most sought-after targets in drug discovery because they are associated with conditions ranging from cardiovascular diseases, autoimmune diseases, inflammation, cancer, and diseases of the nervous system. Moreover, they are one of the most amenable targets for drug discovery because they can be modulated by small molecules, peptides, proteins, and antibodies. Therefore it may not come as a surprise that close to 40 % of the drugs that are currently on the market are targeting GPCRs. It has become evident that GPCR signaling is highly complex and may involve multiple or a subset of pathways depending on the interaction of a GPCR with an agonist or antagonist. It is imperative that any functional screening for GPCR activity integrates this complexity. In this assay protocol, we describe how the xCELLigence RTCA HT impedance-based platform which can be used for functional cell-based GPCR assays can be utilized for GPCR screening.

  8. Paper-Based Digital Microfluidic Chip for Multiple Electrochemical Assay Operated by a Wireless Portable Control System

    DEFF Research Database (Denmark)

    Ruecha, Nipapan; Lee, Jumi; Chae, Heedo


    The printing and modular fabrication of a paper-based active microfluidic lab on a chip implemented with electrochemical sensors (ECSs) is developed and integrated on a portable electrical control system. The electrodes of a chip plate for active electrowetting actuation of digital drops and an ECS...... for multiple analysis assays are fabricated by affordable printing techniques. For enhanced sensitivity of the sensor, the working electrode is modified through the electrochemical method, namely by reducing graphene with voltammetry and coating gold nanoparticles by amperometry. Detachable sensor and absorber...... designed portable power supply and wireless control system, the active paper-based chip platform can be utilized as an advanced point-of-care device for multiple assays in digital microfluidics....

  9. A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Dyhring, Tino


    domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters...... (Z'˜0.7 and Z˜0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 µM to ~193 µM. Binding to the PICK1 PDZ domain...... was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 µM) in the FP assay, and was according...

  10. A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens. (United States)

    Xue, Wenfei; Peng, Jingfu; Yu, Xiaoli; Zhang, Shulin; Zhou, Boping; Jiang, Danqing; Chen, Jianbo; Ding, Bingbing; Zhu, Bin; Li, Yao


    The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.

  11. Colloidal Gold Probe-Based Immunochromatographic Strip Assay for the Rapid Detection of Microbial Transglutaminase in Frozen Surimi

    Directory of Open Access Journals (Sweden)

    Daming Fan


    Full Text Available Adding microbial transglutaminase (MTGase to frozen surimi to enable the surimi to be sold as a higher-grade product at a higher price defrauds surimi product manufacturers and undercuts legitimate industry prices. Therefore, it is important to develop an accurate method of detecting the presence of MTGase in surimi. In this study, an immunochromatographic strip assay with a colloidal gold antibody probe was successfully developed and used to rapidly and qualitatively detect MTGase in surimi samples. The results were obtained in less than 10 min. The limit for the qualitative detection of MTGase using the immunochromatographic strip assay was identified as 1.0 μg/mL. The results of the immunochromatographic strip analysis of frozen surimi samples were verified by comparison with the results of a sandwich enzyme-linked immunosorbent assay. The colloidal gold probe-based immunochromatographic strip assay was thus found to be a rapid, economical, and user friendly method of detecting MTGase in surimi.

  12. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization. (United States)

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J


    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  13. Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry. (United States)

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M


    The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.

  14. A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

    Directory of Open Access Journals (Sweden)

    Xian Zhang


    Full Text Available A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA was developed for rapid and sensitive detection of zearalenone (ZEN. The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904. The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283. We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

  15. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays. (United States)

    Hart, Traver; Zhao, Alice; Garg, Ankit; Bolusani, Swetha; Marcotte, Edward M


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFkappaB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon), and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  16. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Directory of Open Access Journals (Sweden)

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  17. Development of a Filtration-Based Bioluminescence Assay for Detection of Microorganisms in Tea Beverages. (United States)

    Shinozaki, Yohei; Igarashi, Toshinori; Harada, Yasuhiro


    The market for tea drinks as healthy beverages has been steadily expanding, and ready-to-drink beverages in polyethylene terephthalate bottles have been popular. To more rapidly and accurately test tea beverages bottled in polyethylene terephthalate for microbial contamination, a newly developed filtration device and a washing method with a commercial bioluminescence assay were combined to detect low numbers of bacterial spores, fungal conidia, and ascospores. Washing buffers were formulated with nonionic detergents from the Tween series. Commercially available tea beverages were used to evaluate the filtration capacity of the filtration device, the effect of washing buffers, and the performance of the assay. The assay was tested with serially diluted suspensions of colonies of two bacterial strains, spores of three Bacillus strains, conidia of five fungal strains, and ascospores of four fungal strains. The filtration device enabled filtration of a large sample volume (100 to 500 ml), and the washing buffer significantly decreased the background bioluminescence intensity of tea samples when compared with the no-washing method. Low numbers (1 to 10 CFU/100 ml) of the tested strains of bacteria were detected within 8 to 18 h of cultivation, and fungi were detected within 24 to 48 h. Furthermore, a whole bottle (500 ml) of mixed tea was filtered through the filtration device and microbes were detected. This method could be used for quality control of bottled beverages without preincubation.

  18. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots. (United States)

    Ullal, Anirudh J; Millington, David S; Bali, Deeksha S


    Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.

  19. Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics (United States)

    The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

  20. Assessment of Chemical Skin-Sensitizing Potency by an In Vitro Assay Based on Human Dendritic Cells


    Lambrechts, Nathalie; Vanheel, Hanne; Nelissen, Inge; Witters, Hilda; VAN DEN HEUVEL Rosette; Van Tendeloo, Viggo; Schoeters, Greet; HOOYBERGHS, Jef


    The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemica...

  1. Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells (United States)

    Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan


    We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining

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

    Energy Technology Data Exchange (ETDEWEB)

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


    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS

  3. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods (United States)


    inoculated together in 3 deer13. The results from our studies were presented at the Prion2006 meeting and elicited strong interest especially on the part...the ORIGEN technology (the instrument is produced by BioVeris, Gaithersburg, MD) to measure PrP in brain and plasma of scrapie infected animals. We...the ORIGEN assay. To improve the PrPc assay sensitivity an aliquot of the PK-treated plasma was mixed with the 3F4 immuno affinity resin. The resin

  4. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana


    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  5. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana


    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors. PMID:28287182

  6. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

    Directory of Open Access Journals (Sweden)

    Steven L Walker

    Full Text Available Reporter-based assays underlie many high-throughput screening (HTS platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv. ARQiv differs from current "high-content" (e.g., confocal imaging-based whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1 Rapid; achieving true HTS capacities (i.e., >50,000 units per day, 2 Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5, and 3 Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1 Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors, 2 Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3 Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

  7. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study (United States)

    Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; ,; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.


    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

  8. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays (United States)

    Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto


    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the dete