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Sample records for activity assay based

  1. ABAP: antibody-based assay for peptidylarginine deiminase activity.

    PubMed

    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

    2007-10-15

    Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) 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.

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

    SciTech Connect

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

    1984-01-01

    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.

  3. Cell based assays for anti-Plasmodium activity evaluation.

    PubMed

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

    2016-03-10

    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.

  4. FRET-based optical assay for monitoring riboswitch activation.

    PubMed

    Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Davidson, Molly; Narayanan, Latha; Trott, Sandra; Chushak, Yaroslav G; Stone, Morley O

    2009-05-11

    Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

  5. GTP-specific fab fragment-based GTPase activity assay.

    PubMed

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

    2015-03-17

    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.

  6. Activity-based assay for ricin-like toxins

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2007-02-06

    A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

  7. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays.

    PubMed

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

    2008-08-01

    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.

  8. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

    PubMed Central

    Vosyka, Oliver; Vinothkumar, Kutti R.; Wolf, Eliane V.; Brouwer, Arwin J.; Liskamp, Rob M. J.; Verhelst, Steven H. L.

    2013-01-01

    Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design. PMID:23359682

  9. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    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.

  10. A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology.

    PubMed

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

    2017-05-01

    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.

  11. A barium based coordination polymer for the activity assay of deoxyribonuclease I.

    PubMed

    Song, Chan; Wang, Guan-Yao; Wang, Ya-Ling; Kong, De-Ming; Wang, Yong-Jian; Li, Yue; Ruan, Wen-Juan

    2014-10-04

    A new coordination polymer which shows an unusual 2D inorganic connectivity was constructed. This compound exhibits distinct fluorescence quenching ability to the dye-labeled single-stranded DNA probes with different lengths, based on which an analytical method was developed for the activity assay of deoxyribonuclease I.

  12. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

    PubMed Central

    Ouellette, Steven B.; Noel, Brett M.; Parker, Laurie L.

    2016-01-01

    Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. PMID:27598410

  13. Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

    SciTech Connect

    Kuo, Chun-Chen; Chu, Chi-Yuan; Lin, Jing-Jer; Lo, Lee-Chiang

    2010-01-01

    Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

  14. A Malachite Green-Based Assay to Assess Glucan Phosphatase Activity

    PubMed Central

    Sherwood, Amanda R.; Paasch, Bradley C.; Worby, Carolyn A.; Gentry, Matthew S.

    2012-01-01

    With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that in contrast to a general phosphatase assay utilizing a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family. PMID:23201267

  15. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

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

    2016-04-15

    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 Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4

    PubMed Central

    Krishna, Sankar N.; Luan, Chi-Hao; Mishra, Rama K.; Xu, Li; Scheidt, Karl A.; Anderson, Wayne F.; Bergan, Raymond C.

    2013-01-01

    Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. PMID:24339940

  18. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    PubMed

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

    2015-05-15

    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.

  19. High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.

    PubMed

    Bin Zhang; Xiubin Gu; Uppalapati, Uma; Ashwell, Mark A; Leggett, David S; Li, Chiang J

    2008-07-01

    Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

  20. "Reagent-free" L-asparaginase activity assay based on CD spectroscopy and conductometry.

    PubMed

    Kudryashova, Elena V; Sukhoverkov, Kirill V

    2016-02-01

    A new method to determine the catalytic parameters of L-asparaginase using circular dichroism spectroscopy (CD spectroscopy) has been developed. The assay is based on the difference in CD signal between the substrate (L-asparagine) and the product (L-aspartic acid) of enzymatic reaction. CD spectroscopy, being a direct method, enables continuous measurement, and thus differentiates from multistage and laborious approach based on Nessler's method, and overcomes limitations of conjugated enzymatic reaction methods. In this work, we show robust measurements of L-asparaginase activity in conjugates with PEG-chitosan copolymers, which otherwise would not have been possible. The main limitation associated with the CD method is that the analysis should be performed at substrate saturation conditions (V max regime). For K M measurement, the conductometry method is suggested, which can serve as a complimentary method to CD spectroscopy. The activity assay based on CD spectroscopy and conductometry was successfully implicated to examine the catalytic parameters of L-asparaginase conjugates with chitosan and its derivatives, and for optimization of the molecular architecture and composition of such conjugates for improving biocatalytic properties of the enzyme in the physiological conditions. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not enable direct measurement with absorption or fluorescence spectroscopy. This may include a number of amino acid or glycoside-transforming enzymes.

  1. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm.

    PubMed

    Mariscal, Alberto; Lopez-Gigosos, Rosa M; Carnero-Varo, Manuel; Fernandez-Crehuet, Joaquin

    2009-03-01

    A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.

  2. Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

    PubMed Central

    Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

    2014-01-01

    Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

  3. Microplate assay for screening the antibacterial activity of Schiff bases derived from substituted benzopyran-4-one.

    PubMed

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

    2012-09-01

    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.

  4. Sensitive colorimetric assays for α-glucosidase activity and inhibitor screening based on unmodified gold nanoparticles.

    PubMed

    Chen, Hongxia; Zhang, Jiangjiang; Wu, Heng; Koh, Kwangnak; Yin, Yongmei

    2015-05-22

    A colorimetric sensor has been developed in this work to sensitively detect α-glucosidase activity and screen α-glucosidase inhibitors (AGIs) utilizing unmodified gold nanoparticles (AuNPs). The sensing strategy is based on triple-catalytic reaction triggered by α-glucosidase. In the presence of α-glucosidase, aggregation of AuNPs is prohibited due to the oxidation of cysteine to cystine in the system. However, with addition of AGIs, cysteine induced aggregation of AuNPs occurs. Thus, a new method for α-glucosidase activity detection and AGIs screening is developed by measuring the UV-vis absorption or visually distinguishing. A well linear relation is presented in a range of 0.0025-0.05 U mL(-1). The detection limit is found to be 0.001 U mL(-1) for α-glucosidase assay, which is one order of magnitude lower than other reports. The IC50 values of four kinds of inhibitors observed with this method are in accordance with other reports. The using of unmodified AuNPs in this work avoids the complicated and time-consuming modification procedure. This simple and efficient colorimetric method can also be extended to other enzymes assays.

  5. An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

    PubMed

    Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

    2017-02-01

    Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.

  6. Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity.

    PubMed

    Park, Ki Soo; Lee, Chang Yeol; Kang, Kyoung Suk; Park, Hyun Gyu

    2017-02-15

    We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target-triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe. Utilizing this design principle, we have successfully detected the activities of two model enzymes, exonuclease I and uracil DNA glycosylase with high sensitivity and selectivity. Since this strategy is composed of separated target recognition and signal transduction modules, it could be universally employed for the sensitive determination of numerous different target enzymes by simply redesigning the overhang sequence of detection probe, while keeping TaqMan probe-based signal transduction module as a universal signaling tool.

  7. Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

    PubMed Central

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

    2016-01-01

    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

  8. FRET-based protein-DNA binding assay for detection of active NF-kappa B

    SciTech Connect

    Giannetti, Ambra; Baldini, Francesco; Wabuyele, Musundi B; Vo Dinh, Tuan

    2006-01-01

    A novel method to detect the active form of NF-{kappa}B, a transcription factor regulating a battery of inflammatory genes and playing a fundamental role in the development of numerous pathological states, has been developed. In the present work, we used fluorescence resonance energy transfer (FRET) to study DNA-protein binding interaction taking place between double-strand (ds) DNA immobilized in a glass capillary wall and p50 proteins. For this purpose, we developed a regenerable FRET-based system comprising of a single-strand (ss) DNA with auto-complementary sequence that is end-labeled with Cy5 dye and is highly specific for p50 proteins. The proteins were labeled with a Black Hole Quencher (BHQ-3) to be used as FRET pair. The interaction of p50/p50 homodimer active form with its DNA binding site was demonstrated by both electrophoretic mobility shift assays and FRET studies. These preliminary results demonstrated the feasibility of the FRET-based DNA technique to detect the active form of NF-{kappa}B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.

  9. Artifactual prolongation of activated partial thromboplastin time with PEGylated compounds in silica-based assays.

    PubMed

    Murphy, Bethanne; Parrula, Maria C M; Perigard, Christopher J; Li, Jinze

    2014-12-01

    Conjugation of polyethylene glycol (PEG) to another molecule prolongs its half-life in the body, but has a potential to artifactually increase the activated partial thromboplastin time (aPTT) as measured with certain assays. Studies conducted in-house at Bristol-Myers Squibb using the STA-PTT Automate 5 assay, the routine assay used to measure aPTT, demonstrated prolongation of aPTT in plasma samples spiked in vitro with 40-kDa branched PEG (PEG40) conjugated compounds or PEG40 alone, but not in samples spiked with vehicle or non-PEGylated compound, suggesting that the interference is because of the PEG40 moiety. To investigate the cause of this phenomenon, cynomolgus monkey and rat plasma samples were spiked with different concentrations of PEG40 and the aPTT was measured using different proprietary assays. With one exception, prolongation of aPTT was observed with all assays containing silica as the contact activator. No changes in aPTT were noted in assays using kaolin as a contact activator. The findings indicated that the observed prolongation of aPTT is largely because of interference of PEG40 with the silica, but other features of the reagent mixture may also influence aPTT times.

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

    PubMed

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

    2014-06-15

    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. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    PubMed

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    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.

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

    PubMed Central

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

    2008-01-01

    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This “soft” immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is 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 β-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-β-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced γ-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. PMID:18319341

  13. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

    SciTech Connect

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F.

    2016-09-15

    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

  14. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    PubMed

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 10(5) times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.

  15. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    PubMed

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

    2013-01-01

    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.

  16. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    PubMed

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

  17. An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays.

    PubMed

    Qureshi, Anjum; Roci, Irena; Gurbuz, Yasar; Niazi, Javed H

    2012-04-15

    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.

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

    SciTech Connect

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

    2008-03-10

    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) enzymatic (Nimzyme

  19. Cell-based assays to support the profiling of small molecules with histone methyltransferase and demethylase modulatory activity

    PubMed Central

    Martinez, Natalia J.; Simeonov, Anton

    2015-01-01

    Histone methylation is a prevalent and dynamic chromatin modification, executed by the action of histone methyltransferases (HMTs) and demethylases (HDMs). Aberrant activity of many of these enzymes is associated with human disease, hence, there is a growing interest in identifying corresponding small molecule inhibitors with therapeutic potential. To date, most of the technologies supporting the identification of these inhibitors constitute in vitro biochemical assays which, although robust and sensitive, do not study HMTs and HDMs in their native cellular state nor provide information of inhibitor’s cell permeability and toxicity. The evident need for complementary cellular approaches has recently propelled the development of cell-based assays that enable screening of HMT and HDM enzymes in a more relevant environment. Here, we highlight current cellular methodologies for HMT and HDM drug discovery support. We anticipate that implementation of these cell-based assays will positively impact the discovery of pharmacologically potent HMT and HDM inhibitors. PMID:26723887

  20. Assay of FAAH Activity.

    PubMed

    Bari, Monica; Feole, Monica; Maccarrone, Mauro

    2016-01-01

    Fatty acid amide hydrolase (FAAH) is an intracellular enzyme responsible for the hydrolysis of endogenous anandamide (AEA), a reaction that terminates the biological effects of this lipid mediator. The final products of this reaction are arachidonic acid and ethanolamine. In the method described herein, FAAH activity is measured through the use of a radioactive substrate by quantification of reaction products, that is, [(14)C]-ethanolamine from [(14)C-ethanolamine]-AEA.

  1. Fluorescence-based blood coagulation assay device for measuring activated partial thromboplastin time.

    PubMed

    Dudek, Magdalena M; Kent, Nigel; Gustafsson, Kerstin M; Lindahl, Tomas L; Killard, Anthony J

    2011-01-01

    The measurement of blood clotting time is important in a range of clinical applications such as assessing coagulation disorders and controlling the effect of various anticoagulant drug therapies. Clotting time tests essentially measure the onset of clot formation which results from the formation of fibrin fibers in the blood sample. However, such assays are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. This work describes a clotting time measurement assay which uses a fluorescent probe to very precisely detect the onset of fibrin clot formation. It uses a microstructured surface which enhances the formation of multiple localized clot loci and which results in the abrupt redistribution of the fluorescent label at the onset of clot formation in both whole blood and plasma. This methodology was applied to the development of an activated partial thromboplastin time (aPTT) test in a lateral flow microfluidic platform and used to monitor the effect of heparin dosage where it showed linearity from 0 to 2 U/mL in spiked plasma samples (R(2)=0.996, n = 3), correlation against gold standard coagulometry of 0.9986, and correlation against standard hospital aPTT in 32 patient samples of 0.78.

  2. Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

    PubMed

    Gilmore, Marcella A; Williams, Dudley; Okawa, Yumiko; Holguin, Bret; James, Nicholas G; Ross, Justin A; Roger Aoki, K; Jameson, David M; Steward, Lance E

    2011-06-01

    The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

  3. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  4. Development of a high-throughput rubella virus infectivity assay based on viral activation of caspases.

    PubMed

    Bose, Pulkit S; Naspinski, Jennifer; Kartha, Girish; Bennett, Philip S; Wang, Christopher J; Haynes, John I; Benetti, Luca

    2010-08-01

    Rubella virus (RV, German measles) is a teratogenic agent that can lead to serious congenital defects after maternal infection during early pregnancy. Currently, the disease can be prevented effectively by available live attenuated vaccines. An important requisite for the manufacture and release of a safe and potent live virus vaccine is the measurement of the vaccine titer (potency), to ensure the correct dose and efficacy of the vaccine. One historical method for measuring potency is the endpoint dilution TCID(50) assay. Traditionally, RV TCID(50) titers are calculated after visual inspection of cells for presence of cytopathic effect (CPE). Such visual scoring is tedious and labor intensive. The development of a new TCID(50) readout method, based on a fluorescent molecular marker of RV-induced apoptosis, is described in this report. Further, in order to calculate TCID(50) potency a novel mathematical model was established to convert the numerical fluorescence measurements into categorical data. Finally, the assay parameters such as signal-to-noise ratio, robustness, variability and bias were optimized. This new readout method demonstrated strong concordance with the standard manual scoring of CPE, and therefore can provide a practical, objective and higher-throughput alternative to the traditional TCID(50) readout used for calculating titers of rubella virus.

  5. Highly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates.

    PubMed

    Sueyoshi, Kenji; Nogawa, Yuto; Sugawara, Kasumi; Endo, Tatsuro; Hisamoto, Hideaki

    2015-01-01

    In this study, a simple and highly sensitive enzyme activity assay based on reagent-release capillary-isoelectric focusing is described. Reagent-release capillaries containing a fluorescent substrate, which produces fluorescent products possessing an isoelectric point after reaction with enzymes, provides a simple procedure. This is because it allows to spontaneously inject a sample solution into the capillary by capillary action, mixing reagents, and subsequently concentrating the fluorescent products based on isoelectric focusing. Fluorescent rhodamine 110 and its monoamide derivative, which were generated as a final product and an intermediate, respectively, were then focused and separated by reagent-release capillary-isoelectric focusing. After 30 min of enzyme reactions, two focused fluorescent bands were clearly isolated along the prepared capillaries. Employing the focused band of rhodamine 110 monoamide allowed for highly sensitive detection of enzyme activity in the 10 pg mL(-1) order, while that of the conventional assay using a microplate was in the ng mL(-1) order. Furthermore, arraying reagent-release capillaries of different substrates on a chip allowed for simultaneous multi-assay of enzyme activity with good sensitivity in the pg mL(-1) order for each protein.

  6. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    PubMed

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.

  7. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    PubMed Central

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-01-01

    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

  8. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    NASA Astrophysics Data System (ADS)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    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.

  9. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna.

    PubMed

    Ørsted, Michael; Roslev, Peter

    2015-08-01

    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, the authors 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 or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2 Cr2 O7 or the herbicide formulation Roundup®. Toxicant-induced changes in hydrolytic enzyme activity were compared with changes in mobility (International Organization for Standardization standard 6341). The results showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna and the fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup resulted in loss of whole body enzyme activity and release of cell constituents, including enzymes and DNA. Roundup caused comparable inhibition of mobility and alkaline phosphatase activity with median effective concentration values at 20 °C of 8.7 mg active ingredient (a.i.)/L to 11.7 mg a.i./L. Inhibition of alkaline phosphatase activity by Roundup was lowest at 14 °C and greater at 20 °C and 26 °C. The results suggest 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.

  10. Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator.

    PubMed

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

    2011-07-13

    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.

  11. Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay.

    PubMed

    Cleenewerck, Matthias; Grootaert, Mandy O J; Gladysz, Rafaela; Adriaenssens, Yves; Roelandt, Ria; Joossens, Jurgen; Lambeir, Anne-Marie; De Meyer, Guido R Y; Declercq, Wim; Augustyns, Koen; Martinet, Wim; Van der Veken, Pieter

    2016-11-10

    Atg4B is a cysteine hydrolase that plays a key role in autophagy. Although it has been proposed as an attractive drug target, inhibitor discovery has proven highly challenging. The absence of a standardized, easily implementable enzyme activity/inhibition assay for Atg4B most likely contributes to this situation. Therefore, three different assay types for Atg4B activity/inhibition quantification were first compared: (1) an approach using fluorogenic Atg4B-substrates, (2) an in-gel densitometric quantification assay and (3) a thermal shift protocol. The gel-based approach showed the most promising results and was validated for screening of potential Atg4B inhibitors. A set of 8 literature inhibitors was included. Remarkably, in our hands only 2 literature references were found to have measurable Atg4B affinity. Furthermore, a fragment library (n = 182) was tested for Atg4B inhibition. One library member showed inhibition at high micromolar concentration and was found fit for further, fragment-based inhibitor design.

  12. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    PubMed

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  13. Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands.

    PubMed

    Lomin, Sergey N; Krivosheev, Dmitry M; Steklov, Mikhail Yu; Arkhipov, Dmitry V; Osolodkin, Dmitry I; Schmülling, Thomas; Romanov, Georgy A

    2015-04-01

    Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents β6-β7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.

  14. A peptide array-based serological protein kinase A activity assay and its application in cancer diagnosis.

    PubMed

    Kong, Deok-Hoon; Jung, Se-Hui; Jeon, Hye-Yoon; Kim, Woo-Jin; Kim, Young-Myeong; Ha, Kwon-Soo

    2015-10-07

    Protein kinase A (PKA) plays a crucial role in several biological processes; however, there is no assay with sufficient sensitivity and specificity to determine serological PKA (sPKA) activity. Here we present an on-chip activity assay that employs cysteine-modified kemptide arrays to determine specific sPKA activity in human sera that eliminates the potential contributions of other kinases with a protein kinase peptide inhibitor. The sensitivity of the on-chip sPKA activity assay was greatly enhanced by Triton X-100, with a 0.01 U mL(-1) detection limit. sPKA activity was determined by subtracting nonspecific sPK activity from total sPK activity. Our assay provided greater sensitivity and specificity and more accurate area under the curve values for gastric cancer compared to the total sPK activity assay. sPKA activities in human sera from patients with hepatic (n = 30), gastric (n = 30), lung (n = 30), and colorectal (n = 30) cancers were significantly higher than those in controls (n = 30, p < 10(-4)), but no significant difference in sPKA activities between normal and inflammation groups was observed. These results demonstrate that the on-chip assay accurately measures sPKA activity in human sera and that the sPKA activity may be a potential biomarker for cancer diagnosis.

  15. A fluorometric assay for acetylcholinesterase activity and inhibitor detection based on DNA-templated copper/silver nanoclusters.

    PubMed

    Li, Wenhua; Li, Wang; Hu, Yufang; Xia, Yalin; Shen, Qinpeng; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2013-09-15

    A novel label-free, rapid, cost-effective, and highly sensitive fluorometric sensor has been constructed for the detection of acetylcholinesterase (AChE) activity and its inhibitor based on the fluorescence quenching of DNA-templated copper/silver nanoclusters (DNA-Cu/AgNCs). In this assay, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine which induces fluorescence quenching of DNA-Cu/AgNCs. The AChE activity could be detected as low as 0.05mU/mL and with a linear range from 0.05 to 2.0mU/mL. This assay offers a very convenient "mix and detect" approach for AChE activity. On the other hand, tacrine and organophosphorus pesticides (OPPs) were employed to inhibit the hydrolysis of ATCh, which could eliminate the fluorescence quenching of DNA-Cu/AgNCs. The IC50 of tacrine and methamidophos were estimated to be 16.9nM and 0.075mg/L, respectively. This method was also used to detect spiked OPPs in agricultural products successfully. The present work may expand the use of DNA-Cu/AgNCs to the field of enzyme sensors.

  16. Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates.

    PubMed

    Miners, James Scott; Kehoe, Patrick Gavin; Love, Seth

    2008-03-30

    Internally quenched fluorogenic substrates are commonly used for measuring enzyme activity in biological samples and allow high sensitivity and continuous real-time measurement that is well suited for high throughput analysis. We describe the development and optimisation of an immunocapture-based assay that uses the fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)) and allows the specific measurement of insulin-degrading enzyme (IDE) activity in brain tissue homogenates. This fluorogenic substrate can be cleaved by a number of enzymes including neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1) and angiotensin-converting enzyme (ACE), as well as IDE, and we have previously shown that discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. We tested a panel of IDE antibodies to isolate and capture IDE from brain tissue homogenates and found that immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive (linear at 156-2500ng/ml) and specific measurement of IDE activity and negligible cross-reactivity with NEP, ACE or ECE-1. This assay should allow the measurement of IDE enzyme levels in a variety of biological tissues and may be useful in study of diseases such as Alzheimer's disease and insulin-dependent diabetes.

  17. SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase.

    PubMed

    Kokkula, Chakradhar; Palanisamy, Navaneethan; Ericstam, Malin; Lennerstrand, Johan

    2016-10-01

    There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.

  18. A multiplex assay based on encoded microbeads conjugated to DNA NanoBeacons to monitor base excision repair activities by flow cytometry.

    PubMed

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier

    2014-08-15

    We reported here a new assay to detect base excision repair activities from purified enzymes, as well as in cell-free extracts. The multiplex format rests upon the encoding of magnetic beads with the fluorophore Alexa 488, thanks to a fast and original procedure. Fluorescently encoded microbeads are subsequently functionalized by lesion-containing DNA NanoBeacons labeled with the fluorophore/quencher pair Cyanine 5/BHQ2. Probes cleavage, induced by targeted enzymes leads to Cyanine 5 signal enhancement, which is finally quantified by flow cytometry. The multiplex assay was applied to the detection of restriction enzymes activities as well as base excision repair processes. Each test requires only 500fmol of DNA substrate, which constitutes great sensitivity compared to other BER functional assays. The present biosensor is able to detect both uracil DNA N-glycosylase (UNG) and AP-endonuclease 1 (APE1) within few nanograms of nuclear extract. Additionally, we demonstrated that the corresponding assay has potential application in DNA repair inhibitor search. Finally, the current multiplexed tool shows several advantages in comparison to other functional BER assays with no need of electrophoretic separation, straightforward, easy and reproducible functionalization of encoded microbeads and a high stability of DNA probes in cell-free extracts.

  19. In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning. They function both as toxins that cause gastroenteritis after ingestion and as superantigens that non-specifically activate large numbers of T cells. Monkey or kitten bioassays were historically developed for analysis of SE act...

  20. Sensitive fluorimetric assays for α-glucosidase activity and inhibitor screening based on β-cyclodextrin-coated quantum dots.

    PubMed

    Liu, Si-Yao; Wang, Huan; He, Tian; Qi, Liang; Zhang, Zhi-Qi

    2016-02-01

    A fluorescence method was established for a α-glucosidase activity assay and inhibitor screening based on β-cyclodextrin-coated quantum dots. p-Nitrophenol, the hydrolysis product of the α-glucosidase reaction, could quench the fluorescence of β-cyclodextrin-coated quantum dots via an electron transfer process, leading to fluorescence turn-off, whereas the fluorescence of the system turned on in the presence of α-glucosidase inhibitors. Taking advantage of the excellent properties of quantum dots, this method provided a very simple, rapid and sensitive screening method for α-glucosidase inhibitors. Two α-glucosidase inhibitors, 2,4,6-tribromophenol and acarbose, were used to evaluate the feasibility of this screening model, and IC50 values of 24 μM and 0.55 mM were obtained respectively, which were lower than those previously reported. The method may have potential application in screening α-glucosidase inhibitors.

  1. An Activity-Dependent Assay for Ricin and Related RNA N-Glycosidases Based on Electrochemiluminescence

    DTIC Science & Technology

    2006-01-01

    orders of magnitude. Activities were detected with other adenine-specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin...specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin, and abrin II. The substrate that provided the greatest sensitivity...sequence in the hairpin loop (d, 2-deoxyribosyl moiety; Table 1). (A) Ricin (RT, 5 ng/ml; white bars) vs Ricinus communis agglutinin (RCA, 25 ng/ml; gray

  2. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins

    DTIC Science & Technology

    2016-04-01

    edema . The US Army Research Laboratory (ARL) is invested in developing technology to limit the primary effect of concussion by reducing the initial...of secondary injury cascades after a concussive event: fever and edema . Based on official data (US Department of Defense [DOD] 2016), more than 80

  3. SNPs altering ammonium transport activity of human Rhesus factors characterized by a yeast-based functional assay.

    PubMed

    Deschuyteneer, Aude; Boeckstaens, Mélanie; De Mees, Christelle; Van Vooren, Pascale; Wintjens, René; Marini, Anna Maria

    2013-01-01

    Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG(R202C) may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.

  4. SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

    PubMed Central

    Deschuyteneer, Aude; Boeckstaens, Mélanie; De Mees, Christelle; Van Vooren, Pascale; Wintjens, René; Marini, Anna Maria

    2013-01-01

    Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants. PMID:23967154

  5. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  6. Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

    PubMed Central

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J.; de Vries, Antoine A. F.

    2014-01-01

    Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS−) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS− both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS− while at low acceptor-to-donor cell ratios FLPeNLS− was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for

  7. Development of a lentivirus vector-based assay for non-destructive monitoring of cell fusion activity.

    PubMed

    Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J; de Vries, Antoine A F

    2014-01-01

    Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS-) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying

  8. Assessment of SYBR green I dye-based fluorescence assay for screening antimalarial activity of cationic peptides and DNA intercalating agents.

    PubMed

    Bhatia, Rakesh; Gautam, Ankur; Gautam, Shailendra K; Mehta, Divya; Kumar, Vinod; Raghava, Gajendra P S; Varshney, Grish C

    2015-05-01

    The SYBR green I (SG) dye-based fluorescence assay for screening antimalarial compounds is based on direct quantitation of parasite DNA. We show that DNA-interacting cationic cell-penetrating peptides (CPPs) and intercalating agents compete with SG dye to bind to DNA. Therefore, readouts of this assay, unlike those of the [(3)H]hypoxanthine incorporation assay, for the antimalarial activity of the above DNA binding agents may be erroneous. In the case of CPPs, false readouts can be improved by the removal of excess peptides.

  9. Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

    PubMed Central

    Sykes, Melissa L.; Avery, Vicky M.

    2015-01-01

    We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed. PMID:27120069

  10. Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes.

    PubMed

    Sykes, Melissa L; Avery, Vicky M

    2015-12-01

    We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed.

  11. Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity.

    PubMed

    Wang, Shasha; Chen, Zhaopeng; Choo, Jaebum; Chen, Lingxin

    2016-02-01

    A naked-eye sensitive ELISA-like assay was developed based on gold-enhanced peroxidase-like activity of gold nanoparticles (AuNPs). Using human IgG (H-IgG) as an analytical model, goat anti-human IgG antibody (anti-IgG) adsorbed on microtiter plate and AuNPs-labeled anti-IgG acted as capture antibody and detection antibody, respectively. Because the surfaces of AuNPs were blocked by protein molecules, the peroxidase-like activity of AuNPs was almost inhibited, evaluated by the catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB), which could produce a bright blue color in the presence of H2O2. Fortunately, the catalytic ability of AuNPs was dramatically increased by the deposition of gold due to the formation of a new gold shell on immunogold. Under optimal reaction conditions, the colorimetric immunoassay presented a good linear relationship in the range of 0.7-100 ng/mL and the limit of detection (LOD) of 0.3 ng/mL calculated by 3σ/S for UV-vis detection, and obtained LOD of 5 ng/mL for naked-eye detection. The obtained results were competitive with conventional sandwich ELISA with the LOD of 1.6 ng/mL. Furthermore, this developed colorimetric immunoassay was successfully applied to diluted human serum and fetal bovine serum samples, and predicted a broad prospect for the use of peroxidase-like activity involving nanomaterials in bioassay and diagnostics.

  12. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  13. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2012-03-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  14. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2011-11-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  15. Development of a selected reaction monitoring mass spectrometry-based assay to detect asparaginyl endopeptidase activity in biological fluids

    PubMed Central

    Walker, Michael J.; Gray, Oliver J.; Parker, Catriona; Holland, Mark; Williamson, Andrew J.K.; Pierce, Andrew; Unwin, Richard D.; Krishnan, Shekhar

    2016-01-01

    Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease. PMID:27683124

  16. A "turn-on" and label-free fluorescent assay for the rapid detection of exonuclease III activity based on Tb(3+)-induced G-quadruplex conjugates.

    PubMed

    Yang, WeiJuan; Ruan, YaJuan; Wu, WeiHua; Chen, PingPing; Xu, LiangJun; Fu, FengFu

    2014-07-01

    A "turn-on" and label-free fluorescent assay for the specific, rapid, and sensitive detection of 3' → 5' exonuclease III activity is reported in this study. The assay is based on the Tb(3+)-promoted G-quadruplex, which lead to the enhancement of Tb(3+) fluorescence due to the energy transfer from guanines. The proposed assay is highly simple, rapid, and cost-effective, and does not require sophisticated experimental techniques such as gel-based equipment or radioactive labels. It can be used for the rapid detection of exonuclease III activity with a detection limit of 0.8 U and a RSD (n = 6) <5 %. Notably, no dye was covalently conjugated to the DNA strands, which offers the advantages of low-cost and being interference-free.

  17. New cell-based assay indicates dependence of antioxidant biological activity on the origin of reactive oxygen species.

    PubMed

    Dimitrov, Martin D; Pesheva, Margarita G; Venkov, Pencho V

    2013-05-08

    The mobility of the Ty1 transposon in Saccharomyces cerevisiae was found to vary proportionally with the level of ROS generated in cells, which provides the possibility to determine antioxidant activity by changes in a cellular process instead of using chemical reactions. The study of propolis, royal jelly, and honey with the newly developed Ty1antiROS test reveals an inverse exponential dependence of antioxidant activity on increased concentrations. This dependence can be transformed to proportional by changing the source of ROS: instead of cell-produced to applied as hydrogen peroxide. The different test responses are not due to excess of added hydrogen peroxide, as evidenced by the exponential dependence found by usage of yap1Δ tester cells accumulating cell-generated ROS. Results indicate that the activity of antioxidants to oxidative radicals depends on the origin of ROS, and this activity is elevated for cell-generated ROS compared to ROS added as reagents in the assay.

  18. A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-based assay of chitinase activity.

    PubMed

    Price, Neil P J; Naumann, Todd A

    2011-04-01

    A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) assay is described for determination of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates and is readily achievable on a microliter scale (2μl of total volume containing 2μg of substrate and 1ng of protein). The speed and sensitivity of the assay make it potentially well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately 2min, as opposed to typically 30-40min for a single run with a high-performance liquid chromatography (HPLC)-based assay. By using the multiple-place MALDI MS targets, we estimate that 100 assays could be run in approximately 2-3h without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are visualized simultaneously in the TOF spectrum, this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay was used to monitor the purification and transgenic expression of plant class IV chitinases. By performing the assay with chitomer substrates and C-glycoside chitomer analogs, the enzyme mechanism of the class IV chitinases is described for the first time.

  19. Biochemical assays on plasminogen activators and hormones from kidney sources

    NASA Technical Reports Server (NTRS)

    Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

    1988-01-01

    Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

  20. Natural product extracts of the Canadian prairie plant, Thermopsis rhombifolia, have anti-cancer activity in phenotypic cell-based assays.

    PubMed

    Kernéis, Sophie; Swift, Lucy H; Lewis, Cody W; Bruyère, Céline; Oumata, Nassima; Colas, Pierre; Ruchaud, Sandrine; Bain, John; Golsteyn, Roy M

    2015-01-01

    Many plant species within the terrestrial ecological zones of Canada have not yet been investigated for anti-cancer activity. We examined the scientific literature describing the endemic flora from the prairie ecological zone and selected the species, Thermopsis rhombifolia, locally known as the buffalo bean, for investigation of its anti-cancer potential. We tested it in cell-based assays using phenotypic screens that feature some of the hallmarks of cancer. An ethanolic extract prepared from T. rhombifolia was cytotoxic to HT-29 (colon) and SH-SY5Y (brain) cancer cell lines, and showed little cytotoxicity to a normal human cell line (WI-38). In phenotypic assays, we identified activities in the extracts that target cell death, cell cycle and cell adhesion. These data highlight the anti-cancer potential of previously untested plants found in northern ecological zones and the feasibility of using pertinent phenotypic assays to examine the anti-cancer potential of natural product extracts.

  1. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  2. Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe.

    PubMed

    Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin

    2015-05-01

    An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

  3. A label-free near-infrared fluorescent assay for the determination of deoxyribonuclease I activity based on malachite green/G-quadruplexes.

    PubMed

    Sun, Shao-Kai; Wang, Bei-Bei; Yan, Xiu-Ping

    2013-05-07

    Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.

  4. Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay.

    PubMed

    Adcock, Robert S; Chu, Yong-Kyu; Golden, Jennifer E; Chung, Dong-Hoon

    2017-02-01

    Recent studies have clearly underscored the association between Zika virus (ZIKV) and severe neurological diseases such as microcephaly and Guillain-Barre syndrome. Given the historical complacency surrounding this virus, however, no significant antiviral screenings have been performed to specifically target ZIKV. As a result, there is an urgent need for a validated screening method and strategy that is focused on highlighting potential anti-ZIKV inhibitors that can be further advanced via rigorous validation and optimization. To address this critical gap, we sought to test whether a cell-based assay that measures protection from the ZIKV-induced cytopathic effect could serve as a high-throughput screen assay for discovering novel anti-ZIKV inhibitors. Employing this approach, we tested the anti-ZIKV activity of previously known broad-spectrum antiviral compounds and discovered several compounds (e.g., NITD008, SaliPhe, and CID 91632869) with anti-ZIKV activity. Interestingly, while GTP synthesis inhibitors (e.g., ribavirin or mycophenolic acid) were too toxic or showed no anti-ZIKV activity (EC50 > 50 μM), ZIKV was highly susceptible to pyrimidine synthesis inhibitors (e.g., brequinar) in the assay. We amended the assay into a high-throughput screen (HTS)-compatible 384-well format and then screened the NIH Clinical Compound Collection library, which includes a total of 727 compounds organized, using an 8-point dose response format with two Zika virus strains (MR766 and PRVABC59, a recent human isolate). The screen discovered 6-azauridine and finasteride as potential anti-ZIKV inhibitors with EC50 levels of 3.18 and 9.85 μM for MR766, respectively. We further characterized the anti-ZIKV activity of 6-azauridine and several pyrimidine synthesis inhibitors such as brequinar in various secondary assays including an antiviral spectrum test within flaviviruses and alphaviruses, Western blot (protein), real-time PCR (RNA), and plaque reduction assays (progeny

  5. Development of a photosensitive, high-throughput chip-based superoxide dismutase (SOD) assay to explore the radioprotective activity of herbal plants.

    PubMed

    Naoghare, Pravin K; Kwon, Ho Taik; Song, Joon Myong

    2009-08-15

    Appropriate pharmacological interventions and modalities are needed to protect humans against the deleterious effects of ionizing radiation. We disclose a rapid chip-based approach to elucidate the radioprotective/antioxidant potential of herbal plants using a photodiode array (PDA) microchip system. Red light absorption property of nitroblue tetrazolium (NBT) formazan was applied to chip-based superoxide dismutase (SOD) activity measurements of six herbal plant extracts in a high-throughput manner. SOD activities obtained via gel-based assays were in line with the data obtained through the chip-based assay and hence validated our approach. Compared to amifostine, all the tested herbal plant extracts, except apricot kernel, demonstrated greater radioprotective properties. Among the tested herbal extracts, pueraria root showed the highest antioxidant/radioprotective activity and can be considered a preferred radioprotector candidate. Low standard deviations and high statistical confidence obtained during the assay prove the sensitivity and consistency of this approach. The developed approach has several advantages (simplicity, rapidness and portability) over existing methods and can be applied to high-throughput screening of the radioprotective properties of various unexplored plants species.

  6. Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay

    PubMed Central

    Aggarwal, Megha; Sharma, Rajesh; Kumar, Pravindra; Parida, Manmohan; Tomar, Shailly

    2015-01-01

    Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV. PMID:26439734

  7. Rho family and Rap GTPase activation assays.

    PubMed

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  8. A simple microplate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method).

    PubMed

    Visvanathan, Rizliya; Jayathilake, Chathuni; Liyanage, Ruvini

    2016-11-15

    For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of α-amylase inhibitory activity using the glucose assay kit was developed. The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The test is done in microtitre plates with a total volume of 260μL and an assay time of 40min including the pre-incubation steps. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining α-amylase activity.

  9. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    PubMed

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  10. A non-isotopic assay for histone deacetylase activity.

    PubMed

    Hoffmann, K; Brosch, G; Loidl, P; Jung, M

    1999-05-01

    Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. To study the activity of HD and the effect of potential inhibitors in vitro so far only radio-active assays have existed. For the search of new inhibitors and for the use in HD identification and purification we established a simple, non-radioactive assay that allows screening of large numbers of compounds. The assay is based on an aminocoumarin derivative of an Omega-acetylated lysine as enzyme substrate.

  11. High-resolution colorimetric assay for rapid visual readout of phosphatase activity based on gold/silver core/shell nanorod.

    PubMed

    Gao, Zhuangqiang; Deng, Kaichao; Wang, Xu-Dong; Miró, Manuel; Tang, Dianping

    2014-10-22

    Nanostructure-based visual assay has been developed for determination of enzymatic activity, but most involve in poor visible color resolution and are not suitable for routine utilization. Herein, we designed a high-resolution colorimetric protocol based on gold/silver core/shell nanorod for visual readout of alkaline phosphatase (ALP) activity by using bare-eyes. The method relied on enzymatic reaction-assisted silver deposition on gold nanorod to generate significant color change, which was strongly dependent on ALP activity. Upon target ALP introduction into the substrate, the ascorbic acid 2-phosphate was hydrolyzed to form ascorbic acid, and then, the generated ascorbic acid reduced silver ion to metal silver and coated on the gold nanorod, thereby resulting in the blue shift of longitudinal localized surface plasmon resonance peak of gold nanorod accompanying a perceptible color change from red to orange to yellow to green to cyan to blue and to violet. Under optimal conditions, the designed method exhibited the wide linear range 5-100 mU mL(-1) ALP with a detection limit of 3.3 mU mL(-1). Moreover, it could be used for the semiquantitative detection of ALP from 20 to 500 mU mL(-1) by using the bare-eyes. The coefficients of variation for intra- and interassay were below 3.5% and 6.2%, respectively. Finally, this method was validated for the analysis of real-life serum samples, giving results matched well with those from the 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)-based standard method. In addition, the system could even be utilized in the enzyme-linked immunosorbent assay (ELISA) to detect IgG at picomol concentration. With the merits of simplification, low cost, user-friendliness, and sensitive readout, the gold nanorod-based colorimetric assay has the potential to be utilized by the public and opens a new horizon for bioassays.

  12. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    PubMed

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  13. Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells

    PubMed Central

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae. PMID:25658121

  14. 4-Hydroxy-N-propyl-1,8-naphthalimide esters: New fluorescence-based assay for analysing lipase and esterase activity.

    PubMed

    Nalder, Tim D; Ashton, Trent D; Pfeffer, Frederick M; Marshall, Susan N; Barrow, Colin J

    2016-01-01

    Research using 1,8-naphthalimide derivatives has expanded rapidly in recent years owing to their cell-permeable nature, ability to target certain cellular locations and fluorescent properties. Here we describe the synthesis of three new esters of 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) and the development of a simple and sensitive assay protocol to measure the activity of carboxylester hydrolases. The NAP fluorophore was esterified with short (butyrate), medium (octanoate) and long (palmitate) chain fatty acids. The esters were spectroscopically characterised and their properties investigated for their suitability as assay substrates. The esters were found to be relatively stable under the conditions of the assay and levels of spontaneous hydrolysis were negligible. Non-specific hydrolysis by proteins such as bovine serum albumin was also minimal. A simple and rapid assay methodology was developed and used to analyse a range of commercially available enzymes that included enzymes defined as lipases, esterases and phospholipases. Clear differences were observed between the enzyme classes with respect to the hydrolysis of the various chain length esters, with lipases preferentially hydrolysing the medium chain ester, whereas esterases reacted more favourably with the short ester. The assay was found to be highly sensitive with the fluorophore detectable to the low nM range. These esters provide alternate substrates from established coumarin-based fluorophores, possessing distinctly different excitation (447 nm) and emission (555 nm) optima. Absorbing at 440-450 nm also offers the flexibility of analysis by UV-visible spectrophotometry. This represents the first instance of a naphthalimide-derived compound being used to analyse these enzymes.

  15. A Rapid and Quantitative Recombinase Activity Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  16. An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.

    PubMed

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro.

  17. In silico exploratory study using structure-activity relationship models and metabolic information for prediction of mutagenicity based on the Ames test and rodent micronucleus assay.

    PubMed

    Kamath, P; Raitano, G; Fernández, A; Rallo, R; Benfenati, E

    2015-12-01

    The mutagenic potential of chemicals is a cause of growing concern, due to the possible impact on human health. In this paper we have developed a knowledge-based approach, combining information from structure-activity relationship (SAR) and metabolic triggers generated from the metabolic fate of chemicals in biological systems for prediction of mutagenicity in vitro based on the Ames test and in vivo based on the rodent micronucleus assay. In the first part of the work, a model was developed, which comprises newly generated SAR rules and a set of metabolic triggers. These SAR rules and metabolic triggers were further externally validated to predict mutagenicity in vitro, with metabolic triggers being used only to predict mutagenicity of chemicals, which were predicted unknown, by SARpy. Hence, this model has a higher accuracy than the SAR model, with an accuracy of 89% for the training set and 75% for the external validation set. Subsequently, the results of the second part of this work enlist a set of metabolic triggers for prediction of mutagenicity in vivo, based on the rodent micronucleus assay. Finally, the results of the third part enlist a list of metabolic triggers to find similarities and differences in the mutagenic response of chemicals in vitro and in vivo.

  18. An exploration of the estrogen receptor transcription activity of capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays.

    PubMed

    Li, Juan; Ma, Duo; Lin, Yuan; Fu, Jianjie; Zhang, Aiqian

    2014-06-16

    Capsaicin has been considered as an alternative template of dichlorodiphenyl trichloroethane (DDT) in antifouling paint. However, information regarding the estrogenic activity of capsaicin analogues is rather limited in comparison to that of DDT analogues and their metabolites. We here explore the ER transcription activity of selected capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays. Molecular simulation and the agonist/antagonist differential-docking screening identified 6-iodonordihydrocapsaicin (6-I-CPS) as a weak ERα agonist, while anti-estrogenicity was expected for N-arachidonoyldopamine, capsazepine, dihydrocapsaicin, trichostatin A, and capsaicin. On the contrary, the large volume of analogues, such as phorbol 12-phenylacetate 13-acetate 20-homovanillate and phorbol 12,13-dinonanoate 20-homovanillate, cannot fit well with the ER cavity. The result of MVLN assay was in accord with the in silico prediction. 6-I-CPS was demonstrated to induce luciferase gene expression, while the other analogues of relatively small molecular volume reduced luciferase gene expression in MVLN cells, both in the absence and presence of estradiol. This finding suggested that the ER transcription activity of capsaicin analogues is generated at least partly through the ERα-mediated pathway. Moreover, receptor polymorphism analysis indicated that capsaicin analogues may exhibit diverse species selectivity for human beings and marine species.

  19. Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

    2014-11-01

    Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

  20. Assessment of the aryl hydrocarbon receptor-mediated activities of polycyclic aromatic hydrocarbons in a human cell-based reporter gene assay.

    PubMed

    Vondráček, Jan; Pěnčíková, Kateřina; Neča, Jiří; Ciganek, Miroslav; Grycová, Aneta; Dvořák, Zdeněk; Machala, Miroslav

    2017-01-01

    Activation of the aryl hydrocarbon receptor (AhR)-mediated activity is one of key events in toxicity of polycyclic aromatic hydrocarbons (PAHs). Although various classes of AhR ligands may differentially activate human and rodent AhR, there is presently a lack of data on the human AhR-inducing relative potencies (REPs) of PAHs. Here, we focused on estimation of the AhR-mediated activities of a large set of environmental PAHs in human gene reporter AZ-AhR cell line, with an aim to develop the human AhR-based REP values with potential implications for risk assessment of PAHs. The previously identified weakly active PAHs mostly failed to activate the AhR in human cells. The order for REPs of individual PAHs in human cells largely corresponded with the available data from rodent-based experimental systems; nevertheless, we identified differences up to one order of magnitude in REP values of PAHs between human and rodent cells. Higher REP values were found in human cells for some important environmental contaminants or suspected carcinogens, such as indeno[1,2,3-cd]pyrene, benz[a]anthracene or benzo[b]fluoranthene, while lower REP values were determined for methyl-substituted PAHs. Our results also indicate that a different rate of metabolism for individual PAHs in human vs. rodent cells may affect estimation of REP values in human cell-based assay, and potentially alter toxicity of some compounds, such as benzofluoranthenes, in humans. We applied the AZ-AhR assay to evaluation of the AhR-mediated activity of complex mixtures of organic compounds associated with diesel exhaust particles, and we identified the polar compounds present in these mixtures as being particularly highly active in human cells, as compared with rodent cells. The present data suggest that differences may exist between the AhR-mediated potencies of PAHs in human and rodent cells, and that the AhR-mediated effects of polar PAH derivatives and metabolites in human cell models deserve further

  1. A fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon quantum dots through host-guest recognition.

    PubMed

    Tang, Cong; Qian, Zhaosheng; Huang, Yuanyuan; Xu, Jiamin; Ao, Hang; Zhao, Meizhi; Zhou, Jin; Chen, Jianrong; Feng, Hui

    2016-09-15

    A convenient, reliable and highly sensitive assay for alkaline phosphatase (ALP) activity in the real-time manner is developed based on β-cyclodextrin-modified carbon quantum dots (β-CD-CQDs) nanoprobe through specific host-guest recognition. Carbon quantum dots were first functionalized with 3-aminophenyl boronic acid to produce boronic acid-functionalized CQDs, and then further modified with hydropropyl β-cyclodextrins (β-CD) through B-O bonds to form β-CD-CQDs nanoprobe. p-Nitrophenol phosphate disodium salt is used as the substrate of ALP, and can hydrolyze to p-nitrophenol under the catalysis of ALP. The resulting p-nitrophenol can enter the cavity of β-CD moiety in the nanoprobe due to their specific host-guest recognition, where photoinduced electron transfer process between p-nitrophenol and CQDs takes place to efficiently quench the fluorescence of the probe. The correlation between quenched fluorescence and ALP level can be used to establish quantitative evaluation of ALP activity in a broad range from 3.4 to 100.0U/L with the detection limit of 0.9U/L. This assay shows a high sensitivity to ALP even in the presence of a very high concentration of glucose. This study demonstrates a good electron donor/acceptor pair, which can be used to design general detection strategy through PET process, and also broadens the application of host-guest recognition for enzymes detection in clinical practice.

  2. A miniaturized fibrinolytic assay for plasminogen activators

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

    1991-01-01

    This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

  3. Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing.

    PubMed

    He, Kaiyu; Li, Wang; Nie, Zhou; Huang, Yan; Liu, Zhuoliang; Nie, Lihua; Yao, Shouzhuo

    2012-03-26

    The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

  4. Development of a Novel NMR-based Rheb GTPase Assay and Molecular Characterization of TSC2 GAP Activity

    DTIC Science & Technology

    2010-05-01

    pH 8, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 10 mM imidazole, 10 mM b-mercaptoethanol, 0.1% Nonidet P - 40 , 0.1 mMGDP, 1 mM phenylmethylsulfonyl...p21. Biochemistry 29, 6058–6065 (1990). 40 . P . H. Seeburg, W. W. Colby, D. J. Capon, D. V. Goeddel, A. D. Levinson, Biological properties of human c...T I C L EG T P H Y D R O L Y S I SCharacterization of the Intrinsic and TSC2-GAP–Regulated GTPase Activity of Rheb by Real-Time NMR Christopher B

  5. A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

    PubMed

    Kim, Yong Hyun; Sathiyanarayanan, Ganesan; Kim, Hyun Joong; Bhatia, Shashi Kant; Seo, Hyung-Min; Kim, Jung-Ho; Song, Hun-Seok; Kim, Yun-Gon; Park, Kyungmoon; Yang, Yung-Hun

    2015-12-28

    A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

  6. Application of intact cell-based NFAT-β-lactamase reporter assay for Pasteurella multocida toxin-mediated activation of calcium signaling pathway

    PubMed Central

    Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A.

    2009-01-01

    Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C β1 (PLCβ1) signal transduction through its selective action on the alpha subunit of the Gq protein. Here, we describe the application of an NFAT-β-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCβ1-IP3-Ca2+ signaling pathway. Use of the NFAT-β-lactamase reporter assay with a cell-permeable fluorogenic substrate provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. This assay system was optimized for cell density, dose and time exposure of PMT stimulation. It is suited for quantitative characterization of PMT activity in mammalian cells and for use as a high-throughput screening method for PMT deletion and point mutants suitable for vaccine development. This method has application for diagnostic screening of clinical isolates of toxinogenic P. multocida. PMID:18190943

  7. A Cell-Based Assay to Assess Hemichannel Function

    PubMed Central

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

    2017-01-01

    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. PMID:28356896

  8. Sensitive electrochemical assay of alkaline phosphatase activity based on TdT-mediated hemin/G-quadruplex DNAzyme nanowires for signal amplification.

    PubMed

    Liu, Yunqing; Xiong, Erhu; Li, Xiaoyu; Li, Junjing; Zhang, Xiaohua; Chen, Jinhua

    2017-01-15

    Taking TdT-mediated hemin/G-quadruplex DNAzyme nanowires as NADH oxidase and HRP-mimicking DNAzyme, a novel DNA-based electrochemical method has been developed for sensitive and selective assay of alkaline phosphatase (AP) activity. The double-stranded DNA (dsDNA) probe consisted of thiol-functionalized DNA1 and 3'-phosphorylated DNA2, was immobilized on a gold nanoparticles (AuNPs) modified glassy carbon (GC) electrode. In the presence of AP, 3'-phosphoryl end of DNA2 was dephosphorylated. Terminal deoxynucletidyl transferase (TdT) catalyzed the sequential addition of deoxynucleotides (dTTPs) at 3'-OH end of DNA2 to extend DNA2 with a poly-T sequence. Then, G-rich DNA3 strand hybridized with the poly-T sequence of DNA2. Upon addition of hemin, the hemin/G-quadruplex DNAzyme was formed. In the presence of NADH, the hemin/G-quadruplex DNAzyme oxidased NADH to NAD(+), accompanied by the formation of H2O2 which was further catalyzed by hemin/G-quadruplex DNAzyme (served as a HRP-mimicking DNAzyme) with the thionine (Thi) as electron transfer mediator, leading to the amplified electrochemical signal. Under optimized conditions, the response peak current was linear with the concentration of AP in the range from 0.1UL(-1) to 5UL(-1) with the detection limit of 0.03UL(-1). Also, the developed biosensor possessed good selectivity, reproducibility and stability, and simple sensing structure, showing promising practical applications in AP activity assay.

  9. Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

    PubMed

    Miners, James Scott; Verbeek, Marcel M; Rikkert, Marcel Olde; Kehoe, Patrick Gavin; Love, Seth

    2008-01-30

    Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.

  10. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-08-21

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

  11. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  12. Paper-based assay of antioxidant activity using analyte-mediated on-paper nucleation of gold nanoparticles as colorimetric probes.

    PubMed

    Choleva, Tatiana G; Kappi, Foteini A; Giokas, Dimosthenis L; Vlessidis, Athanasios G

    2015-02-20

    With the increasing interest in the health benefits arising from the consumption of dietary products rich in antioxidants, there exists a clear demand for easy-to-use and cost-effective tests that can be used for the identification of the antioxidant power of food products. Paper-based analytical devices constitute a remarkable platform for such expedient and low-cost assays with minimal external resources but efforts in this direction are still scarce. In this work we introduce a new paper-based device in the form of a sensor patch that enables the determination of antioxidant activity through analyte-driven on-paper formation of gold nanoparticles. The principle of detection capitalizes, for the first time, on the on-paper nucleation of gold ions to its respective nanoparticles, upon reduction by antioxidant compounds present in an aqueous sample. The ensuing chromatic transitions, induced on the paper surface, are used as an optical "signature" of the antioxidant strength of the solution. The response of the paper-based sensor was evaluated against a large variety of antioxidant species and the respective dose response curves were constructed. On the basis of these data, the contribution of each species according to its chemical structure was elucidated. For the analysis of real samples, a concentration-dependent colorimetric response was established against Gallic acid equivalents over a linear range of 10 μM-1.0 mM, with detection limits at the low and ultra-low μM levels (i.e. <1.0 μM) and satisfactory precision (RSD=3.6-12.6%). The sensor has been tested for the assessment of antioxidant activity in real samples (teas and wines) and the results correlated well with commonly used antioxidant detection methods. Importantly, the sensor performed favorably for long periods of time when stored at moisture-free and low temperature conditions without losing its activity thus posing as an attractive alternative to the assessment of antioxidant activity without

  13. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  14. DEVELOPMENT OF CRASSPHAGE-BASED QPCR ASSAYS ...

    EPA Pesticide Factsheets

    A newly discovered bacteriophage, “crAssphage”, is predicted to be both highlyabundant and predominantly human-associated, both ideal characteristics for a human-specific fecal indicator. A total of 384 end-point PCR primers were designed along the length of the crAssphage genome, eliminating regions suspected to be hypervariable or react with other animal sources. The primer pairs were rigorously tested in three rounds of screening for specificity, geographic variability, limit of detection, and environmental water performance. The two best performing assays, crAss056 and crAss064, were adapted to a qPCR platform and exhibited a specificity of 98.0% and 98.9%, respectively. The markers’ abundance was compared with two bacterial based assays and were found at concentrations at or above the bacterial based assays in wastewater influent and impacted environmental waters. This poster will present the methodology of the novel marker development and the potential uses for this technology in maintaining sustainable waterways in the future. To inform the public.

  15. A new assay system for guinea pig interferon biological activity.

    PubMed

    Yamamoto, Toshiko; Jeevan, Amminikutty; Ohishi, Kazue; Nojima, Yasuhiro; Umemori, Kiyoko; Yamamoto, Saburo; McMurray, David N

    2002-07-01

    We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

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

    PubMed

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B

    2015-01-01

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

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

    PubMed Central

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare. PMID:26658480

  18. [Crude extraction and activity assay of CEL I].

    PubMed

    Han, Suo-Yi; Yang, Ma-Li; Gai, Jun-Yi; Yu, De-Yue

    2006-09-01

    CEL I, extracted from celery, is the first known eukaryotic nuclease that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. It is a key enzyme for TILLING research. Here we reported a crude extraction method and activity assay of CEL I. Incision at mismatches of single nucleotide suggested that CEL I can effectively detect DNA at G-->A base substitution and the result can be obtained from an ABI377 Sequencer. Therefore, the extracted enzyme can be used in TILLING.

  19. Mass-based readout for agglutination assays

    NASA Astrophysics Data System (ADS)

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

    2007-11-01

    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.

  20. 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.

    PubMed

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

    2015-05-22

    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.

  1. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    PubMed

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-03-04

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1(-/-) mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  2. A Highly Scalable Peptide-Based Assay System for Proteomics

    PubMed Central

    Kozlov, Igor A.; Thomsen, Elliot R.; Munchel, Sarah E.; Villegas, Patricia; Capek, Petr; Gower, Austin J.; K. Pond, Stephanie J.; Chudin, Eugene; Chee, Mark S.

    2012-01-01

    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. PMID:22701568

  3. A DNA-based assay for toxic chemicals in wastewater.

    PubMed

    Foreman, Amy L; Phillips, Leo; Kanellis, Vangelis G; Hammoudeh, Daoud; Naumann, Christoph; Wong, Henri; Chisari, Robert; Hibbert, D Brynn; Lee, Garry S H; Patra, Ronald; Julli, Moreno; Chapman, John; Cooke, A Roger; dos Remedios, Cristobal G

    2011-08-01

    Chemical toxicants, particularly metal ions, are a major contaminant in global waterways. Live-organism bioassays used to monitor chemical toxicants commonly involve measurements of activity or survival of a freshwater cladoceran (Ceriodaphnia dubia) or light emitted by the marine bacterium Vibrio fischeri, used in the commercial Microtox® bioassay. Here we describe a novel molecule-based assay system employing DNA as the chemical biosensor. Metals bind to DNA, causing structural changes that expel a bound (intercalated) fluorescent reporter dye. Analyses of test data using 48 wastewater samples potentially contaminated by metal ions show that the DNA-dye assay results correlate with those from C. dubia and Microtox bioassays. All three assays exhibit additive, antagonistic, and synergistic responses that cannot be predicted by knowing individual metal concentrations. Analyses of metals in these samples imply the presence of chemical toxicants other than metal ions. The DNA-dye assay is robust, has a 12-month shelf life, and is only slightly affected by sample pH in the range 4 to 9. The assay is completed in a matter of minutes, and its portability makes it well suited as a screening assay for use in the field. We conclude that the DNA-dye test is a surrogate bioassay suitable for screening chemical toxicity.

  4. Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

    PubMed Central

    Lopez-Gigosos, Rosa M.; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin

    2015-01-01

    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R2 = 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques. PMID:26276114

  5. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    PubMed

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  6. Electrochemical biosensor for protein kinase A activity assay based on gold nanoparticles-carbon nanospheres, phos-tag-biotin and β-galactosidase.

    PubMed

    Zhou, Yunlei; Yin, Huanshun; Li, Xue; Li, Zhi; Ai, Shiyun; Lin, Hai

    2016-12-15

    A sensitive and selective electrochemical biosensor was fabricated for protein kinase A (PKA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres (Au@C), the biocomposite of SiO2 and streptavidin (SiO2-SA), the composite of AuNPs and biotinylated β-galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p-aminophenol. After peptides were assembled on Au@C modified electrode surface, they were phosphorylated by PKA in the presence of ATP. Then, biotinylated Phos-tag was modified on electrode surface through the specific interaction between Phos-tag and phosphate group. Finally, SiO2-SA and AuNPs-B-Gal were captured through the specific interaction between biotin and streptavidin. Because the electrochemical response of p-aminophenol was directly related to PKA concentration, an innovative electrochemical assay could be realized for PKA detection. The detection limit was 0.014unit/mL. The developed method showed high detection sensitivity and selectivity. In addition, the fabricated biosensor can be also applied to detect PKA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results.

  7. RAB21 Activity Assay Using GST-fused APPL1

    PubMed Central

    Jean, Steve; Kiger, Amy A.

    2016-01-01

    The Rab family of small GTPases are essential regulators of membrane trafficking events. As with other small GTPase families, Rab GTPases cycle between an inactive GDP-bound state and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) promote Rab activation with the exchange of bound GDP for GTP, while GTPase-activating proteins (GAPs) regulate Rab inactivation with GTP hydrolysis. Numerous methods have been established to monitor the activation status of Rab GTPases. Of those, FRET-based methods are used to identify when and where a Rab GTPase is activated in cells. Unfortunately, the generation of such probes is complex, and only a limited number of Rabs have been probed this way. Biochemical purification of activated Rabs from cell or tissue extracts is easily achievable through the use of a known Rab effector domain to pull down a specific GTP-bound Rab form. Although this method is not ideal for detailed subcellular localization, it can offer temporal resolution of Rab activity. The identification of a growing number of specific effectors now allows tests for activation levels of many Rab GTPases in specific conditions. Here, we described an affinity purification approach using GST fused APPL1 (a known RAB21 effector) to test RAB21 activation in mammalian cells. This method was successfully used to assay changes in RAB21 activation status under nutrient rich versus starved conditions and to test the requirement of the MTMR13 RAB21 GEF in this process. PMID:28251173

  8. Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.

    PubMed

    Lorincz, M C; Parente, M K; Roederer, M; Nolan, G P; Diwu, Z; Martin, D I; Herzenberg, L A; Wolfe, J H

    1999-01-08

    Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

  9. Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging.

    PubMed

    Swain, Umakanta; Subba Rao, Kalluri

    2011-08-01

    Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as "comet" assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

  10. An assay for α 1,6-fucosyltransferase (FUT8) activity based on the HPLC separation of a reaction product with fluorescence detection.

    PubMed

    Ihara, Hideyuki; Tsukamoto, Hiroki; Taniguchi, Naoyuki; Ikeda, Yoshitaka

    2013-01-01

    N-Glycans with an α-fucose unit linked to the 6-position of the innermost GlcNAc are widely distributed among the animal kingdom, from worms and insects to human. This α1,6-linked fucosyl residue, frequently referred to as a core fucose, is formed via the action of an α1,6-fucosyltransferase, the mammalian ortholog which is systematically called FUT8. In mammals, it is well known that the extent of core-fucosylation in cellular and secreted glycoproteins varies, e.g., according to differentiation and carcinogenesis of the cells. This chapter describes a method for the sensitive and quantitative assay of FUT8 activity using a fluorescence-labeled oligosaccharyl asparagine derivative as the glycosyl acceptor substrate.

  11. The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems

    PubMed Central

    Fu, Huiying; Cheng, Hongqiang; Cao, Gang; Zhang, Xingde; Tu, Jue; Sun, Mingjiao; Mou, Xiaozhou; Shou, Qiyang; Ke, Yuehai

    2016-01-01

    Chinese herbs have long been used to treat allergic disease, but recently the development was greatly impeded by the lack of good methods to explore the mechanism of action. Here, we showed the effects of Chinese herb Radix Paeoniae alba were identified and characterized by a mast cell activation assay that involves electronic impedance readouts for dynamic monitoring of cellular responses to produce time-dependent cell responding profiles (TCRPs), and the anti-allergic activities were further confirmed with various conventional molecular and cell biology tools. We found Radix P. alba can dose-dependently inhibit TCPRs, and have anti-allergic function in vitro and in vivo. Radix P. alba suppressed mast cell degranulation not only inhibiting the translocation of granules to the plasma membrane, but also blocking membrane fusion and exocytosis; and that there may be other anti-allergic components in addition to paeoniflorin. Our results suggest that Radix P. alba regulated mast cell activation with multiple targets, and this approach is also suitable for discovering other mast cell degranulation-targeting Chinese herbs and their potential multi-target mechanisms. PMID:27195739

  12. A Rapid Fluorescence-based Assay for Soluble Methane Monooxgyenase

    SciTech Connect

    Miller, Amber Reese; Keener, William Kelvin; Roberto, Francisco Figueroa; Watwood, Maribeth E.

    2002-01-01

    A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be Km(app)=262 µM and Vmax(app)=821 nmol 7-hydroxycoumarin min–1 mg protein–1. The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.

  13. Enzyme activity assays within microstructured optical fibers enabled by automated alignment

    PubMed Central

    Warren-Smith, Stephen C.; Nie, Guiying; Schartner, Erik P.; Salamonsen, Lois A.; Monro, Tanya M.

    2012-01-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women’s health. PMID:23243579

  14. Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats.

    PubMed

    Halpin, Rachel E; Saunders, Rebecca S; Thompson, Beverly J; Rohde Newgent, Allison S; Amorim, Juliana; Melillo, Gabrielle N; DeClue, Amy E

    2016-05-01

    OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples. SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats. PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups. RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection. CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.

  15. A simple assay for measuring catalase activity: a visual approach.

    PubMed

    Iwase, Tadayuki; Tajima, Akiko; Sugimoto, Shinya; Okuda, Ken-ichi; Hironaka, Ippei; Kamata, Yuko; Takada, Koji; Mizunoe, Yoshimitsu

    2013-10-30

    In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20-300 units (U) (y = 0.3794x - 2.0909, r(2) = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli, catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli, and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells.

  16. Screening with a novel cell-based assay for TAZ activators identifies a compound that enhances myogenesis in C2C12 cells and facilitates muscle repair in a muscle injury model.

    PubMed

    Yang, Zeyu; Nakagawa, Kentaro; Sarkar, Aradhan; Maruyama, Junichi; Iwasa, Hiroaki; Bao, Yijun; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; Hata, Shoji; Nishina, Hiroshi; Abe, Shinya; Kitagawa, Masanobu; Hata, Yutaka

    2014-05-01

    The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy.

  17. Screening with a Novel Cell-Based Assay for TAZ Activators Identifies a Compound That Enhances Myogenesis in C2C12 Cells and Facilitates Muscle Repair in a Muscle Injury Model

    PubMed Central

    Yang, Zeyu; Nakagawa, Kentaro; Sarkar, Aradhan; Maruyama, Junichi; Iwasa, Hiroaki; Bao, Yijun; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; Hata, Shoji; Nishina, Hiroshi; Abe, Shinya; Kitagawa, Masanobu

    2014-01-01

    The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy. PMID:24550007

  18. Multiplexed assays by high-content imaging for assessment of GPCR activity.

    PubMed

    Ross, D A; Lee, S; Reiser, V; Xue, J; Alves, K; Vaidya, S; Kreamer, A; Mull, R; Hudak, E; Hare, T; Detmers, P A; Lingham, R; Ferrer, M; Strulovici, B; Santini, F

    2008-07-01

    G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor alone has limitations: relying on a single mechanistic step of beta-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multiplexed the Transfluor assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization.

  19. Diosgenin does not express estrogenic activity: a uterotrophic assay.

    PubMed

    Medigović, Ivana; Ristić, Nataša; Živanović, Jasmina; Šošić-Jurjević, Branka; Filipović, Branko; Milošević, Verica; Nestorović, Nataša

    2014-04-01

    This study assessed the effects of diosgenin on estrogenic activity using a uterotrophic assay. Immature female rats received diosgenin orally at doses of 200, 100, or 20 mg/kg body mass; and 17α ethynylestradiol at doses of 1 or 0.3 μg/kg, daily, for 3 consecutive days from day 19 to day 21. Controls were distributed among 2 groups: an intact control group and a vehicle control group. Animals were sacrificed 24 h after the last application of diosgenin, estradiol, or vehicle (22nd day of life). Uterine wet weight, stereological and histomorphometrical changes, immunohistochemical expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and the expression of lactoferrin (LF) were examined. Diosgenin did not affect the uterine wet weight, epithelium height, volume densities of endometrium, endometrial epithelia, number of endometrial glands, or histological appearance of vaginal epithelia. ERα, PR, and LF immunostaining intensity were not altered in the animals that received diosgenin. High-potency reference ER agonist 17α-ethynylestradiol induced a significant increase in all of the measured parameters, and as expected, decreased ERα immunostaining intensity. Based on these data, it can be concluded that diosgenin, at doses of 20-200 mg/kg, did not act as an estrogen agonist in the immature rat uterotrophic assay.

  20. A sensitive quartz crystal microbalance assay of adenosine triphosphate via DNAzyme-activated and aptamer-based target-triggering circular amplification.

    PubMed

    Song, Weiling; Zhu, Zheng; Mao, Yaning; Zhang, Shusheng

    2014-03-15

    In this work, a simple and novel quartz crystal microbalance (QCM) assay is demonstrated to selectively and sensitively detect the adenosine triphosphate (ATP). The amplification process consists of circular nucleic acid strand-displacement polymerization, aptamer recognition strategy and nanoparticle signal amplification. With the involvement of an aptamer-based complex, two amplification reaction templates and AuNP-functionalized probes, the whole circle amplification process is triggered by the target recognition of ATP. As an efficient mass amplifier, AuNP-functionalized probes are introduced to enhance the QCM signals. As a result of DNA multiple amplification, a large number of AuNP-functionalized probes are released and hybridized with the capture probes on the gold electrode. Therefore the QCM signals are significantly enhanced, reaching a detection limit of ATP as low as 1.3 nM. This strategy can be conveniently used for any aptamer-target binding events with other biological detection such as protein and small molecules. Moreover, the practical determination of ATP in cancer cells demonstrates the feasibility of this QCM approach and potential application in clinical diagnostics.

  1. Urolithins display both antioxidant and pro-oxidant activities depending on assay system and conditions.

    PubMed

    Kallio, Tuija; Kallio, Johanna; Jaakkola, Mari; Mäki, Marianne; Kilpeläinen, Pekka; Virtanen, Vesa

    2013-11-13

    The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

  2. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    SciTech Connect

    Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng; Nelson, Ornella D.; Li, Zhi; Lin, Hening; Callister, Stephen J.; Richardson, Ruth E.

    2015-01-01

    Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.

  3. Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Sulpice, Ronan; Tschoep, Hendrik; VON Korff, Maria; Büssis, Dirk; Usadel, Björn; Höhne, Melanie; Witucka-Wall, Hanna; Altmann, Thomas; Stitt, Mark; Gibon, Yves

    2007-09-01

    D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the first step in photosynthetic carbon assimilation and represents the largest sink for nitrogen in plants. Improvement of its kinetic properties or the efficiency with which it is used in planta would benefit photosynthesis, nitrogen and water use efficiency, and yield. This paper presents a new non-radioactive microplate-based assay, which determines the product [3-phosphoglycerate (3-PGA)] in an enzymic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase. High sensitivity permits use of highly diluted extracts, and a short reaction time to avoid problems due to fall-off. Throughput was several hundreds of samples per person per day. Sensitivity and convenience compared favourably with radioisotopic assays, which were previously used to assay Rubisco. Its use is illustrated in three applications. (1) Maximal and initial activities and the K(m) for ribulose-1,5-bisphosphate were determined in raw extracts of leaves from several species. Similar values were obtained from those in the literature. (2) Diurnal changes were compared in rosettes of wild-type (WT) Arabidopsis and the starchless pgm mutant. Despite these dramatic differences in carbon metabolism, Rubisco activity and activation were similar in both genotypes. (3) A preliminary association mapping study was performed with 118 Arabidopsis accessions, using 183 markers that probably cover approximately 3-8% of the total genome. At a P-value < 0.005, two, two and no quantitative trait loci (QTL) were found for Rubisco maximal activity, initial activity and activation state, respectively. Inspection of the genomic regions that span these markers revealed these QTL involved genes not previously implicated in the regulation of Rubisco expression or activity.

  4. Quantum-dot-based cell motility assay.

    PubMed

    Gu, Weiwei; Pellegrino, Teresa; Parak, Wolfgang J; Boudreau, Rosanne; Le Gros, Mark A; Gerion, Daniele; Alivisatos, A Paul; Larabell, Carolyn A

    2005-06-28

    Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

  5. Nondestructive assay using active and passive computed tomography

    SciTech Connect

    Roberson, G. P. ,LLNL

    1998-07-01

    The United States Department of Energy (DOE) has over 600,000 transuranic (TRU) waste drums temporarily stored at nearly 40 sites within the United States. Contents of these drums must be characterized before they are transported for permanent disposal. Traditional gamma-ray methods used to characterize nuclear waste introduce errors that are related to nonuniform measurement responses associated with unknown radioactive source and matrix material distributions. These errors can be reduced by application of tomographic techniques, that measure these distributions. The Lawrence Livermore National Laboratory (LLNL) has developed two tomographic-based waste assay systems. They use external radioactive sources and tomography-protocol to map the attenuation within a waste drum as a function of mono-energetic gamma-ray energy in waste containers. Passive tomography is used to localize and identify specific radioactive waste contents within the same waste containers. Reconstruction of the passive data via the active images allows internal waste radioactivities in a drum to be corrected for any overlying heterogeneous materials, thus yielding an absolute assay of the waste radioactivities. Calibration of both systems requires only point source measurements and are independent of matrix materials. The first system is housed at LLNL and was developed to study and validate research concepts. The second system is being developed with Bioimaging Research, Inc. (BIR) and is housed within a mobile waste characterization trailer. This system has traveled to three DOE facilities to demonstrate the active and passive computed tomography capability. Both systems have participated in and successfully passed the requirements of formal DOE-sponsored intercomparison studies. The systems have measured approximately 1 to 100 grains of plutonium within a variety of waste matrix materials. Laboratory and field results from these two systems over the past several years show that both systems

  6. A fluorescence-based assay for indoleamine 2,3-dioxygenase.

    PubMed

    Matin, Azadeh; Streete, Isla M; Jamie, Ian M; Truscott, Roger J W; Jamie, Joanne F

    2006-02-01

    A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.

  7. Complement dependent cytotoxicity (CDC) activity of a humanized anti Lewis-Y antibody: FACS-based assay versus the 'classical' radioactive method -- qualification, comparison and application of the FACS-based approach.

    PubMed

    Nechansky, A; Szolar, O H J; Siegl, P; Zinoecker, I; Halanek, N; Wiederkum, S; Kircheis, R

    2009-05-01

    The fully humanized Lewis-Y carbohydrate specific monoclonal antibody (mAb) IGN311 is currently tested in a passive immunotherapy approach in a clinical phase I trail and therefore regulatory requirements demand qualified assays for product analysis. To demonstrate the functionality of its Fc-region, the capacity of IGN311 to mediate complement dependent cytotoxicity (CDC) against human breast cancer cells was evaluated. The "classical" radioactive method using chromium-51 and a FACS-based assay were established and qualified according to ICH guidelines. Parameters evaluated were specificity, response function, bias, repeatability (intra-day precision), intermediate precision (operator-time different), and linearity (assay range). In the course of a fully nested design, a four-parameter logistic equation was identified as appropriate calibration model for both methods. For the radioactive assay, the bias ranged from -6.1% to -3.6%. The intermediate precision for future means of duplicate measurements revealed values from 12.5% to 15.9% and the total error (beta-expectation tolerance interval) of the method was found to be <40%. For the FACS-based assay, the bias ranged from -8.3% to 0.6% and the intermediate precision for future means of duplicate measurements revealed values from 4.2% to 8.0%. The total error of the method was found to be <25%. The presented data demonstrate that the FACS-based CDC is more accurate than the radioactive assay. Also, the elimination of radioactivity and the 'real-time' counting of apoptotic cells further justifies the implementation of this method which was subsequently applied for testing the influence of storage at 4 degrees C and 25 degrees C ('stability testing') on the potency of IGN311 drug product. The obtained results demonstrate that the qualified functional assay represents a stability indicating test method.

  8. A mechanistically relevant cytotoxicity assay based on the detection of cellular GFP.

    PubMed

    Halter, Michael; Almeida, Jamie L; Tona, Alessandro; Cole, Kenneth D; Plant, Anne L; Elliott, John T

    2009-08-01

    Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.

  9. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

    PubMed Central

    Körner, Wolfgang; Vinggaard, Anne Marie; Térouanne, Béatrice; Ma, Risheng; Wieloch, Carise; Schlumpf, Margret; Sultan, Charles; Soto, Ana M

    2004-01-01

    We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%. PMID

  10. Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase).

    PubMed

    Schultis, T; Metzger, J W

    2004-12-01

    In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed. With the LYES-assay the estrogenic activities of natural (17beta-estradiol E2 and estrone), synthetic (17alpha-ethinylestradiol EE2) and pharmaceutical estrogens (diethylstilbestrol DES) as well as xenoestrogens (4-nonylphenol NP and five parabens) were determined and compared with the results obtained by other in vitro-assays namely the conventional YES-assay, the E-Screen-assay (MCF-7 breast tumor cell proliferation) and a receptor binding-assay (RB) with human estrogen receptors hER-alpha and hER-beta. In the case of E2 the LYES-assay had a significantly lower limit of quantification (LOQ) than the conventional YES-assay and even two orders of magnitude lower than the RB-assay. Compared to the E-Screen-assay the LOQ of the LYES-assay was almost one order of magnitude higher. The time required to perform the LYES-assay was as little as seven hours compared to three to five days for the conventional YES-assay. Thus, the LYES-assay is a very good alternative to existing estrogenic in vitro-assays, since it has a good sensitivity, is cheap and much faster than the other assays.

  11. Toxin activity assays, devices, methods and systems therefor

    DOEpatents

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2016-04-05

    Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  12. STRESS PATHWAY-BASED REPORTER ASSAYS TO ASSESS TOXICITY OF ENVIRONMENTAL CHEMICALS.

    EPA Science Inventory

    There is an increasing need for assays for the rapid and efficient assessment of toxicities of large numbers of environmental chemicals. To meet this need, we are developing cell-based reporter assays that measure the activation of key molecular stress pathways. We are using pro...

  13. How Do Detergents Work? A Qualitative Assay to Measure Amylase Activity

    ERIC Educational Resources Information Center

    Novo, M. Teresa; Casanoves, Marina; Garcia-Vallvé, Santi; Pujadas, Gerard; Mulero, Miquel; Valls, Cristina

    2016-01-01

    We present a practical activity focusing on two main goals: to give learners the opportunity to experience how the scientific method works and to increase their knowledge about enzymes in everyday situations. The exercise consists of determining the amylase activity of commercial detergents. The methodology is based on a qualitative assay using a…

  14. Cell-based resorption assays for bone graft substitutes.

    PubMed

    Zhang, Ziyang; Egaña, José T; Reckhenrich, Ann K; Schenck, Thilo Ludwig; Lohmeyer, Jörn A; Schantz, Jan Thorsten; Machens, Hans-Günther; Schilling, Arndt F

    2012-01-01

    The clinical utilization of resorbable bone substitutes has been growing rapidly during the last decade, creating a rising demand for new resorbable biomaterials. An ideal resorbable bone substitute should not only function as a load-bearing material but also integrate into the local bone remodeling process. This means that these bone substitutes need to undergo controlled resorption and then be replaced by newly formed bone structures. Thus the assessment of resorbability is an important first step in predicting the in vivo clinical function of bone substitute biomaterials. Compared with in vivo assays, cell-based assays are relatively easy, reproducible, inexpensive and do not involve the suffering of animals. Moreover, the discovery of RANKL and M-CSF for osteoclastic differentiation has made the differentiation and cultivation of human osteoclasts possible and, as a result, human cell-based bone substitute resorption assays have been developed. In addition, the evolution of microscopy technology allows advanced analyses of the resorption pits on biomaterials. The aim of the current review is to give a concise update on in vitro cell-based resorption assays for analyzing bone substitute resorption. For this purpose models using different cells from different species are compared. Several popular two-dimensional and three-dimensional optical methods used for resorption assays are described. The limitations and advantages of the current ISO degradation assay in comparison with cell-based assays are discussed.

  15. Active and passive computed tomography for nondestructive assay

    SciTech Connect

    Bernardi, R T; Camp, D E; Clard, D; Jackson, J A; Martz, H E, Decman, D J; Roberson, G P

    1998-10-28

    Traditional gamma-ray methods used to characterize nuclear waste introduce errors that are related to non-uniform measurement responses associated with unknown radioactive source and matrix material distributions. These errors can be reduced by applying an active and passive tomographic technique (A&PCT) developed at the Lawrence Livermore National Laboratory (LLNL). The technique uses an external radioactive source and active tomography to map the attenuation within a waste barrel as a function of mono-energetic gamma-ray energy. Passive tomography is used to localize and identify specific radioactive waste within the same container. Reconstruction of the passive data using the attenuation maps at specific energies allows internal waste radioactivity to be corrected for any overlying heterogeneous materials, thus yielding an absolute assay of the waste activity. LLNL and Bio-Imaging Research, Inc. have collaborated in a technology transfer effort to integrate an A&PCT assay system into a mobile waste characterization trailer. This mobile system has participated in and passed several formal DOE-sponsored performance demonstrations, tests and evaluations. The system is currently being upgraded with multiple detectors to improve throughput, automated gamma-ray analysis code to simplify the assay, and a new emission reconstruction code to improve accuracy

  16. Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

    PubMed

    Takenouchi, Osamu; Fukui, Shiho; Okamoto, Kenji; Kurotani, Satoru; Imai, Noriyasu; Fujishiro, Miyuki; Kyotani, Daiki; Kato, Yoshinao; Kasahara, Toshihiko; Fujita, Masaharu; Toyoda, Akemi; Sekiya, Daisuke; Watanabe, Shinichi; Seto, Hirokazu; Hirota, Morihiko; Ashikaga, Takao; Miyazawa, Masaaki

    2015-11-01

    To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance.

  17. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    PubMed

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

    2015-12-01

    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.

  18. A rapid fluorometric assay for the proteolytic activity of SKI-1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus.

    PubMed

    Basak, Ajoy; Chrétien, Michel; Seidah, Nabil G

    2002-03-13

    The subtilase subtilisin kexin isozyme-1 (SKI-1)/site 1 protease (S1P), has been implicated in the processing of Lassa virus glycoprotein C (GP-C) precursor into GP1 and GP2 that are responsible for viral fusion with the host cell membrane. Here, we studied in vitro the kinetics of this cleavage by hSKI-1 using an intramolecularly quenched fluorogenic (IQF) peptide, Q-GPC(251-263) [Abz-(251)Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu/Gly-Thr-Phe-Thr(263)-3-NitroTyr-Ala-CONH(2)], containing the identified site. The measured V(max (app))/K(m (app)) was compared to those for other IQF SKI-substrates. Q-GPC(251-263) is cleaved 10-fold more efficiently than the previously known best SKI-substrate, Q-hproSKI(134-142). This study confirmed the role of SKI-1 in GP-C processing and provides a novel, rapid and efficient enzymatic assay of SKI-1.

  19. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  20. Synthesis and Assay of SIRT1-Activating Compounds.

    PubMed

    Dai, H; Ellis, J L; Sinclair, D A; Hubbard, B P

    2016-01-01

    The NAD(+)-dependent deacetylase SIRT1 plays key roles in numerous cellular processes including DNA repair, gene transcription, cell differentiation, and metabolism. Overexpression of SIRT1 protects against a number of age-related diseases including diabetes, cancer, and Alzheimer's disease. Moreover, overexpression of SIRT1 in the murine brain extends lifespan. A number of small-molecule sirtuin-activating compounds (STACs) that increase SIRT1 activity in vitro and in cells have been developed. While the mechanism for how these compounds act on SIRT1 was once controversial, it is becoming increasingly clear that they directly interact with SIRT1 and enhance its activity through an allosteric mechanism. Here, we present detailed chemical syntheses for four STACs, each from a distinct structural class. Also, we provide a general protocol for purifying active SIRT1 enzyme and outline two complementary enzymatic assays for characterizing the effects of STACs and similar compounds on SIRT1 activity.

  1. Synthesis and Assay of SIRT1-Activating Compounds

    PubMed Central

    Dai, H.; Ellis, J.L.; Sinclair, D.A.; Hubbard, B.P.

    2016-01-01

    The NAD+-dependent deacetylase SIRT1 plays key roles in numerous cellular processes including DNA repair, gene transcription, cell differentiation, and metabolism. Over-expression of SIRT1 protects against a number of age-related diseases including diabetes, cancer, and Alzheimer's disease. Moreover, overexpression of SIRT1 in the murine brain extends lifespan. A number of small-molecule sirtuin-activating compounds (STACs) that increase SIRT1 activity in vitro and in cells have been developed. While the mechanism for how these compounds act on SIRT1 was once controversial, it is becoming increasingly clear that they directly interact with SIRT1 and enhance its activity through an allosteric mechanism. Here, we present detailed chemical syntheses for four STACs, each from a distinct structural class. Also, we provide a general protocol for purifying active SIRT1 enzyme and outline two complementary enzymatic assays for characterizing the effects of STACs and similar compounds on SIRT1 activity. PMID:27423864

  2. Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

    PubMed Central

    Schwartz, David Eric; Gong, Ping; Shepard, Kenneth L.

    2008-01-01

    We present an active oligonucleotide microarray platform for time-resolved Förster resonance energy transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors. PMID:18515059

  3. Photon upconversion in homogeneous fluorescence-based bioanalytical assays.

    PubMed

    Soukka, Tero; Rantanen, Terhi; Kuningas, Katri

    2008-01-01

    Upconverting phosphors (UCPs) are very attractive reporters for fluorescence resonance energy transfer (FRET)-based bioanalytical assays. The large anti-Stokes shift and capability to convert near-infrared to visible light via sequential absorption of multiple photons enable complete elimination of autofluorescence, which commonly impairs the performance of fluorescence-based assays. UCPs are ideal donors for FRET, because their very narrow-banded emission allows measurement of the sensitized acceptor emission, in principle, without any crosstalk from the donor emission at a wavelength just tens of nanometers from the emission peak of the donor. In addition, acceptor dyes emitting at visible wavelengths are essentially not excited by near-infrared, which further emphasizes the unique potential of upconversion FRET (UC-FRET). These characteristics result in favorable assay performance using detection instrumentation based on epifluorometer configuration and laser diode excitation. Although UC-FRET is a recently emerged technology, it has already been applied in both immunoassays and nucleic acid hybridization assays. The technology is also compatible with optically difficult biological samples, such as whole blood. Significant advances in assay performance are expected using upconverting lanthanide-doped nanocrystals, which are currently under extensive research. UC-FRET, similarly to other fluorescence techniques based on resonance energy transfer, is strongly distance dependent and may have limited applicability, for example in sandwich-type assays for large biomolecules, such as viruses. In this article, we summarize the essentials of UC-FRET, describe its current applications, and outline the expectations for its future potential.

  4. [Techniques for assaying the activity of transcription factor NF-κB].

    PubMed

    Ling, Xiao-Qian; Wang, Jin-Ke

    2013-05-01

    NF-κB is a stimulatory transcription factor that is ubiquitous in almost all kinds of cells. When cells are under various stimuli, NF-κB is activated and regulates large numbers of target genes, and thus controls important cellular processes, ranging from cell growth and differentiation to apoptosis and cancer. Therefore, NF-κB is a forefront hotspot transcription factor that is intensively studied in virtually all fields of biomedical sciences, and becomes a promising target for disease therapy and drug screening. The activity detection is the first and inevitable step for the studies of NF-κB activation and function.Therefore, the techniques for detection of NF-κB activity have always been paid more attention and continuously developed. Especially in recent year, along with the development of each disciplines, various new techniques have been developed, including ELISA-like assays based on dsDNA-coupled plate, filter binding assays, FRET assays, fluorescence reporting and nucleic acids amplification assays based on exonuclease and endonuclease, MS and flow cytometry assays based on immunomicrobeads, and other biophysical and electrochemical assays. Some of these techniques have already played important roles in NF-κB studies. This paper reviewed new techniques developed in recent years by classification, in order to provide an overview of NF-κB activity assays, which may be helpful for researchers to select appropriate techniques used in their studies. Moreover, the learning and understanding of these techniques may inspire researchers to improve currently existing techniques and develop novel methods for the studies of NF-κB.

  5. Uranium internal exposure evaluation based on urine assay data

    SciTech Connect

    Lawrence, J.N.P.

    1984-09-01

    The difficulties in assessing internal exposures to uranium from urine assay data are described. A simplified application of the ICRP-30 and ICRP Lung Model concepts to the estimation of uranium intake is presented. A discussion follows on the development of a computer code utilizing the ICRP-30-based uranium elimination model with the existing urine assay information. The calculated uranium exposures from 1949 through 1983 are discussed. 13 references, 1 table.

  6. A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

    PubMed Central

    2014-01-01

    Background Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. Results We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. Conclusion The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional

  7. Nanobeads-based assays. The case of gluten detection

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

    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.

  8. Automated cell-based assay for screening of aquaporin inhibitors.

    PubMed

    Mola, Maria Grazia; Nicchia, Grazia Paola; Svelto, Maria; Spray, David C; Frigeri, Antonio

    2009-10-01

    Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 microM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders.

  9. Automated Cell-Based Assay for Screening of Aquaporin Inhibitors

    PubMed Central

    Mola, Maria Grazia; Nicchia, Grazia Paola; Svelto, Maria; Spray, David C.; Frigeri, Antonio

    2010-01-01

    Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 μM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders. PMID:19705854

  10. Serum-induced platelet procoagulant activity: an assay for the characterization of prothrombotic disorders.

    PubMed

    Warner, M N; Pavord, S; Moore, J C; Warkentin, T E; Hayward, C P; Kelton, J G

    1999-02-01

    Platelets contribute to hemostasis by forming a platelet plug and by providing a procoagulant surface for the assembly and activation of the coagulation factors. The contribution of platelets to prothrombotic disorders has been difficult to analyze. Recently an assay was reported that measured the procoagulant activity of test platelets by making the platelet lipid surface the limiting factor in the production of thrombin. In this report we describe a novel technique, based on this assay, that we used to study patient serum factors that activate control platelets and in turn initiate measurable procoagulant activity. Using this assay we investigated a group of patients with prothrombotic disorders. The patient test serum was incubated with normal platelets in the presence of activated factor Xa. The resultant thrombin was measured in a chromogenic assay. The rate-limiting step was the presence of any potential platelet-activating factors, such as antibodies in the heat-treated test serum, that would allow the Xa to bind to the platelet phospholipid surface. Serum samples from patients with heparin-induced thrombocytopenia (HIT) and the anti-phospholipid antibody syndrome enhanced platelet procoagulant activity, while samples from patients with idiopathic thrombocytopenic purpura and disseminated intravascular coagulation (DIC) did not. HIT serum samples also induced platelet activation, as measured by platelet microparticle shedding, carbon 14-labeled serotonin release, and platelet aggregation. The measurement of serum-induced platelet procoagulant activity provides a method for the investigation of circulating platelet agonists in prothrombotic disorders.

  11. A Random Motility Assay Based on Image Correlation Spectroscopy

    PubMed Central

    Prummer, Michael; Kling, Dorothee; Trefzer, Vanessa; Enderle, Thilo; Zoffmann, Sannah; Prunotto, Marco

    2013-01-01

    We demonstrate the random motility (RAMOT) assay based on image correlation spectroscopy for the automated, label-free, high-throughput characterization of random cell migration. The approach is complementary to traditional migration assays, which determine only the collective net motility in a particular direction. The RAMOT assay is less demanding on image quality compared to single-cell tracking, does not require cell identification or trajectory reconstruction, and performs well on live-cell, time-lapse, phase contrast video microscopy of hundreds of cells in parallel. Effective diffusion coefficients derived from the RAMOT analysis are in quantitative agreement with Monte Carlo simulations and allowed for the detection of pharmacological effects on macrophage-like cells migrating on a planar collagen matrix. These results expand the application range of image correlation spectroscopy to multicellular systems and demonstrate a novel, to our knowledge, migration assay with little preparative effort. PMID:23746508

  12. Construction of a Turn Off-On-Off Fluorescent System Based on Competitive Coordination of Cu2+ between 6,7-Dihydroxycoumarin and Pyrophosphate Ion for Sensitive Assay of Pyrophosphatase Activity

    PubMed Central

    Zhao, Liu; Miao, Yanqing; Liu, Chunye; Zhang, Chenxiao

    2016-01-01

    The detection of pyrophosphatase (PPase) activity is of great significance in diagnosing diseases and understanding the function of PPase-related biological events. This study constructed a turn off-on-off fluorescent system for PPase activity assay based on PPase-regulated competitive coordination of Cu2+ between a water-soluble fluorescent probe 6,7-dihydroxycoumarin (DHC) and pyrophosphate (PPi). The probe DHC can coordinate with Cu2+ and consequently display on-off type fluorescence response. Furthermore, the in situ formed nonfluorescent Cu2+-DHC complex can act as an effective off-on type fluorescent probe for sensing PPi due to the higher coordination reactivity between Cu2+ and PPi than that between Cu2+ and DHC. The subsequent addition of PPase to the mixture containing Cu2+, DHC, and PPi leads to the fluorescence requenching of the system again (an off state) because PPase catalyzes the hydrolysis of PPi into orthophosphate in the reaction system. Under the optimum conditions, the decrease of the fluorescence intensity of DHC-Cu2+-PPi system was linear with the increase of the PPase activity in the range from 0.1 to 0.3 U. The detection limit was down to 0.028 U PPase (S/N = 3). Moreover, the as-established system was also applied to evaluate PPase inhibitor. This study offers a simple yet effective method for the detection of PPase activity. PMID:27766179

  13. ORGANOPHOSPHORUS HYDROLASE-BASED ASSAY FOR ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    We report a rapid and versatile Organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), ...

  14. EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.

    PubMed

    Wehrman, Tom; Nguyen, Mimi; Feng, Wei; Bader, Benjamin

    2013-05-01

    Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.

  15. Novel assay for direct fluorescent imaging of sialidase activity

    NASA Astrophysics Data System (ADS)

    Tomin, A.; Shkandina, T.; Bilyy, R.

    2011-07-01

    Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 3.2.1.18, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.

  16. Assay and characterization of the NO dioxygenase activity of flavohemoglobins.

    PubMed

    Gardner, Paul R

    2008-01-01

    A variety of hemoglobins, including several microbial flavohemoglobins, enzymatically dioxygenate the free radical nitric oxide (*NO) to form nitrate. Many of these *NO dioxygenases have been shown to control *NO toxicity and signaling. Furthermore, *NO dioxygenation appears to be an ancient and intrinsic function for members of the hemoglobin superfamily found in Archaea, eukaryotes, and bacteria. Yet for many hemoglobins, a function remains to be elucidated. Methods for the assay and characterization of the *NO dioxygenase (EC 1.14.12.17) activity and function of flavohemoglobins are described. The methods may also be applied to the discovery and design of inhibitors for use as antibiotics or as modulators of *NO signaling.

  17. SPR-based assays enable the full functional analysis of bispecific molecules.

    PubMed

    Meschendoerfer, W; Gassner, C; Lipsmeier, F; Regula, J T; Moelleken, J

    2017-01-05

    The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins raises new challenges for functional characterization. When compared to standard antibodies, two individual interactions and the inter-dependency of binding events need to be considered for bispecific antibodies. We have previously described an SPR-based assay setup, which enables us to assess the binding activity of a bivalent-bispecific molecule to both targets simultaneously and - in addition to one individual target - in a single setup. However, there might be some pitfalls when applying the bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an alternative SPR-based assay principle, which allows the individual assessment of both targets in solution. Comparison of data between the assays showed that simultaneous binding can be calculated based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay principles allow a "full" functional analysis of a bispecific CrossMab in only one assay. The assay principles can be qualified and enable an efficient drug development.

  18. A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching.

    PubMed

    Raturi, Arun; Vacratsis, Panayiotis O; Seslija, Dana; Lee, Lana; Mutus, Bulent

    2005-10-15

    PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV-visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent K(m) of PDI for diabz-GSSG was estimated to be approx. 15 muM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.

  19. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  20. A paper-based lateral flow assay for morphine.

    PubMed

    Teerinen, Tuija; Lappalainen, Timo; Erho, Tomi

    2014-09-01

    Morphine was used as a model analyte to examine the possibility of using cellulose, physically modified by papermaking and converting techniques, as a capillary matrix in a lateral flow type of diagnostic assay. This research was directed toward low-cost, disposable, and portable paper-based diagnostics, with the aim of addressing the analytical performance of paper as a substrate in the analysis for drugs of abuse. Antibody Fab fragments were used as sensing molecules, and gold nanoparticle detection was employed. Inkjet printing was used to pattern sensing biomolecules as detection zones on paper. To validate the usefulness of paper as a diagnostic platform, the principle of a direct sandwich assay, based on immunocomplex formation between morphine and the anti-morphine Fab fragment and detection of the formed immunocomplex by another Fab fragment, was implemented. Results were compared with that achieved by using nitrocellulose as a reference material. Possible interfering from the sample matrix on assay quality was investigated with spiked oral fluid samples. Under optimized conditions, a visually assessed limit of detection for the sandwich assay was 1 ng/mL, indicating that the paper-based test devices developed in this work can perform screening for drugs of abuse and can fulfill the requirement for a sensitive assay in diagnostically relevant ranges.

  1. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  2. High content cell-based assay for the inflammatory pathway

    NASA Astrophysics Data System (ADS)

    Mukherjee, Abhishek; Song, Joon Myong

    2015-07-01

    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.

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

    PubMed

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

    2013-01-01

    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.

  4. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    PubMed Central

    Otwell, A.E.; Sherwood, R.W.; Zhang, S.; Nelson, O.D.; Li, Z.; Lin, H.; Callister, S.J.; Richardson, R.E.

    2015-01-01

    Summary Understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. Using non-denaturing separations and mass spectrometry identification, in combination with a colorimetric screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins from the D. reducens proteome not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins identified are NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. This study is the first functional proteomic analysis of D. reducens and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium. PMID:25389064

  5. Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays.

    PubMed

    López-Alarcón, Camilo; Denicola, Ana

    2013-02-06

    Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been developed including the popular ORAC that evaluates the potential of a sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening, they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that allows comparing and ordering different products. More recently, nanoparticles-based assays have been developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal models and human studies are more appropriate but also more expensive and time-consuming, making the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity (CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of antioxidant capacity of natural products.

  6. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    PubMed

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  7. Immunomagnetic nanoparticle-based assays for detection of biomarkers

    PubMed Central

    Park, Hoyoung; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    The emergence of biomarkers as key players in the paradigm shift towards preventative medicine underscores the need for their detection and quantification. Advances made in the field of nanotechnology have played a crucial role in achieving these needs, and have contributed to recent advances in the field of medicine. Nanoparticle-based immunomagnetic assays, in particular, offer numerous advantages that utilize the unique physical properties of magnetic nanoparticles. In this review, we focus on recent developments and trends with regards to immunomagnetic assays used for detection of biomarkers. The various immunomagnetic assays are categorized into the following: particle-based multiplexing, signal control, microfluidics, microarray, and automation. Herein, we analyze each category and discuss their advantages and disadvantages. PMID:24285924

  8. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  9. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  10. Plasma drug activity assay for treatment optimization in tuberculosis patients.

    PubMed

    Heysell, Scott K; Mtabho, Charles; Mpagama, Stellah; Mwaigwisya, Solomon; Pholwat, Suporn; Ndusilo, Norah; Gratz, Jean; Aarnoutse, Rob E; Kibiki, Gibson S; Houpt, Eric R

    2011-12-01

    Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C(2 h)). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C(2 h) plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C(2 h) rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ~1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (≤ 2.0) TDA was significantly associated with both lower isoniazid and rifampin C(2 h) levels, and very low (≤ 1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.

  11. The capture proteasome assay: A method to measure proteasome activity in vitro.

    PubMed

    Vigneron, Nathalie; Abi Habib, Joanna; Van den Eynde, Benoît J

    2015-08-01

    Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i-β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.

  12. [Detection of viable metabolically active yeast cells using a colorimetric assay].

    PubMed

    Růzicka, F; Holá, V

    2008-02-01

    The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

  13. pHluorin-based in vivo assay for hydrolase screening.

    PubMed

    Schuster, Sascha; Enzelberger, Markus; Trauthwein, Harald; Schmid, Rolf D; Urlacher, Vlada B

    2005-05-01

    pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.

  14. Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

    PubMed Central

    Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao

    2016-01-01

    From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field. PMID:27768770

  15. Liquid chromatographic assay based on microextraction by packed sorbent for therapeutic drug monitoring of carbamazepine, lamotrigine, oxcarbazepine, phenobarbital, phenytoin and the active metabolites carbamazepine-10,11-epoxide and licarbazepine.

    PubMed

    Ferreira, Ana; Rodrigues, Márcio; Oliveira, Paula; Francisco, Joana; Fortuna, Ana; Rosado, Luísa; Rosado, Pedro; Falcão, Amílcar; Alves, Gilberto

    2014-11-15

    A new, sensitive and fast high-performance liquid chromatography-diode-array detection assay based on microextraction by packed sorbent (MEPS/HPLC-DAD) is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in human plasma. Chromatographic separation of analytes and ketoprofen, used as internal standard (IS), was achieved in less than 15min on a C18-column, at 35°C, using acetonitrile (6%) and a mixture (94%) of water-methanol-triethylamine (73.2:26.5:0.3, v/v/v; pH 6.5) pumped at 1mL/min. The analytes and IS were detected at 215, 237 or 280nm. The method showed to be selective, accurate [bias ±14.8% (or ±17.8% in the lower limit of quantification)], precise [coefficient variation ≤9.7% (or ≤17.7% in the lower limit of quantification)] and linear (r(2)≥0.9946) over the concentration ranges of 0.1-15μg/mL for CBZ; 0.1-20μg/mL for LTG; 0.1-5μg/mL for OXC and CBZ-E; 0.2-40μg/mL for PB; 0.3-30μg/mL for PHT; and 0.4-40μg/mL for LIC. The absolute extraction recovery of the analytes ranged from 57.8 to 98.1% and their stability was demonstrated in the studied conditions. This MEPS/HPLC-DAD assay was successfully applied to real plasma samples from patients, revealing to be a cost-effective tool for routine therapeutic drug monitoring of CBZ, LTG, OXC, PB and/or PHT.

  16. Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.

    PubMed

    Li, Yijun; Ptolemy, Adam S; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T

    2011-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) μmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

  17. Ultra Fast and Sensitive Liquid Chromatography Tandem Mass Spectrometry Based Assay for Galactose-1-Phosphate Uridylyltransferase and Galactokinase Deficiencies

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([13C6]-uridine diphosphate galactose in GALT assay and [13C6]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4 ± 4.2 and GALK activity of 1.8 ± 0.47 (mean ± SD) µmol·(g Hgb) −1·hr−1. Erythrocyte GALT activities in a cohort of 16 patients with classic galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analzyed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. PMID:20863731

  18. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

    PubMed Central

    Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R.; Alter, Galit

    2011-01-01

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. PMID:21192942

  19. Plant compounds enhance the assay sensitivity for detection of active Bacillus cereus toxin.

    PubMed

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-03-11

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

  20. Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

    PubMed Central

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-01-01

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety. PMID:25767986

  1. Mutagenic activity of isoxazolylnaphthoquinoneimines assayed by micronucleus bone marrow test.

    PubMed

    Sicardi, S M; Ferrato, E

    1995-05-01

    Studies were undertaken to evaluate the ability of various quinoneimines to induce micronuclei in bone marrow cells as a measure of their genotoxicity. Accordingly, 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I), its 2-acetyl derivative (II) and 2-[(5-methyl-3-isoxazolyl)amino]-N-(5-methyl-3-isoxazolyl)-1 ,4- naphthoquinone-4-imine (III), as well as two of their precursors, 2-hydroxynaphthoquinone (NQ-2-OH) and 3,4-dimethyl-5-aminoisoxazole (DMAI) were given by intraperitoneal injection at 5, 50, 100 and 200 mg/Kg doses to S.J.L. Swiss mice with 24 h sampling time. Compounds I and II displayed highly significant differences at 50, 100 and 200 mg/kg doses (p < 0.01) and their mutagenic dose response curves correlated closely with an inverted U-shaped form whose interpretation is still the subject of controversy. NQ-2-OH only produced a significant increase in micronucleus frequency at 50 mg/kg, whereas no mutagenic activity was found for compound III and DMAI at the doses assayed. At 50 mg/kg the order of relative mutagenic potencies was I > II > NQ-2-OH. Mechanisms advanced to explain loss of drug activity at high doses include capture saturation, enzymatic induction during metabolism and participation of an independent defense system.

  2. A new effective assay to detect antimicrobial activity of filamentous fungi.

    PubMed

    Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

    2013-01-15

    The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.

  3. Identification of ATP diphosphohydrolase activity in human term placenta using a novel assay for AMP.

    PubMed

    Papamarcaki, T; Tsolas, O

    1990-09-03

    Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN3 were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase. A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05-0.8 microM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates. The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.

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

    SciTech Connect

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. )

    1990-06-15

    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.

  5. Cell-based potassium ion channel screening using the FluxOR assay.

    PubMed

    Beacham, Daniel W; Blackmer, Trillium; O' Grady, Michael; Hanson, George T

    2010-04-01

    FluxOR technology is a cell-based assay used for high-throughput screening measurements of potassium channel activity. Using thallium influx as a surrogate indicator of potassium ion channel activity, the FluxOR Potassium Ion Channel Assay is based on the activation of a novel fluorescent dye. This indicator reports channel activity with a large fluorogenic response and is proportional to the number of open potassium channels on the cell, making it extremely useful for studying K(+) channel targets. In contrast to BTC-AM ester, FluxOR dye is roughly 10-fold more thallium sensitive, requiring much lower thallium for a larger signal window. This also means that the assay is carried out in a physiological, normal-chloride saline. In this article, the authors describe how they used BacMam gene delivery to express Kv7.2 and 7.3 (KCNQ), Kir2.1, or Kv11.1 (hERG) potassium ion channels in U2-OS cells. Using these cells, they ran the FluxOR assay to identify and characterize channel-specific inhibitory compounds discovered within the library (Tocriscreen Mini 1200 and Sigma Sodium/Potassium Modulators Ligand set). The FluxOR assay was able to identify several known specific inhibitors of Kv7.2/7.3 or hERG, highlighting its potential to identify novel and more efficacious small-molecule modulators.

  6. A naked-eye based strategy for semiquantitative immunochromatographic assay.

    PubMed

    Zhang, Daohong; Li, Peiwu; Zhang, Qi; Li, Ran; Zhang, Wen; Ding, Xiaoxia; Li, Chang Ming

    2012-08-31

    It is critical to develop a cost-effective quantitative/semiquantitative assay for rapid diagnosis and on-site detection of toxic or harmful substances. Here, a naked-eye based semiquantitative immunochromatographic strip (NSI-strip) was developed, on which three test lines (TLs, TL-I, TL-II and TL-III) were dispensed on a nitrocellulose membrane to form the test zone. Similar as the traditional strip assay for small molecule, the NSI-strip assay was also based on the competitive theory, difference was that the analyte competed three times with the capture reagent for the limited number of antibody binding sites. After the assay, the number of TLs developed in the test zone was inversely proportional to the analyte concentration, thus analyte content levels could be determined by observing the appeared number of TLs. Taking aflatoxin B(1) as the model analyte, visual detection limit of the NSI-strip was 0.06 ng mL(-1) and threshold concentrations for TL-I-III were 0.125, 0.5, and 2.0 ng mL(-1), respectively. Therefore, according to the appeared number of TLs, the following concentration ranges would be detectable by visual examination: 0-0.06 ng mL(-1) (negative samples), and 0.06-0.125 ng mL(-1), 0.125-0.5 ng mL(-1), 0.5-2.0 ng mL(-1) and >2.0 ng mL(-1) (positive samples). That was to say, compared to traditional strips the NSI-strip could offer more parameter information of the target analyte content. In this way, the NSI-strip improved the qualitative presence/absence detection of traditional strips by measuring the content (range) of target analytes semiquantitatively.

  7. Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc)

    EPA Science Inventory

    The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity i...

  8. Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc).

    EPA Science Inventory

    The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity i...

  9. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  10. Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

    PubMed

    Ichibangase, Tomoko; Ohba, Yoshihito; Kishikawa, Naoya; Nakashima, Kenichiro; Kuroda, Naotaka

    2004-01-01

    A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.

  11. Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.

    PubMed

    Jones, Laurie J; Haugland, Richard P; Singer, Victoria L

    2003-04-01

    We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.

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

    PubMed

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

    2011-12-01

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

  13. Evaluation of estrogenic activity in diets for experimental animals using in vitro assay.

    PubMed

    Kato, Hideo; Iwata, Toshio; Katsu, Yoshinao; Watanabe, Hajime; Ohta, Yasuhiko; Iguchi, Taisen

    2004-03-10

    We used a modified yeast-based human estrogen receptor alpha (ER alpha) bioassay to determine the estrogenic activity in 22 kinds of diets for experimental animals. The estrogenic activity of each diet was reevaluated by comparison with a calibration curve of 17 beta-estradiol. Almost all of the diets had estrogenic activity. The diets for rabbits and guinea pigs had the highest estrogenic activity compared to any other diets, including those for rats and mice. Estrogenic activity was found in dried skim milk, fishmeal, soybean meal, and alfalfa meal. In the NIH-07 diet opened for the ingredients, estrogenic activity was nearly all derived from the alfalfa meal. Multiple assays were performed to evaluate potential seasonal variations in the estrogenic potency in the raw materials of the rat and mouse diets. We found that the estrogenic activity in these raw materials changed throughout the year.

  14. Enhanced detection of lipid transfer inhibitor protein activity by an assay involving only low density lipoprotein.

    PubMed

    Morton, R E; Greene, D J

    1994-11-01

    Lipid transfer inhibitor protein (LTIP) activity has been typically quantitated by its ability to suppress lipid transfer protein-mediated lipid movement between low density lipoprotein (LDL) and high density lipoprotein (HDL). In an attempt to establish an LTIP activity assay that is more sensitive, we have exploited the reported preference of the inhibitor protein to interact with LDL. A lipid transfer assay was established that involves LDL as both the donor and the acceptor; LDL in one of these two pools was biotinylated to facilitate its removal with immobilized avidin. Compared to the standard LDL to HDL assay, LTIP inhibited lipid transfer from radiolabeled LDL to biotin-LDL 7-fold more. In the absence of LTIP, lipid transfer activity was the same in both assays. An added benefit of this assay was the near linearity (up to 85%) of the inhibitory response, in contrast to the highly curvilinear response of LTIP in LDL to HDL transfer assays. The high sensitivity of the LDL to biotin-LDL transfer assay in measuring LTIP activity could not be duplicated by other transfer assays including assays containing only HDL (HDL to biotin-HDL), assays between liposomes and LDL, or assays between LDL and HDL where the concentration of lipoproteins was reduced 10-fold. Thus, LTIP activity is most effectively measured in homologous lipid transfer assays involving only LDL (and its biotin derivative). This increased sensitivity to LTIP suggests that the inhibitor binds more avidly to the LDL surface than does lipid transfer protein.

  15. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    PubMed

    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

    2015-11-01

    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.

  16. Validation of paper-based assay for rapid blood typing.

    PubMed

    Al-Tamimi, Mohammad; Shen, Wei; Zeineddine, Rania; Tran, Huy; Garnier, Gil

    2012-02-07

    We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μL blood sample on a paper surface where grouping antibodies have already been introduced. A thin film chromatograph tank was used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate, resulting in a high optical density of the spot, with no visual trace in the buffer wicking path. Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper substrates had comparable ability to fix agglutinated blood, while a more porous substrate like Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized conditions, a rapid assay for detection of blood groups was developed by spotting blood to antibodies absorbed to paper and eluted with 200 μL of 0.9% NaCl buffer directly by pipetting. RBCs fixation on paper accurately detected blood groups (ABO and RhD) using ascending buffer for 10 min or using a rapid elution step in 100/100 blood samples including 4 weak AB and 4 weak RhD samples. The assay has excellent reproducibility where the same blood group was obtained in 26 samples assessed in 2 different days. Agglutinated blood fixation on porous paper substrate provides a new, simple, and sensitive assay for rapid detection of blood group for point-of-care applications.

  17. A Continuous Spectrophotometric Assay for APS Reductase Activity with Sulfite-Selective Probes

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) adenosine 5′-phosphosulfate (APS) reductase (EC number 1.8.4.10), (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of TB infection. Despite the importance of APR to Mtb, and other bacterial pathogens, current assay methods depend on use of [35S]-labeled APS or shunt AMP to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off-on” fluorescent levulinate-based probes. The APR activity can thus be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michelis-Menten kinetic constants (Km, kcat, kcat/Km) and apparent inhibition constant (Ki) for adenosine 5′-diphosphate (ADP), which compared favorably to values obtained in the gold-standard radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer. PMID:23711725

  18. Dynamic optical tweezers based assay for monitoring early drug resistance

    NASA Astrophysics Data System (ADS)

    Wu, Xiaojing; Zhang, Yuquan; Min, Changjun; Zhu, Siwei; Feng, Jie; Yuan, X.-C.

    2013-06-01

    In this letter, a dynamic optical tweezers based assay is proposed and investigated for monitoring early drug resistance with Pemetrexed-resistant non-small cell lung cancer (NSCLC) cell lines. The validity and stability of the method are verified experimentally in terms of the physical parameters of the optical tweezers system. The results demonstrate that the proposed technique is more convenient and faster than traditional techniques when the capability of detecting small variations of the response of cells to a drug is maintained.

  19. FluxCTTX: A LIMS-based tool for management and analysis of cytotoxicity assays data

    PubMed Central

    2015-01-01

    Background Cytotoxicity assays have been used by researchers to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds or screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical. These assays may be used as an alternative to animal experimentation and are becoming increasingly important in modern laboratories. However, the execution of these assays in large scale and different laboratories requires, among other things, the management of protocols, reagents, cell lines used as well as the data produced, which can be a challenge. The management of all this information is greatly improved by the utilization of computational tools to save time and guarantee quality. However, a tool that performs this task designed specifically for cytotoxicity assays is not yet available. Results In this work, we have used a workflow based LIMS -- the Flux system -- and the Together Workflow Editor as a framework to develop FluxCTTX, a tool for management of data from cytotoxicity assays performed at different laboratories. The main work is the development of a workflow, which represents all stages of the assay and has been developed and uploaded in Flux. This workflow models the activities of cytotoxicity assays performed as described in the OECD 129 Guidance Document. Conclusions FluxCTTX presents a solution for the management of the data produced by cytotoxicity assays performed at Interlaboratory comparisons. Its adoption will contribute to guarantee the quality of activities in the process of cytotoxicity tests and enforce the use of Good Laboratory Practices (GLP). Furthermore, the workflow developed is complete and can be adapted to other contexts and different tests for management of other types of data. PMID:26696462

  20. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  1. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides

    NASA Astrophysics Data System (ADS)

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

    1999-02-01

    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.

  2. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    PubMed

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms.

  3. Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    Roberts, Julia; Ling, Yan; Quezada, Landys Lopez; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Warren, J. David; Nathan, Carl

    2015-01-01

    The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic. PMID:26239979

  4. Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer.

    PubMed

    Zauner, Thomas; Berger-Hoffmann, Renate; Müller, Katrin; Hoffmann, Ralf; Zuchner, Thole

    2011-10-01

    Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.

  5. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

    PubMed

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

    2016-08-01

    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.

  6. Miniaturisation and validation of a cell-based assay for screening of Ca2+ channel modulators.

    PubMed

    Tammela, Päivi; Vuorela, Pia

    2004-06-30

    Voltage-operated calcium channels (VOCCs) play a significant role in the regulation of intracellular calcium concentrations in cardiovascular, neuronal and skeletal tissues. Therefore, physiologically relevant screening methods for calcium channel modulators are required. A 45Ca2+ uptake assay based on clonal rat pituitary cell line GH4C1, possessing L-type VOCCs, was miniaturised into a 96-well plate format. The assay was validated by known Ca2+ channel blockers, verapamil and nimodipine (IC50 values 3.4 and 0.007 microM, respectively) and by a set of natural compounds and their synthetic derivatives. The results were consistent with our previous data and demonstrated the reliability of the assay. The signal-to-background ratio was 3.9 +/- 0.4, signal-to-noise ratio 10.3 +/- 2.3, Z' factor 0.59 +/- 0.10, and day-to-day variability in positive control values 5%. Furthermore, experiments were also made on a Biomek FX workstation to evaluate the suitability of the assay for automation. With minor modifications the assay is applicable, e.g. for studying possible Ca2+ channel activators in detail. The established 96-well plate assay modification for screening of calcium channel modulators reduces considerably the time, labour and resources needed for cell culture and experiments, and has significant advantages in terms of automation suitability and overall cost-efficiency.

  7. In Vitro Ubiquitination Activity Assays in Plant Immune Responses.

    PubMed

    Furlan, Giulia; Trujillo, Marco

    2017-01-01

    Ubiquitination is a central posttranslational modification that impinges on the fate of proteins. While attachment of K48-linked chains onto soluble proteins marks them for proteolysis via the 26S proteasome, mono-ubiquitination or K63-linked chains result in the endocytosis and sorting through the endomembrane system of integral membrane proteins, such as pattern recognition receptors. In vitro ubiquitination assays allow the biochemical analysis of all individual components of the ubiquitination machinery and its potential substrates. Here, we describe how to reconstitute the ubiquitination cascade in vitro and detail different variations of the assay, the required controls and how to interpret the obtained results.

  8. High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity

    PubMed Central

    Abdiche, Yasmina Noubia; Miles, Adam; Eckman, Josh; Foletti, Davide; Van Blarcom, Thomas J.; Yeung, Yik Andy; Pons, Jaume; Rajpal, Arvind

    2014-01-01

    Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending

  9. A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.

    PubMed

    Yu, Kai; Hu, Sheng; Huang, Jun; Mei, Le-He

    2011-08-10

    A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK(a) to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

  10. Development of a keratinase activity assay using recombinant chicken feather keratin substrates

    PubMed Central

    Jin, Hyeon-Su; Park, Seon Yeong; Kim, Kyungmin; Lee, Yong-Jik; Nam, Gae-Won; Kang, Nam Joo; Lee, Dong-Woo

    2017-01-01

    Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers. PMID:28231319

  11. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel . E-mail: octave@nchm.ucl.ac.be

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.

  12. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    SciTech Connect

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C.; Miller, R.T.

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  13. Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

    SciTech Connect

    Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.; Bolton, Harvey

    2011-02-01

    Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was added to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.

  14. Cell-based assays in GPCR drug discovery.

    PubMed

    Siehler, Sandra

    2008-04-01

    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.

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

    PubMed Central

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

    2015-01-01

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

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

    PubMed

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

    2014-12-01

    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 list of positive and negative developmental exposures, with exposure defined by toxicokinetic data, specifically maternal plasma Cmax . We selected a series of 20 chemicals that caused developmental toxicity and for which there were appropriate toxicokinetic data. Where possible, we used the same chemical for both positive and negative exposures, the positive being the Cmax at a dose level that produced significant teratogenicity or embryolethality, the negative being the Cmax at a dose level not causing developmental toxicity. It was not possible to find toxicokinetic data at the no-effect level for all positive compounds, and the negative exposure list contains Cmax values for some compounds that do not have developmental toxicity up to the highest dose level tested. This exposure-based reference list represents a fundamentally different approach to the evaluation of alternative tests and is proposed as a step toward application of alternative tests in quantitative risk assessment.

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

    PubMed

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

    2015-03-30

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

  18. A simple liposome assay for the screening of zinc ionophore activity of polyphenols.

    PubMed

    Clergeaud, Gael; Dabbagh-Bazarbachi, Husam; Ortiz, Mayreli; Fernández-Larrea, Juan B; O'Sullivan, Ciara K

    2016-04-15

    An efficient liposomal system for screening the zinc ionophore activity of a selected library consisting of the most relevant dietary polyphenols is presented. The zinc ionophore activity was demonstrated by exploring the use of zinc-specific fluorophore FluoZin-3 loaded liposomes as simple membrane tools that mimic the cell membrane. The zinc ionophore activity was demonstrated as the capacity of polyphenols to transport zinc cations across the liposome membrane and increase the zinc-specific fluorescence of the encapsulated fluorophore FluoZin-3. In addition, the zinc chelation strength of the polyphenols was also tested in a competition assay based on the fluorescence quenching of zinc-dependent fluorescence emitted by zinc-FluoZin-3 complex. Finally, the correlation between the chelation capacity and ionophore activity is demonstrated, thus underlining the sequestering or ionophoric activity that the phenolic compounds can display, thus, providing better knowledge of the importance of the structural conformation versus their biological activity. Furthermore, the assays developed can be used as tools for rapid, high-throughput screening of families of polyphenols towards different biometals.

  19. Automated microfluidic screening assay platform based on DropLab.

    PubMed

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

    2010-12-01

    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.

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

    PubMed

    Hendren, Christine Ogilvie; Lowry, Gregory V; Unrine, Jason M; Wiesner, Mark R

    2015-12-01

    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.

  1. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    PubMed

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins.

  2. Development and evaluation of a new kinetic assay for the quantitation of fibronectin gelatin-binding activity.

    PubMed

    Gelder, F B; Brown, S T; Moore, C A

    1985-10-01

    A new rapid and sensitive kinetic assay that measures the gelatin-binding activity of fibronectin has been developed. This assay is based on the rate of fibronectin-mediated aggregation of covalently coupled latex-gelatin particles. The addition of human plasma and serum resulted in aggregation rates proportional to the concentration of fibronectin in the test sample. This assay was inhibited by the addition of gelatin, demonstrating substrate specificity. This new assay requires 50 microliter of sample and can be performed within 5 minutes. Particle aggregation rates were affected by pH, heparin, and coupled gelatin concentration per milligram of latex. Maximum aggregation rates were observed at pH 8.0. Heparin was not an absolute requirement for particle aggregation but enhanced rates up to 1 U heparin/ml with little additive effect at greater concentrations. Heparin had a relatively greater effect on assays performed in acidic buffers. The concentration of gelatin per milligram of latex was rate limiting up to approximately 50 micrograms gelatin/mg latex with little change in aggregation rates at higher concentrations. Good correlation between total antigenic fibronectin (electroimmunoassay) and fibronectin gelatin-binding activity (latex-gelatin kinetic aggregation assay) was demonstrated in plasma from normal blood donors. This new assay will allow further definition of the relationship between fibronectin gelatin-binding activity and antigenic fibronectin in normal and pathophysiologic states.

  3. Hybridization assay based on evanescent fluorescence excitation and collection

    NASA Astrophysics Data System (ADS)

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

    2003-08-01

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

  4. A homogeneous fluorometric assay platform based on novel synthetic proteins

    SciTech Connect

    Vardar-Schara, Goenuel; Krab, Ivo M.; Yi, Guohua; Su, Wei Wen . E-mail: wsu@hawaii.edu

    2007-09-14

    Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step 'mix and read' noncompetitive homogeneous assay process, based on modulating the Foerster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications.

  5. Sensitive, coupled assay for plasminogen activator using a thiol ester substrate for plasmin

    SciTech Connect

    Coleman, P L; Green, G D.J.

    1980-01-01

    Several assays for plasminogen activator employ a direct assay method. These are remarkably sensitive methods, yet they suffer in comparison to the sensitivity of coupled methods. Coupling the assay with plasminogen not only amplifies the sensitivity by the multiplicative effect of plasmin, but insures that only those proteases specific for plasminogen are assayed. The choice of substrate for plasmin is critical. A thiol ester substrate, thiobenzyl benzyloxy-carbonyl-L-lysinate (Z-Lys-SBzl), has been synthesized which combines high k/sub cat/ with alkaline stability. In an effort to characterize the plasminogen activator from hepatoma tissue culture (HTC) and its hormonally-controlled inhibitor, Z-Lys-SBzl was used in a coupled approach providing an assay which is superior to the /sup 125/I-fibrinolytic assay. It is also extremely sensitive to plasminogen activator and can be used for routine analysis of purification as well as kinetic and binding studies. (ERB)

  6. Development of Tyrosinase Promoter-Based Fluorescent Assay for Screening of Anti-melanogenic Agents.

    PubMed

    Lee, JaeHo; Lee, SeungJun; Lee, ByungMan; Roh, KyungBaeg; Park, DeokHoon; Jung, EunSun

    2015-01-01

    For screening of skin-whitening ingredients that modulate inhibition of melanogenesis, tyrosinase promoter-based assay using a three-dimensional (3D) spheroid culture technique is a beneficial tool to improve the accuracy of raw material screening in cosmetics through mimicking of the in vivo microenvironment. Although the advantages of high-throughput screening (HTS) are widely known, there has been little focus on specific cell-based promoter assays for HTS in identifying skin-whitening ingredients that inhibit accumulation of melanin. The aim of this study was therefore to develop a large-scale compatible assay through pTyr-EGFP, an enhanced green fluorescent protein (EGFP)-based tyrosinase-specific promoter, to seek potential melanogenesis inhibitors for cosmetic use. Herein, a stably transfected human melanoma cell line expressing EGFP under the control of a 2.2-kb fragment derived from the tyrosinase gene was generated. Spontaneous induction of the tyrosinase promoter by 3D spheroid culture resulted in increased expression of EGFP, providing a significant correlation with the tyrosinase mRNA level, and subsequent inhibition of tyrosinase activity. Importantly, the pTyr-EGFP system provided successful tracking of the changes in the live image and real-time monitoring. Thus tyrosinase promoter-based fluorescent assay using a 3D spheroid culture can be useful as a screening system for exploring the efficiency of anti-melanogenesis ingredients.

  7. Survey of estrogenic activity in fish feed by yeast estrogen-screen assay.

    PubMed

    Matsumoto, Takeru; Kobayashi, Makito; Moriwaki, Toshihisa; Kawai, Shin'ichiro; Watabe, Shugo

    2004-10-01

    Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.

  8. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  9. A MEMBRANE FILTER PROCEDURE FOR ASSAYING CYTOTOXIC ACTIVITY IN HETEROTROPHIC BACTERIA ISOLATED FROM DRINKING WATER

    EPA Science Inventory

    Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Wate...

  10. A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.).

    PubMed

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud'homme, Marie-Pascale; van der Graaff, Eric; Roitsch, Thomas

    2015-01-01

    Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.

  11. A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.)

    PubMed Central

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud’homme, Marie-Pascale; Roitsch, Thomas

    2015-01-01

    Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding. PMID:26734049

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

    PubMed

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne; Linde, Viggo; Pedersen, Hans-Christian; Krog-Jensen, Christian; Rosenkilde, Mette M; Pagliaro, Len

    2005-06-01

    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 will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used 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 discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

  13. Estrogenic activity of constituents of underarm deodorants determined by E-Screen assay.

    PubMed

    Lange, Claudia; Kuch, Bertram; Metzger, Jörg W

    2014-08-01

    The purpose of this study was to ascertain whether different kinds of underarm deodorants commercially available in Germany might contain substances with estrogenic potential which after use enter the aquatic environment via wastewater. Twenty five deodorants produced by ten different manufacturers in the form of sprays, roll-ons and sticks were investigated using an in vitro-test system (E-Screen assay) for the determination of estrogenic activity based on the human breast cancer cell line MCF-7. Seven out of ten spray deodorant samples showed a quantifiable estrogenic activity. In the case of the sticks and roll-ons it was only one out of six and one out of nine, respectively. The 17β-estradiol equivalent concentrations (EEQs) of the samples ranged from 0.1 ng g(-1) to 9 ng g(-1) deodorant. Spray deodorant samples showed the highest activities in the E-Screen assay compared to the stick and roll-on deodorants. In order to identify substances possibly contributing to the observed biological activity the samples were additionally analyzed by GC/MS. The obtained results of this non-target screening led to the selection of 62 single substances present in the deodorants which for their part were analyzed by E-Screen assay. Eight of these single substances, all of them fragrances, showed estrogenic effects with estradiol equivalence factors (EEFs) similar to parabens, a group of 4-hydroxybenzoic acid esters commonly used as preservatives in personal care products, which are known to have a slight estrogenic effect. Thus, these fragrances are obviously responsible to a substantial degree for the observed estrogenic activity of the deodorants.

  14. Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout.

    PubMed

    Cabezas, Maria D; Mirkin, Chad A; Mrksich, Milan

    2017-03-08

    Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption-ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies.

  15. TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

    PubMed Central

    Madiraju, Charitha; Welsh, Kate; Cuddy, Michael P.; Godoi, Paulo; Pass, Ian; Ngo, Tram; Vasile, Stefan; Sergienko, Eduard A.; Diaz, Paul; Matsuzawa, Shu-Ichi; Reed, John C.

    2014-01-01

    UBC13 is a non-canonical Ubiquitin Conjugating Enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of Lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair, and thus a potential radiosensitizer and chemosensitizer target for oncology. We developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. We defined conditions for optimized performance of the TR-FRET assay in both 384 and 1536-well formats. Chemical library screens (total 456,865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically > 0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13. PMID:22034497

  16. A fluorescence-based assay for measuring the redox potential of 5-lipoxygenase inhibitors.

    PubMed

    Lee, Sangchul; Park, Youngsam; Kim, Junghwan; Han, Sung-Jun

    2014-01-01

    The activities and side effects of 5-lipoxygenase (5-LO) inhibitors can be predicted by identifying their redox mechanisms. In this study, we developed a fluorescence-based method to measure the redox potential of 5-LO inhibitors and compared it to the conventional, absorbance-based method. After the pseudo-peroxidase reaction, the amount of remaining lipid peroxide was quantified using the H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence dye. Our method showed large signal windows and provided comparable redox potential values. Importantly, the redox mechanisms of known inhibitors were accurately measured with the fluorescence assay, whereas the conventional, absorbance-based method showed contradictory results. Our findings suggest that our developed method is a better alternative for classifying the redox potential of 5-LO inhibitors, and the fluorescence assay can be effectively used to study the mechanisms of action that are related to redox cycling.

  17. In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

    PubMed Central

    Zufferey, Rachel

    2016-01-01

    Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme. PMID:26780155

  18. A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

    PubMed

    Chakrabarti, Subhra; Bhattacharya, Sumana; Bhattacharya, Sanjoy K

    2002-01-01

    A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.

  19. A novel in vitro image-based assay identifies new drug leads for giardiasis.

    PubMed

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

    2017-04-01

    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.

  20. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    PubMed

    Langer, Gernot

    2016-01-01

    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

  1. A cell-based phenotypic assay to identify cardioprotective agents

    PubMed Central

    Guo, Stephanie; Olm-Shipman, Adam; Walters, Andrew; Urciuoli, William R.; Devito, Stefanie; Nadtochiy, Sergiy M.; Wojtovich, Andrew P.; Brookes, Paul S.

    2012-01-01

    Rationale Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). Objective To develop a model of IR injury that is both physiologically relevant and amenable to HTS. Methods and Results A micro-plate based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument’s mechanical systems, yielded a 24 well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. Following initial validation with known protective molecules, the model was used to screen a 2000 molecule library, with post IR cell death as an endpoint. pO2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental and inert molecules from the screen were subsequently tested in a Langendorff perfused heart model of IR injury, revealing strong correlations between the screening endpoint and both recovery of cardiac function (negative r2=0.66), and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics, and protection against IR injury, were also studied. Conclusion This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules. PMID:22394516

  2. The Hornworm Assay: Useful in Mathematically-Based Biological Investigations

    ERIC Educational Resources Information Center

    Rice, Stanley A.; Griffin, Jennifer R.

    2004-01-01

    Hornworms are good assay organisms for leaf toxins, and can be raised on an artificial medium ("chow"), consisting of corn meal, soy flour, dry milk, yeast and other additives and preservatives. The hornworm assay is less useful in ecological and toxicological research, but is very useful in learning about experimental design and hypothesis…

  3. A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases.

    PubMed

    Li, Shu; Gu, X Justin; Hao, Qin; Fan, Hong; Li, Ling; Zhou, Shaolian; Zhao, Kehao; Chan, Ho Man; Wang, Y Karen

    2013-12-15

    Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.

  4. Fluorescence-based assay as a new screening tool for toxic chemicals

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  5. Fluorescence-based assay as a new screening tool for toxic chemicals.

    PubMed

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

    2016-09-22

    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.

  6. Fluorescence-based assay as a new screening tool for toxic chemicals

    PubMed Central

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

    2016-01-01

    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. PMID:27653274

  7. A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.

    PubMed

    Obayashi, Yusuke; Iino, Ryota; Noji, Hiroyuki

    2015-08-07

    Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

  8. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

    PubMed Central

    Suauam, Pitipreya; Yingyongnarongkul, Boon-ek; Palaga, Tanapat; Miyakawa, Tokichi; Yompakdee, Chulee

    2015-01-01

    Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant. PMID:26313553

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

    PubMed

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

    2012-08-21

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

  10. Antiviral activity of a Rac GEF inhibitor characterized with a sensitive HIV/SIV fusion assay

    SciTech Connect

    Pontow, Suzanne; Harmon, Brooke; Campbell, Nancy; Ratner, Lee

    2007-11-10

    A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

  11. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    PubMed Central

    Pugholm, Lotte Hatting; Revenfeld, Anne Louise Schacht; Søndergaard, Evo Kristina Lindersson; Jørgensen, Malene Møller

    2015-01-01

    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. PMID:26770974

  12. A label-free, fluorescence based assay for microarray

    NASA Astrophysics Data System (ADS)

    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

  13. CFU-GM assay for evaluation of drug myelotoxic activity.

    PubMed

    Pessina, Augusto; Bonomi, Arianna

    2007-11-01

    To study hematotoxicity of compounds on the myeloid cell compartment, the authors describe a standard procedure developed as a workable good laboratory practices-compliant protocol to determine the in vitro myelotoxic effect of drugs and chemicals. Specific protocols are presented to prepare human and murine myeloid progenitors (CFU-GM) for testing in a validated CFU-GM assay. Details are given for performing a screening test when toxicity data are not available and for passing on to an accurate inhibitory concentration-determination phase. To quantify the potential hematotoxicity of xenobiotics from their direct adverse effects on CFU-GM, the unit describes how to manage the results by means of an algorithm able to predict the acute xenobiotic exposure levels that cause maximum tolerated decreases (MTD) in absolute neutrophil count (ANC). A protocol describes a miniaturized application of the procedure in 96-well plates for high-throughput screening of compounds or for testing compounds that are available in very small quantities.

  14. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES

    EPA Science Inventory

    A simple and rapid assay to detect DNA damage is reported. This assay is based on the ability of certain dyes to fluoresce upon intercalation with dsDNA. Damage caused by ultraviolet (UV) radiation, chemicals or restriction enzymes is detected using this assay. UV radiation at...

  15. Disk Diffusion Assay to Assess the Antimicrobial Activity of Marine Algal Extracts.

    PubMed

    Desbois, Andrew P; Smith, Valerie J

    2015-01-01

    Marine algae are a relatively untapped source of bioactive natural products, including those with antimicrobial activities. The ability to assess the antimicrobial activity of cell extracts derived from algal cultures is vital to identifying species that may produce useful novel antibiotics. One assay that is used widely for this purpose is the disk diffusion assay due to its simplicity, rapidity, and low cost. Moreover, this assay gives output data that are easy to interpret and can be used to screen many samples at once irrespective of the solvent used during preparation. In this chapter, a step-by-step protocol for performing a disk diffusion assay is described. The assay is particularly well suited to testing algal cell extracts and fractions resulting from separation through bioassay-guided approaches.

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

    PubMed

    Daksis, Jasmine I; Erikson, Glen H

    2007-03-21

    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.

  17. Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

    PubMed Central

    Caers, Jelle; Peymen, Katleen; Suetens, Nick; Temmerman, Liesbet; Janssen, Tom; Schoofs, Liliane; Beets, Isabel

    2014-01-01

    For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their

  18. Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay

    PubMed Central

    Normandin, Karine; Lavallée, Jean-François; Futter, Marie; Beautrait, Alexandre; Duchaine, Jean; Guiral, Sébastien; Marinier, Anne; Archambault, Vincent

    2016-01-01

    Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries. PMID:27874094

  19. A Cell-based PDE4 Assay in 1536-well Plate format for High Throughput Screening

    PubMed Central

    Titus, Steven A.; Li, Xiao; Southall, Noel; Lu, Jianming; Inglese, James; Brasch, Michael; Austin, Christopher P.; Zheng, Wei

    2009-01-01

    The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases including asthma, cardiovascular disease, ADHD, Parkinson’s disease, and Alzheimer’s disease. Though biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. Here we report the development and validation of a new cell-based PDE4 assay using a constitutively active GPCR as a driving force for cAMP production and a cyclic nucleotide gated (CNG) cation channel as a biosensor in 1536-well plates. PMID:18591513

  20. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.

    PubMed

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

    2016-04-01

    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.

  1. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors.

    PubMed

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

    2014-04-01

    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.

  2. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

    PubMed Central

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

    2016-01-01

    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. PMID:26820057

  3. An improvement of Barter's method for assaying plasma cholesterol ester transfer activity: experimental and clinical applications.

    PubMed

    Harvengt, C; Desager, J P; Mailleux, P; Heller, F R

    1989-01-01

    The use of a discontinuous density gradient and of a vertical rotor to separate plasma lipoproteins are modifications of Barter's described method for assaying cholesteryl ester transfer activity (CETA) in plasma. The original feature of our approach is the fast preparation of the labeled substrate by a physiologic-like process, which renders the assay easy and suitable for measurement of this activity in both man and animals.

  4. Microchip-based ultrafast serodiagnostic assay for tuberculosis

    PubMed Central

    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

    2016-01-01

    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

  5. Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

    PubMed

    Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

    2016-11-01

    The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids.

  6. EC50 estimation of antioxidant activity in DPPH· assay using several statistical programs.

    PubMed

    Chen, Zheng; Bertin, Riccardo; Froldi, Guglielmina

    2013-05-01

    DPPH(·) assay is a reliable method to determine the antioxidant capacity of biological substrates. The DPPH(·) radical scavenging activity is generally quantified in terms of inhibition percentage of the pre-formed free radical by antioxidants, and the EC(50) (concentration required to obtain a 50% antioxidant effect) is a typically employed parameter to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC(50) estimation was performed using a comparative approach based on various regression models implemented in six specialized computer programs: GraphPad Prism® version 5.01, BLeSq, OriginPro® 8.5.1, SigmaPlot® 12, BioDataFit® 1.02, and IBM SPSS Statistics® Desktop 19.0. For this project, quercetin, catechin, ascorbic acid, caffeic acid, chlorogenic acid and acetylcysteine were screened as antioxidant standards with DPPH(·) assay to define the EC(50) parameters. All the statistical programs gave similar EC(50) values, but GraphPad Prism® five-parameter analysis pointed out a best performance, also showing a minor variance in relation to the actual EC(50).

  7. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

    PubMed Central

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O.; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  8. A spectrophotometric assay for lipase activity utilizing immobilized triacylglycerols.

    PubMed

    Safarík, I

    1991-01-01

    New substrates for the determination of lipase activity have been developed. Triacylglycerols were immobilized by adsorption on an appropriate carrier or adsorbent yielding a lipase substrate in a powder form. The adsorbed triacylglycerols were easily hydrolyzed by lipases present in a reaction mixture. The released fatty acids were extracted with benzene and converted to the corresponding Cu (II) salts (copper soaps) which were measured spectrophotometrically.

  9. Fluorescence-based retention assays reveals sustained release of vascular endothelial growth factor from bone grafts.

    PubMed

    Kang, Wonmo; Yun, Ye-Rang; Lee, Dong-Sung; Kim, Tae-Hyun; Kim, Joong-Hyun; Kim, Hae-Won; Jang, Jun-Hyeog

    2016-01-01

    The sustained release of growth factors following their implantation in vivo is essential for successful outcomes in bone tissue engineering. In this study, we evaluated the release kinetics and delivery efficacies of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, incorporated into calcium phosphate bone grafts (BGs). We evaluated the release profile of VEGF from BGs using a novel fluorescence-based retention assay, which revealed that VEGF loaded on BGs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. In contrast, an ELISA-based release assay showed VEGF to have an early burst-release profile for the first week. However, the biological activity of VEGF released from the BGs was preserved over the 7-week release period, which is consistent with the sustained-release profile observed in the fluorescence-based retention assay. Furthermore, the in vivo bone-forming action of the VEGF-loaded BGs was well demonstrated in a rat subcutaneous model. Taken together, the sustained release of VEGF loaded onto BGs was effective in stimulating proliferation, angiogenesis and osteogenesis, suggesting the ultimate value of VEGF-engineered BGs for bone tissue engineering.

  10. Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

    PubMed Central

    Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

    2014-01-01

    The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

  11. Field-based multiplex and quantitative assay platforms for diagnostics

    NASA Astrophysics Data System (ADS)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  12. Expanding the available assays: adapting and validating In-Cell Westerns in microfluidic devices for cell-based assays.

    PubMed

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

    2010-10-01

    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.

  13. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

    PubMed Central

    Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.

    2017-01-01

    Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

  14. [Thrombotic Thrombocytopenic Purpura --Pathophysiology and Assays of ADAMTS13 Activity].

    PubMed

    Kato, Seiji; Fujimura, Yoshihiro

    2015-10-01

    Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder classified with a type of thrombotic microangiopathy (TMA). TTP is caused by a deficiency of von Willebrand factor-cleaving protease called ADAMTS13 (a disintegrin-like and metalloprotease with a thrombospondin type1 motif 13). Low ADAMTS13 levels result in increased ultra-large von Willebrand factor multimers (UL-VWFM), which induce platelet adhesion and thrombosis. Congenital TTP (Upshaw-Schulman syndrome: USS) is an inherited disorder of ADAMTS13, and the other more commonly is an acquired TTP caused by autoantibodies against ADAMTS13. This article reviews the progress of ADAMTS13 activity measurement and the resulting changes in the diagnosis and treatment of TTP.

  15. Active nondestructive assay of nuclear materials: principles and applications

    SciTech Connect

    Gozani, Tsahi

    1981-01-01

    The purpose of this book is to present, coherently and comprehensively, the wealth of available but scattered information on the principles and applications of active nondestructive analysis (ANDA). Chapters are devoted to the following: background and overview; interactions of neutrons with matter; interactions of ..gamma..-rays with matter; neutron production and sources; ..gamma..-ray production and sources; effects of neutron and ..gamma..-ray transport in bulk media; signatures of neutron- and photon-induced fissions; neutron and photon detection systems and electronics; representative ANDA systems; and instrument analysis, calibration, and measurement control for ANDA. Each chapter has an introductory section describing the relationship of the topic of that chapter to ANDA. Each chapter ends with a section that summarizes the main results and conclusions of the chapter, and a reference list.

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

    PubMed

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    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.

  17. Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.

    PubMed

    Kim, Joungmok; Park, Hae-Chul; Gedi, Vinayakumar; Park, Hye-Yeon; Roberts, Arthur G; Atkins, William M; Yoon, Moon-Young

    2011-01-07

    Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.

  18. A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol.

    PubMed

    Hassan, Rabeay Y A; Bilitewski, Ursula

    2011-12-01

    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.

  19. Cell-based assays for screening androgen receptor ligands

    PubMed Central

    Campana, Carmela; Pezzi, Vincenzo; Rainey, William E

    2015-01-01

    The androgen receptor (AR, NR3C4), mediates the majority of androgen effects on target cells. The AR is activated following ligand binding that result in activation of target gene transcription. Several cell based model systems have been developed that allow sensitive detection and monitoring of steroids or other compounds with AR bioactivity. Most cell based AR reporter models use transgenic gene constructs that include an androgen response element (ARE) that controls reporter gene expression. The DNA cis-regulatory elements that respond to AR share sequence similarity with cis-regulatory elements for glucocorticoid (GR, NR3C1), mineralocorticoid (MR, NR3C2) and progesterone (PGR, NR3C3) receptors, which has compromised AR selectivity for some models. In recent years, the sensitivity and selectivity of AR bioassays have been significantly improved through careful selection of cell models, utilization of improved reporter genes and the use of yeast two hybrid AR systems. This review summarizes and compares the currently available androgen-responsive cell model systems. PMID:26036905

  20. Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

    PubMed

    Castaman, G; Hillarp, A; Goodeve, A

    2014-05-01

    The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

  1. A fluorescent microsphere-based method for assay of multiple analytes in plasma.

    PubMed

    Bernhard, Oliver K; Mathias, Rommel A; Barnes, Thomas W; Simpson, Richard J

    2011-01-01

    Measurement of multiple analytes can provide increased sensitivity and specificity for the detection and management of disease. The enzyme-linked immunosorbent assay (ELISA) is currently the "gold standard" for protein quantification; however, individual assays for each analyte must be performed, placing demand on sample volume. On the contrary, multiplex assays using microsphere-based technologies allow for multiple analytes to be simultaneously assayed within a single sample. Here, we present a protocol for the preparation and development of a multiple-analyte assay in human plasma using the BioPlex 200 platform (Bio-Rad), which incorporates xMAP technology (Luminex).

  2. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    PubMed

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

  3. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

    PubMed Central

    Jońca, Joanna; Żuk, Monika; Wasąg, Bartosz; Janaszak-Jasiecka, Anna; Lewandowski, Krzysztof; Wielgomas, Bartosz; Waleron, Krzysztof; Jasiecki, Jacek

    2015-01-01

    Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. PMID:26444431

  4. An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

    PubMed

    King, Brian C; Donnelly, Marie K; Bergstrom, Gary C; Walker, Larry P; Gibson, Donna M

    2009-03-01

    Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

  5. Photography by Cameras Integrated in Smartphones as a Tool for Analytical Chemistry Represented by an Butyrylcholinesterase Activity Assay.

    PubMed

    Pohanka, Miroslav

    2015-06-11

    Smartphones are popular devices frequently equipped with sensitive sensors and great computational ability. Despite the widespread availability of smartphones, practical uses in analytical chemistry are limited, though some papers have proposed promising applications. In the present paper, a smartphone is used as a tool for the determination of cholinesterasemia i.e., the determination of a biochemical marker butyrylcholinesterase (BChE). The work should demonstrate suitability of a smartphone-integrated camera for analytical purposes. Paper strips soaked with indoxylacetate were used for the determination of BChE activity, while the standard Ellman's assay was used as a reference measurement. In the smartphone-based assay, BChE converted indoxylacetate to indigo blue and coloration was photographed using the phone's integrated camera. A RGB color model was analyzed and color values for the individual color channels were determined. The assay was verified using plasma samples and samples containing pure BChE, and validated using Ellmans's assay. The smartphone assay was proved to be reliable and applicable for routine diagnoses where BChE serves as a marker (liver function tests; some poisonings, etc.). It can be concluded that the assay is expected to be of practical applicability because of the results' relevance.

  6. Photography by Cameras Integrated in Smartphones as a Tool for Analytical Chemistry Represented by an Butyrylcholinesterase Activity Assay

    PubMed Central

    Pohanka, Miroslav

    2015-01-01

    Smartphones are popular devices frequently equipped with sensitive sensors and great computational ability. Despite the widespread availability of smartphones, practical uses in analytical chemistry are limited, though some papers have proposed promising applications. In the present paper, a smartphone is used as a tool for the determination of cholinesterasemia i.e., the determination of a biochemical marker butyrylcholinesterase (BChE). The work should demonstrate suitability of a smartphone-integrated camera for analytical purposes. Paper strips soaked with indoxylacetate were used for the determination of BChE activity, while the standard Ellman’s assay was used as a reference measurement. In the smartphone-based assay, BChE converted indoxylacetate to indigo blue and coloration was photographed using the phone’s integrated camera. A RGB color model was analyzed and color values for the individual color channels were determined. The assay was verified using plasma samples and samples containing pure BChE, and validated using Ellmans’s assay. The smartphone assay was proved to be reliable and applicable for routine diagnoses where BChE serves as a marker (liver function tests; some poisonings, etc.). It can be concluded that the assay is expected to be of practical applicability because of the results’ relevance. PMID:26110404

  7. The C-Circle Assay for alternative-lengthening-of-telomeres activity.

    PubMed

    Henson, Jeremy D; Lau, Loretta M; Koch, Sylvia; Martin La Rotta, Nancy; Dagg, Rebecca A; Reddel, Roger R

    2017-02-01

    The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.

  8. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized.

  9. Nondestructive assay of TRU waste using gamma-ray active and passive computed tomography

    SciTech Connect

    Roberson, G.P.; Decman, D.; Martz, H.; Keto, E.R.; Johansson, E.M.

    1995-10-04

    The authors have developed an active and passive computed tomography (A and PCT) scanner for assaying radioactive waste drums. Here they describe the hardware components of their system and the software used for data acquisition, gamma-ray spectroscopy analysis, and image reconstruction. They have measured the performance of the system using ``mock`` waste drums and calibrated radioactive sources. They also describe the results of measurements using this system to assay a real TRU waste drum with relatively low Pu content. The results are compared with X-ray NDE studies of the same TRU waste drum as well as assay results from segmented gamma scanner (SGS) measurements.

  10. Nondestructive assay of spent boiling-water-reactor fuel by active neutron interrogation

    SciTech Connect

    Blakeman, E.D.; Ricker, C.W.; Ragan, G.L.; Difilippo, F.C.; Slaughter, G.G.

    1981-01-01

    Spent boiling water reactor (BWR) fuel from Dresden I was assayed for total fissile mass, using the active neutron interrogation method. The nondestructive assay (NDA) system used has four Sb-Be sources for interrogation of the fuels; the induced fission neutrons from the fuel are counted by four lead-shielded methane-filled proportional counters biased above the energy of the source neutrons. Results agreed with results from the chemical analyses to within 2 to 3%. Similar agreement was obtained when two combinations of canned spent fuel were used as standards for the nondestructive assays.

  11. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays.

    PubMed

    Hayat, Akhtar; Cunningham, Jessica; Bulbul, Gonca; Andreescu, Silvana

    2015-07-23

    Nanomaterial-based enzyme mimics have attracted considerable interest in chemical analysis as alternative catalysts to natural enzymes. However, the conditions in which such particles can replace biological catalysts and their selectivity and reactivity profiles are not well defined. This work explored the oxidase like properties of nanoceria particles in the development of colorimetric assays for the detection of dopamine and catechol. Selectivity of the system with respect to several phenolic compounds, the effect of interferences and real sample analysis are discussed. The conditions of use such as buffer composition, selectivity, pH, reaction time and particle type are defined. Detection limits of 1.5 and 0.2μM were obtained with nanoceria for dopamine and catechol. The same assay could be used as a general sensing platform for the detection of other phenolics. However, the sensitivity of the method varies significantly with the particle type, buffer composition, pH and with the structure of the phenolic compound. The results demonstrate that nanoceria particles can be used for the development of cost effective and sensitive methods for the detection of these compounds. However, the selection of the particle system and experimental conditions is critical for achieving high sensitivity. Recommendations are provided on the selection of the particle system and reaction conditions to maximize the oxidase like activity of nanoceria.

  12. Application of gamma-ray active and passive computed tomography to nondestructively assay TRU waste

    SciTech Connect

    Martz, H.E.; Decman, D.J.; Roberson, G.P.; Johansson, E.M.; Keto, E.R.

    1996-05-01

    The authors have developed an active and passive computed tomography scanner for assaying radioactive waste drums. They describe the hardware and software components of the system used for data acquisition, gamma-ray spectroscopy analysis, and image reconstruction. They have measured the performance of the system using mock waste drums and calibrated radioactive sources. They describe the results of measurements using this system to assay a real TRU waste drum with relatively low Pu content.

  13. Pseudotype-Based Neutralization Assays for Influenza: A Systematic Analysis

    PubMed Central

    Carnell, George William; Ferrara, Francesca; Grehan, Keith; Thompson, Craig Peter; Temperton, Nigel James

    2015-01-01

    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 hemagglutinin (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 toward 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–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 HI, single radial hemolysis, and microneutralization. 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. PMID:25972865

  14. Factors affecting a cyanogen bromide-based assay of thiamin.

    PubMed

    Wyatt, D T; Lee, M; Hillman, R E

    1989-11-01

    We analyzed extensively a modified thiochrome method for thiamin analysis. Acid phosphatase (EC 3.1.3.2) from potato was superior to either alpha-amylase or acid phosphatase from wheat germ as a dephosphorylating agent. Timing of cyanogen bromide exposure was important, but the assay had good precision and accuracy. The standard curve was linear from 10 to 3000 nmol/L. The within-run and between-run coefficients of variation for total thiamin in whole blood were 3.6% and 7.4%, respectively. Analytical recoveries for low, intermediate, and high additions of thiamin to whole blood were 93-109%. Sample yield was increased by 41% (+/- 29% SD) with pre-assay freezing. Samples were stable for two days at room temperature, for seven days when refrigerated, and for two years when frozen. Previously unreported interference was seen with penicillin derivatives, and with several commonly used diuretic and antiepileptic medications. This assay may be suitable for population screening; 200 samples could be analyzed weekly at a cost of +0.20 per sample.

  15. Selection and Separation of Viable Cells Based on a Cell-Lethal Assay

    PubMed Central

    Xu, Wei; Herman, Annadele; Phillips, Colleen; Pai, Jeng-Hao; Sims, Christopher E.; Allbritton, Nancy L.

    2010-01-01

    A method to select and separate viable cells based on the results of a cell-lethal assay was developed. Cells were plated on an array of culture sites with each site composed of closely spaced, releasable micropallets. Clonal colonies spanning multiple micropallets on individual culture sites were established within 72 h of plating. Adjacent sites were widely spaced with 100% of the colonies remaining sequestered on a single culture site during expansion. A laser-based method mechanically released a micropallet underlying a colony to segment the colony into two genetically identical colonies. One portion of the segmented colony was collected with 90% efficiency while viability of both fractions was 100%. The segmented colonies released from the array were fixed and subjected to immunofluorescence staining of intracellular phospho-ERK kinase to identify colonies that were highly resistant or sensitive to phorbol ester-induced activation of ERK. These resistant and sensitive cells were then matched to the corresponding viable colonies on the array. Sensitive and resistant colonies on the array were released and cultured. When these cultured cells were reanalyzed for phorbol ester-induced ERK activity, the cells retained the sensitive or resistant phenotype of the originally screened subcolony. Thus cells were separated and collected based using the result of a cell-lethal assay as selection criteria. These microarrays enabling clonal colony segmentation permitted sampling and manipulation of the colonies at very early times and at small cell numbers to reduce reagent, time and manpower requirements. PMID:21142138

  16. High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria

    PubMed Central

    2016-01-01

    In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages. PMID:27275010

  17. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots.

    PubMed

    Ullal, Anirudh J; Millington, David S; Bali, Deeksha S

    2014-01-01

    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.

  18. A Robotic MCF-7:WS8 Cell Proliferation Assay to Detect Agonist and Antagonist Estrogenic Activity

    PubMed Central

    Casey, Warren

    2014-01-01

    Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program’s Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17β-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development. PMID:24213142

  19. A robotic MCF-7:WS8 cell proliferation assay to detect agonist and antagonist estrogenic activity.

    PubMed

    Yang, Chun Z; Casey, Warren; Stoner, Matthew A; Kollessery, Gayathri J; Wong, Amy W; Bittner, George D

    2014-02-01

    Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program's Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17β-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development.

  20. Comparison of enzyme immunoassay–based assays for environmental Alternaria alternata

    PubMed Central

    Barnes, Charles; Portnoy, Jay; Sever, Michelle; Arbes, Samuel; Vaughn, Ben; Zeldin, Darryl C.

    2007-01-01

    Background Alternaria alternata–derived allergenic materials are causes of human disease. Several immunoassays exist to quantify these materials. Objective To compare methods for evaluating Alternaria content. Methods Four methods, including 1 monoclonal antibody (MAb)–based assay specific for recombinant Alt a 1, 1 MAb-based assay for chromatographically purified Alt a 1, 1 polyclonal antibody (PAb)–based assay for chromatographically purified Alt a 1, and 1 PAb-based assay for whole Alternaria extract, were evaluated. Environmental samples collected as part of the National Survey of Lead and Allergens in Housing were examined. Alternaria spore counts were determined in dust by observation. Results The MAb-based assay for recombinant Alt a 1 detected Alternaria in few samples (25%); the PAb-based assay for whole Alternaria proteins detected antigen in 97% of the samples. The PAb- and MAb-based assays for purified Alt a 1 detected antigen in 100% of the samples. There was a significant positive correlation between the 2 assays directed against purified Alt a 1. There was a positive correlation between the PAb-based assay for whole Alternaria and the PAb-based assay for Alt a 1. Nearly all the dust samples contained Alternaria spores, and there was a strong positive correlation between counts and all assays. Conclusion Because of the multifaceted nature of Alternaria, the disparities between methods for quantifying Alternaria, the cross-reactivity between fungal allergens, and the documented genetic promiscuity of this fungus, enzyme immunoassays using PAbs against a range of Alternaria proteins will probably produce the most reliable estimation of overall Alternaria exposure in house dust. PMID:17042141

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

    SciTech Connect

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

    2014-03-14

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

  2. Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors.

    PubMed Central

    Bold, R J; Donoghue, D J

    1985-01-01

    We have constructed retroviral expression vectors by manipulation of the Moloney murine leukemia virus genome such that an exogenous DNA sequence may be inserted and subsequently expressed when introduced into mammalian cells. A series of N-terminal deletions of the v-mos oncogene was constructed and assayed for biological activity with these retroviral expression vectors. The results of the deletion analysis demonstrate that the region of p37mos coding region upstream of the third methionine codon is dispensable with respect to transformation. However, deletion mutants of v-mos which allow initiation of translation at the fourth methionine codon have lost the biological activity of the parental v-mos gene. Furthermore, experiments were also carried out to define the C-terminal limit of the active region of p37mos by the construction of premature termination mutants by the insertion of a termination oligonucleotide. Insertion of the oligonucleotide just 69 base pairs upstream from the wild-type termination site abolished the focus-forming ability of v-mos. Thus, we have shown the N-terminal limit of the active region of p37mos to be between the third and fourth methionines, while the C-terminal limit is within the last 23 amino acids of the protein. PMID:3018503

  3. Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

    PubMed

    Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

    2017-03-15

    Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity.

  4. Cell-based assay protocol for the prognostic prediction of idiopathic scoliosis using cellular dielectric spectroscopy.

    PubMed

    Akoume, Marie-Yvonne; Franco, Anita; Moreau, Alain

    2013-10-16

    This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

  5. A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

    EPA Science Inventory

    In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently...

  6. EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis.

    PubMed

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

    2017-01-01

    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.

  7. Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

    PubMed

    Lumor, Stephen E; Fredrickson, Neal R; Ronningen, Ian; Deen, Bronwyn D; Smith, Kenneth; Diez-Gonzalez, Francisco; Labuza, Theodore P

    2012-06-01

    This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

  8. Analytical Performance and Clinical Utility of a Nucleic Acid Sequence-Based Amplification Assay for Detection of Cytomegalovirus Infection

    PubMed Central

    Witt, Donald J.; Kemper, M.; Stead, Andrew; Sillekens, P.; Ginocchio, Christine C.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.; Roeles, Frits; Caliendo, Angela M.

    2000-01-01

    A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection. PMID:11060058

  9. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro.

    PubMed

    Sabokbar, A; Millett, P J; Myer, B; Rushton, N

    1994-10-01

    Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

  10. Evaluation of a research use only luminex based assay for measurement of procalcitonin in serum samples

    PubMed Central

    Garrigan, Charles; Han, Jennifer; Tolomeo, Pam; Johnson, Katherine J; Master, Stephen R; Lautenbach, Ebbing; Nachamkin, Irving

    2016-01-01

    Research use only (RUO) assays do not undergo a validation process similar to test kits used for clinical purposes. Several studies have suggested that RUO assays need to be validated prior to use in any research studies. We evaluated a research use only Luminex platform based assay for measuring serum procalcitonin levels (Bio-Plex ProTM Human Acute Phase Multiplex Assay, Bio-Rad Laboratories, Hercules, CA) for comparability with an FDA cleared assay for procalcitonin (VIDAS B.R.A.H.M.S. PCT Assay, bioMérieux, Durham, NC). We tested 1,072 serum samples collected from patients with suspected sepsis in an intensive care unit setting for the comparison. There was poor correlation of the luminex based assay (r=0.081) with the VIDAS PCT Assay in the clinically relevant measurement range (<10 ng/mL). Additionally the Bio-Plex assay showed poor precision. Mass-spectrometry analysis of material eluted from PCT beads did not reveal any identifiable procalcitonin. The results show that research use only assays need to be validated to determine their suitability for research studies. PMID:27830020

  11. Receptor-based screening assays for the detection of antibiotics residues - A review.

    PubMed

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

    2017-05-01

    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.

  12. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-09-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.

  13. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

    PubMed

    Koshy, P J; Rowan, A D; Life, P F; Cawston, T E

    1999-11-15

    We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.

  14. Activities of the OECD/NEA Expert Group on Assay Data for Spent Nuclear Fuel

    SciTech Connect

    Gauld, Ian C; Rugama, Yolanda

    2009-01-01

    Management of spent nuclear fuel is a key issue for many NEA member countries. In nuclear criticality safety, the decision of many countries to advance burnup credit as part of their licensing strategy has heightened recent interest in experimental data needed to validate computer codes used in burnup credit calculations. This paper discusses recent activities of an Expert Group on assay data, formed under the OECD/NEA/NSC/WPNCS (Working Party on Nuclear Criticality Safety) to help coordinate isotopic assay data activities and facilitate international collaboration between NEA member countries developing or implementing burnup credit methodologies. Recent activities of the Expert Group are described, focusing on the planned expansion of the Spent Fuel Isotopic Composition Database (SFCOMPO), and preparation of a state-of-the-art report on assay data that includes sections on recommended radiochemical analysis methods, techniques, and lessons learned from previous experiments.

  15. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed Central

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-01-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

  16. Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

    PubMed

    Rücker, Hannelore; Amslinger, Sabine

    2015-01-01

    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

  17. A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

    PubMed Central

    Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

    2010-01-01

    Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

  18. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    SciTech Connect

    Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

    2016-03-15

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  19. Comparison of bee products based on assays of antioxidant capacities

    PubMed Central

    Nakajima, Yoshimi; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Mishima, Satoshi; Hara, Hideaki

    2009-01-01

    Background Bee products (including propolis, royal jelly, and bee pollen) are popular, traditional health foods. We compared antioxidant effects among water and ethanol extracts of Brazilian green propolis (WEP or EEP), its main constituents, water-soluble royal jelly (RJ), and an ethanol extract of bee pollen. Methods The hydrogen peroxide (H2O2)-, superoxide anion (O2·-)-, and hydroxyl radical (HO·)- scavenging capacities of bee products were measured using antioxidant capacity assays that employed the reactive oxygen species (ROS)-sensitive probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) or aminophenyl fluorescein (APF). Results The rank order of antioxidant potencies was as follows: WEP > EEP > pollen, but neither RJ nor 10-hydroxy-2-decenoic acid (10-HDA) had any effects. Concerning the main constituents of WEP, the rank order of antioxidant effects was: caffeic acid > artepillin C > drupanin, but neither baccharin nor coumaric acid had any effects. The scavenging effects of caffeic acid were as powerful as those of trolox, but stronger than those of N-acetyl cysteine (NAC) or vitamin C. Conclusion On the basis of the present assays, propolis is the most powerful antioxidant of all the bee product examined, and its effect may be partly due to the various caffeic acids it contains. Pollen, too, exhibited strong antioxidant effects. PMID:19243635

  20. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    PubMed

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

  1. High-throughput asparaginase activity assay in serum of children with leukemia

    PubMed Central

    Fernandez, Christian A; Cai, Xiangjun; Elozory, Allie; Liu, Chengcheng; Panetta, J Carl; Jeha, Sima; Molinelli, Alejandro R; Relling, Mary V

    2013-01-01

    Asparaginase is an antineoplastic agent used in combination therapy for acute lymphoblastic leukemia (ALL). The asparaginase activity measured in serum reflects the effectiveness of the drug. However, the wide inter-individual variability in the pharmacokinetics of asparaginase suggests that the serum activity should be closely monitored in patients during therapy. In order to identify patients with low asparaginase exposure during treatment, a fast, sensitive, and high-throughput assay is required for measuring asparaginase activity in patient sera. In this study, asparaginase activity was determined by monitoring the enzymatically-coupled oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD+ in a 96-well format. The rate of disappearance of NADH (ΔmOD/minute) was directly proportional to the activity of asparaginase, and the linear range of the assay was established from 0.025 to 2.2 IU/mL (R2 = 0.998) with a reportable range that was extended to 4.0 IU/mL by dilution with serum albumin. Inter-assay precision was established (low control CV% = 8.8, high control CV% = 9.0), as was intra-assay precision (low control CV% = 3.3, high control CV% = 2.7). The method is high-throughput and provides a broader linear range of detection compared to previously described assays. The speed, ease, and accuracy of the assay make it suitable for assessing serum asparaginase activity after standard doses of native E. coli, Erwinia, and PEGylated E. coli asparaginase given to children during the treatment of leukemia. PMID:23936585

  2. Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

    PubMed

    Su, Xin; Li, Zehao; Yan, Xinzhong; Wang, Lei; Zhou, Xu; Wei, Lin; Xiao, Lehui; Yu, Changyuan

    2017-03-21

    Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

  3. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  4. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    PubMed Central

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  5. Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

    PubMed

    Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Lejbkowicz, Flavio; Rennert, Hedy S; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

    2015-09-01

    The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

  6. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

    PubMed

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

    2009-08-31

    A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance

  7. A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

    PubMed

    Kage, Karen L; Richardson, Paul L; Traphagen, Linda; Severin, Jean; Pereda-Lopez, Ana; Lubben, Thomas; Davis-Taber, Rachel; Vos, Melissa H; Bartley, Diane; Walter, Karl; Harlan, John; Solomon, Larry; Warrior, Usha; Holzman, Thomas F; Faltynek, Connie; Surowy, Carol S; Scott, Victoria E

    2007-03-30

    Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

  8. A protein chip membrane-capture assay for botulinum neurotoxin activity

    SciTech Connect

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-12-15

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

  9. Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

    PubMed

    Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro

    2016-12-20

    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 <85>). 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.

  10. A facile low-cost enzymatic paper-based assay for the determination of urine creatinine.

    PubMed

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

    2015-11-01

    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.

  11. A competitive and reversible deactivation approach to catalysis-based quantitative assays

    PubMed Central

    Koide, Kazunori; Tracey, Matthew P.; Bu, Xiaodong; Jo, Junyong; Williams, Michael J.; Welch, Christopher J.

    2016-01-01

    Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation. PMID:26891765

  12. Sensitivity of two in vitro assays for evaluating plant activity against the infective stage of Haemonchus contortus strains.

    PubMed

    Al-Rofaai, A; Rahman, W A; Abdulghani, Mahfoudh

    2013-02-01

    The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.

  13. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES

    EPA Science Inventory

    A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) ra...

  14. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY STYRENE OXIDE

    EPA Science Inventory

    A rapid and simple assay to detect DNA damage to calf thymus DNA caused by styrene oxide (SO) is reported. This assay is based on changes observed in the melting and annealing behavior of the damaged DNA. The melting annealing process was monitored using a fluorescence indicat...

  15. Evaluation of estrogenic activity of parabens and their chlorinated derivatives by using the yeast two-hybrid assay and the enzyme-linked immunosorbent assay.

    PubMed

    Terasaki, Masanori; Kamata, Ryo; Shiraishi, Fujio; Makino, Masakazu

    2009-01-01

    We assessed the estrogen agonist activities of 21 parabens and their chlorinated derivatives by using yeast two-hybrid assays incorporating either the human or medaka (Oryzias latipes) estrogen receptor alpha (hERalpha and medERalpha, respectively), and by using hERalpha competitive enzyme-linked immunosorbent assay (ER-ELISA). In the two-hybrid assay with hERalpha, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17beta-estradiol [E2] = 1) ranged from 2.0 x 10(-5) to 2.0 x 10(-4), with the highest activity observed in i-butylparaben. In the medERalpha assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 x 10(-5) to 3.5 x 10(-3), with the highest activity observed in benzylparaben, its monochlorinated derivative, i-butylparaben, and n-butylparaben. Although medERalpha demonstrated an activity to E2 that was three times lower than that demonstrated by hERalpha, medERalpha has a higher sensitivity to parabens than hERa (1.3-8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ERa in the ER-ELISA; of the parabens, i-butylparaben exhibited the strongest binding affinity. The yeast two-hybrid assay and the ER-ELISA also revealed that many of the assayed chlorinated parabens were much weaker than the parent compound. In addition, the results mainly showed that parabens with a bulk substituent (e.g., i-butyl and benzyl groups) had a higher activity than those with a sterically small substituent. It is considered that derivatization masks the apparent estrogenic activity of parabens, but the resulting chlorinated compounds may represent a potential hazard and therefore other toxicity tests should be performed to determine the toxicity of the chlorinated derivatives.

  16. Nondestructive assay of spent boiling water reactor fuel by active neutron interrogation

    SciTech Connect

    Blakeman, E.D.; Ricker, C.W.; Ragan, G.L.; Difilippo, F.C.; Slaughter, G.G.

    1981-01-01

    Spent boiling water reactor (BWR) fuel from Dresden I was assayed for total fissile mass, using the active neutron interrogation method. The nondestructive assay (NDA) system used has four Sb-Be sources for interrogation of the fuels; the induced fission neutrons from the fuel are counted by four lead-shielded methane-filled proportional counters biased above the energy of the source neutrons. Spent fuel rods containing 9 kg of heavy metal were chopped into 5-cm segments and loaded into three 1-liter cans. The three cans were assayed in seven combinations of one, two, or three cans, enabling an evaluation of the precision and accuracy of the NDA system for different amounts of fissile material. The fissile mass in each combination was determined by comparing the induced-fission-neutron counts with the counts obtained from a known standard comprising chopped segments of unirradiated Dresden fuel. These masses were compared to the masses determined by chemical analyses of the spent fuel. The results from the nondestructive assays agreed with results from the chemical analyses to within 2 to 3%. Similar agreement was obtained when two combinations of canned spent fuel were used as standards for the nondesctuctive assays. The assay of BWR spent fuel served as a test of the NDA system which was developed at the Oak Ridge National Laboratory for the assay of spent liquid metal fast breeder reactor (LMFBR) fuel subassemblies at the heat-end of a reprocessing plant. Results of previous experiments and calculations reported earlier using simulated LMFBR fuel subassemblies indicated that the NDA system can measure the fissile masses of spent fuel subassemblies to within an accuracy of 3%. Results of the assays of spent BWR fuel reported herein support this conclusion.

  17. Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions.

    PubMed

    Mani, Helen; Hesse, Christian; Stratmann, Gertrud; Lindhoff-Last, Edelgard

    2013-01-01

    Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

  18. Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

    PubMed Central

    Gogia, Shobhit; Lo, Chi Y.; Neelamegham, Sriram

    2015-01-01

    Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly

  19. Measuring permeability with a whole cell-based biosensor as an alternate assay for angiogenesis: comparison with common in vitro assays.

    PubMed

    Ghosh, Gargi; Mehta, Ishan; Cornette, Abagail L; Anderson, Kimberly W

    2008-02-28

    Angiogenesis plays cardinal role in normal developmental processes as well as in numerous pathologies. Multiple cytokines are released and act simultaneously to activate endothelial cells in vivo. The present study investigated the relative ability of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-alpha) in modulating cell monolayer permeability, migration, proliferation and tube formation individually and in combination. While the common methods for assaying angiogenesis were conducted for studying cell migration, proliferation and differentiation, endothelial cell monolayer permeability studies were carried out using a whole cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell monolayer permeability dysfunction to detect the presence of small quantities of cytokines. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. The response obtained after exposing this sensor to different cytokines for 1 and 3h, can be attributed to the modulation of monolayer permeability by these cytokines. The present study demonstrated that at the concentrations experimented with, the relative change in permeability assay in the presence of cytokines compared to the control was much higher than that observed in other assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis.

  20. Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides.

    PubMed

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

    2015-06-15

    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.

  1. Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

    USGS Publications Warehouse

    Stavreva, Diana A.; Varticovski, Lyuba; Levkova, Ludmila; George, Anuja A.; Davis, Luke; Pegoraro, Gianluca; Blazer, Vicki; Iwanowicz, Luke R.; Hager, Gordon L.

    2016-01-01

    Even though the presence of endocrine disrupting chemicals (EDCs) with thyroid hormone (TH)-like activities in the environment is a major health concern, the methods for their efficient detection and monitoring are still limited. Here we describe a novel cell assay, based on the translocation of a green fluorescent protein (GFP)—tagged chimeric molecule of glucocorticoid receptor (GR) and the thyroid receptor beta (TRβ) from the cytoplasm to the nucleus in the presence of TR ligands. Unlike the constitutively nuclear TRβ, this GFP-GR-TRβ chimera is cytoplasmic in the absence of hormone while translocating to the nucleus in a time- and concentration-dependent manner upon stimulation with triiodothyronine (T3) and thyroid hormone analogue, TRIAC, while the reverse triiodothyronine (3,3′,5′-triiodothyronine, or rT3) was inactive. Moreover, GFP-GR-TRβ chimera does not show any cross-reactivity with the GR-activating hormones, thus providing a clean system for the screening of TR beta-interacting EDCs. Using this assay, we demonstrated that Bisphenol A (BPA) and 3,3′,5,5′-Tetrabromobisphenol (TBBPA) induced GFP-GR-TRβ translocation at micro molar concentrations. We screened over 100 concentrated water samples from different geographic locations in the United States and detected a low, but reproducible contamination in 53% of the samples. This system provides a novel high-throughput approach for screening for endocrine disrupting chemicals (EDCs) interacting with TR beta.

  2. A rapid and simple assay for growth hormone-binding protein activity in human plasma.

    PubMed

    Baumann, G; Shaw, M A; Amburn, K

    1988-12-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

  3. Immunological-based assays for specific detection of shrimp viruses

    PubMed Central

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-01-01

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  4. A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

    PubMed Central

    Ollinger, Juliane; Bailey, Mai Ann; Moraski, Garrett C.; Casey, Allen; Florio, Stephanie; Alling, Torey; Miller, Marvin J.; Parish, Tanya

    2013-01-01

    Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors. PMID:23593234

  5. Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of a biochar made from switchgrass on four soil enzymes (ß- glucosidase, ß-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Inconsistent results from enzyme assays of char-amended soils s...

  6. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

    PubMed

    Wolfe, Kelly L; Liu, Rui Hai

    2007-10-31

    A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

  7. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gold standard assay for measuring the activity and typing of Clostridium botulinum neurotoxins is the mouse bioassay. The mouse bioassay is sensitive, robust and does not require specialized equipment. However, the mouse bioassay is slow, not practical for many settings and results in the death ...

  8. Activity of the human carcinogen MeCCNU in the mouse bone marrow micronucleus assay

    SciTech Connect

    Tinwell, H.; Ashby, J. )

    1991-01-01

    The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.

  9. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    PubMed

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  10. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    PubMed Central

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

  11. Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter.

    PubMed

    Hu, Qiong; Zhou, Baojing; Dang, Pengyun; Li, Lianzhi; Kong, Jinming; Zhang, Xueji

    2017-01-15

    We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe(3+) to Fe(2+). The complexation of Fe(2+) with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS)3(4-) reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0-220 mU mL(-1) with a detection limit of 0.94 mU mL(-1). Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL(-1) being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing.

  12. Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge.

    PubMed

    Sibag, Mark; Kim, Seung Hwan; Kim, Choah; Kim, Hee Jun; Cho, Jinwoo

    2015-06-01

    ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209). We observed a high degree of interference (90%) in the presence of 100 mg/L silica nanoparticles and a low level of ATP being measured (0.01 μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used.

  13. Portable ceria nanoparticle-based assay for rapid detection of food antioxidants (NanoCerac)

    PubMed Central

    Sharpe, Erica; Frasco, Thalia; Andreescu, Daniel; Andreescu, Silvana

    2012-01-01

    With increased awareness of nutrition and the advocacy for healthier food choices, there exists a great demand for a simple, easy-to-use test that can reliably measure the antioxidant capacity of dietary products. We report development and characterization of a portable nanoparticle based-assay, similar to a small sensor patch, for rapid and sensitive detection of food antioxidants. The assay is based on the use of immobilized ceria nanoparticles, which change color after interaction with antioxidants by means of redox and surface chemistry reactions. Monitoring corresponding optical changes enables sensitive detection of antioxidants in which the nanoceria provides an optical ‘signature’ of antioxidant power, while the antioxidants act as reducing agents. The sensor has been tested for the detection of common antioxidant compounds including ascorbic acid, gallic acid, vanilic acid, quercetin, caffeic acid, and epigallocatechin gallate and its function has been successfully applied for the assessment of antioxidant activity in real samples (teas and medicinal mushrooms). The colorimetric response was concentration dependent, with detection limits ranging from 20–400 μM depending on the antioxidant involved. Steady-state color intensity was achieved within seconds upon addition of antioxidants. The results are presented in terms of Gallic Acid Equivalents (GAE). The sensor performed favorably when compared with commonly used antioxidant detection methods. This assay is particularly appealing for remote sensing applications, where specialized equipment is not available, and also for high throughput analysis of a large number of samples. Potential applications for antioxidant detection in remote locations are envisioned. PMID:23139929

  14. Development of a new catalase activity assay for biological samples using optical CUPRAC sensor.

    PubMed

    Bekdeşer, Burcu; Özyürek, Mustafa; Güçlü, Kubilay; Alkan, Fulya Üstün; Apak, Reşat

    2014-11-11

    A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor-based 'cupric reducing antioxidant capacity' (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-colored Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 μM). This optical sensor-based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC.

  15. Development of a new catalase activity assay for biological samples using optical CUPRAC sensor

    NASA Astrophysics Data System (ADS)

    Bekdeşer, Burcu; Özyürek, Mustafa; Güçlü, Kubilay; Alkan, Fulya Üstün; Apak, Reşat

    2014-11-01

    A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor-based ‘cupric reducing antioxidant capacity' (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-colored Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 μM). This optical sensor-based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC.

  16. In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays.

    PubMed

    Chen, Huang-Han; Hsiao, Yu-Chieh; Li, Jie-Ren; Chen, Shu-Hui

    2015-03-20

    Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) viatert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphorylation activity, which was detected to a 1.5-time lesser extent for the point mutated sequence (LRRGSLG) containing the recognition motif (RRXS), and was nearly undetectable for another point mutated sequence (LRLASLG) that lacked the recognition motif. These results indicate that the reported fabrication method is able to yield highly specific peptide sequences on PDMS, leading to a highly motif-sensitive enzyme activity assay.

  17. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    PubMed

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

    2016-05-01

    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.

  18. A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

    PubMed

    Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

    2016-01-01

    The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.

  19. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

  20. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...

  1. Evaluation of Two PCR-Based Swine-Specific Fecal Source Tracking Assays (Poster)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the specificity and distribution of these targets have not been adequately assessed. Consequently, the utility of these assays in identifying swine fecal contamination...

  2. Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

    PubMed

    Lefkowitz, Roy B; Schmid-Schönbein, Geert W; Heller, Michael J

    2010-07-01

    The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

  3. Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses.

    PubMed

    Sudarshana, M R; Reddy, D V

    1989-10-01

    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.

  4. Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores.

    PubMed

    Ikanovic, Milada; Rudzinski, Walter E; Bruno, John G; Allman, Amity; Carrillo, Maria P; Dwarakanath, Sulatha; Bhahdigadi, Suneetha; Rao, Poornima; Kiel, Johnathan L; Andrews, Carrie J

    2007-03-01

    A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.

  5. An optical assay of the transport activity of ClC-7.

    PubMed

    Zanardi, Ilaria; Zifarelli, Giovanni; Pusch, Michael

    2013-01-01

    Osteoporosis, characterized by excessive osteoclast mediated bone resorption, affects millions of people worldwide representing a major public health problem. ClC-7 is a chloride-proton exchanger localized in lysosomes and in the resorption lacuna in osteoclasts where it is essential for bone resorption. Thus, drugs targeted at ClC-7 have been proposed for ameliorating osteoporosis. However, functional assays suited for high throughput screening (HTS) of ClC-7 function are lacking. Here we describe two complementary variants of purely optical assays of the transport activity of ClC-7, redirected to the plasma membrane employing a genetically encoded fluorescent Cl⁻/pH indicator fused to the ClC-7 protein. These simple and robust functional assays of ClC-7 transport are well-suited to be applied in HTS of small-molecule inhibitors and may help to develop drugs suited for the treatment of osteoporosis.

  6. Real-time ratiometric fluorescent assay for alkaline phosphatase activity with stimulus responsive infinite coordination polymer nanoparticles.

    PubMed

    Deng, Jingjing; Yu, Ping; Wang, Yuexiang; Mao, Lanqun

    2015-03-03

    This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb(3+) as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay.

  7. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

    PubMed

    James, Dominic I; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D; Ogilvie, Donald J

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

  8. A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay.

    PubMed

    Emter, Roger; Natsch, Andreas

    2015-06-01

    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.

  9. Solid-phase assay of lectin activity using HRP-conjugated glycoproteins.

    PubMed

    Kojima-Aikawa, Kyoko

    2014-01-01

    Various enzyme-conjugated probes have been widely used for detection of specific interactions between biomolecules. In the case of glycan-protein interaction, horseradish peroxidase (HRP)-conjugated glycoproteins (HRP-GPs) are useful for the detection of carbohydrate-binding activity of plant and animal lectins. In this chapter, a typical solid-phase assay of the carbohydrate-binding activity of Sophora japonica agglutinin I, a Gal/GalNAc-specific lectin, using HRP-conjugated asialofetuin is described. HRP-GPs are versatile tools for probing lectin activities in crude extracts, screening many samples at one time, and applicable not only for solid-phase binding assays but also samples which are dot- or Western-blotted onto the membrane.

  10. An Alternative Procedure for the Glucose Oxidase Assay of Glucose as Applied to the Lactase Activity Assay

    NASA Astrophysics Data System (ADS)

    Corbin Mullis, T.; Winge, Jeffery T.; Deal, S. Todd

    1999-12-01

    The glucose oxidase assay of glucose has been modified to eliminate the use of micropipets. The modification involves the use of disposable Pasteur pipets and a specified number of drops of each reagent. This simplified technique gives accurate and reproducible results.

  11. Estrogenic and androgenic activity of PCBs, their chlorinated metabolites and other endocrine disruptors estimated with two in vitro yeast assays.

    PubMed

    Svobodová, K; Placková, M; Novotná, V; Cajthaml, T

    2009-11-01

    Investigations of environmental pollution by endocrine-disrupting chemicals are now in progress. Up to now, several in vitro bioassays have been developed for evaluation of the endocrine disruptive activity; however, there is still a lack of comparative studies of their sensitivity. In this work comparison of the estrogen screening assay based on beta-galactosidase expression and a bioluminescent estrogen screen revealed differences in the sensitivity and specificity of the two tests. With the beta-galactosidase screen a slight estrogen-like activity of Delor 103, a commercial mixture of PCB congeners, and a fungicide triclosan was measured whereas no activity was detected using the bioluminescent assay. A bioluminescent androgen test negated previously suggested androgenic potential of triclosan. Further, this work demonstrates the androgenic activity of Delor 103, with an EC(50) value of 2.29 x 10(-2)mg/L. On the other hand, chlorobenzoic acids (CBAs), representing potential PCB degradation metabolites, exhibited no androgenic activity but were slightly estrogenic. Their estrogenicity varied with their chemical structure, with 2,3-CBA, 2,3,6-CBA, 2,4,6-CBA and monochlorinated compounds exhibiting the highest activity. Thus the results indicated possible transitions of the hormonal activity of PCBs during bacterial degradation.

  12. Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

    PubMed Central

    Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok

    2005-01-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748

  13. Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

    PubMed

    Crannell, Zachary Austin; Cabada, Miguel Mauricio; Castellanos-Gonzalez, Alejandro; Irani, Ayesha; White, Arthur Clinton; Richards-Kortum, Rebecca

    2015-03-01

    Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

  14. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    PubMed

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

  15. Predicting fish acute toxicity using a fish gill cell line-based toxicity assay.

    PubMed

    Tanneberger, Katrin; Knöbel, Melanie; Busser, Frans J M; Sinnige, Theo L; Hermens, Joop L M; Schirmer, Kristin

    2013-01-15

    The OECD test guideline 203 for determination of fish acute toxicity requires substantial numbers of fish and uses death as an apical end point. One potential alternative are fish cell lines; however, several studies indicated that these appear up to several orders of magnitude less sensitive than fish. We developed a fish gill cell line-based (RTgill-W1) assay, using several measures to improve sensitivity. The optimized assay was applied to determine the toxicity of 35 organic chemicals, having a wide range of toxicity to fish, mode of action and physicochemical properties. We found a very good agreement between in vivo and in vitro effective concentrations. For up to 73% of the tested compounds, the difference between the two approaches was less than 5-fold, covering baseline toxicants but as well compounds with presumed specific modes of action, including reactivity, inhibition of acetylcholine esterase or uncoupling of oxidative phosphorylation. Accounting for measured chemical concentrations eliminated two outliers, the hydrophobic 4-decylaniline and the volatile 2,3-dimethyl-1,3-butadiene, with an outlier being operationally defined as a substance showing a more than 10-fold difference between in vivo/in vitro effect concentrations. Few outliers remained. The most striking were allyl alcohol (2700-fold), which likely needs to be metabolically activated, and permethrin (190-fold) and lindane (63-fold), compounds acting, respectively, on sodium and chloride channels in the brain of fish. We discuss further developments of this assay and suggest its use beyond predicting acute toxicity to fish, for example, as part of adverse outcome pathways to replace, reduce, or refine chronic fish tests.

  16. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    PubMed

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-03

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

  17. A computer program based on parallel line assay for analysis of skin tests.

    PubMed

    Martín, S; Cuesta, P; Rico, P; Cortés, C

    1997-01-01

    A computer program for the analysis of differences or changes in skin sensitivity has been developed. It is based on parallel line assay, and its main features are its ability to conduct a validation process which ensures that the data from skin tests conform to the conditions imposed by the analysis which is carried out (regression, parallelism, etc.), the estimation of the difference or change in skin sensitivity, and the determination of the 95% and 99% confidence intervals of this estimation. This program is capable of managing data from independent groups, as well as paired data, and it may be applied to the comparison of allergen extracts, with the aim of determining their biologic activity, as well as to the analysis of changes in skin sensitivity appearing as a consequence of treatment such as immunotherapy.

  18. A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

    PubMed Central

    Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

    2015-01-01

    A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

  19. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

    PubMed

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga

    2011-06-01

    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.

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

    PubMed

    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

    2015-01-01

    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 <40 to >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.

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

    PubMed Central

    Wargocki, Piotr; Deng, Wei; Anwer, Ayad G.; Goldys, Ewa M.

    2015-01-01

    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. PMID:26007723

  2. A digital microfluidic method for multiplexed cell-based apoptosis assays.

    PubMed

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R

    2012-02-07

    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.

  3. Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation

    PubMed Central

    Pinne, Marija; Ponce, Elsa; Raucy, Judy L.

    2016-01-01

    The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 μM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 μM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 μM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 μM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR. PMID:27732639

  4. Digital magnetic tagging for multiplexed suspension-based biochemical assays

    NASA Astrophysics Data System (ADS)

    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.

    2009-04-01

    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

  5. FRET-based optical assay for selection of artificial riboswitches.

    PubMed

    Harbaugh, Svetlana V; Chapleau, Molly E; Chushak, Yaroslav G; Stone, Morley O; Kelley-Loughnane, Nancy

    2014-01-01

    Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E. coli cells that is based on TEV protease-FRET substrate reporter system.

  6. Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

    PubMed Central

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q.; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

  7. Active and passive CT for waste assay using LaBr3(Ce) detector

    NASA Astrophysics Data System (ADS)

    Roy, Tushar; More, M. R.; Ratheesh, Jilju; Sinha, Amar

    2017-01-01

    An active and passive computed tomography system has been developed that localizes and quantifies 239Pu in a waste drum. The active (transmission) measurement uses an external gamma source and LaBr3(Ce) detector to determine the attenuation map of waste drum contents at different selected energies. The passive (emission) measurement uses multiple LaBr3(Ce) detectors to record the spectra of gamma-rays emitted from within the drum. The active and passive data sets are then coupled to quantitatively assay drum contents for 239Pu.

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

    PubMed

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

    2016-01-15

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

  9. Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test

    PubMed Central

    2013-01-01

    Background Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as “rattle or rattlesnake” and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of the present study was to investigate the estrogenic potential of C. pallida leaves by means of the Recombinant Yeast Assay (RYA), seeking an alternative for estrogen replacement therapy during menopause; and to reflect on the safe use of natural products to assess the mutagenic activity of the crude extract from C. pallida leaves, the dichloromethane fraction and stigmasterol by means of the Ames test. Methods The recombinant yeast assay with the strain BY4741 of Saccharomyces cerevisiae, was performed with the ethanolic extract, dichloromethane fraction and stigmasterol isolated from the leaves of C. pallida. Mutagenic activity was evaluated by the Salmonella/microsome assay (Ames test), using the Salmonella typhimurium tester strains TA100, TA98, TA97 and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. Results All samples showed estrogenic activity, mainly stigmasterol. The ethanolic extract from C. pallida leaves showed mutagenic activity in the TA98 strain (-S9), whereas dichloromethane fraction and stigmasterol were found devoid of activity. Conclusion Considering the excellent estrogenic activity performed by stigmasterol in the RYA associated with the absence of mutagenic activity when evaluated by the Ames test, stigmasterol becomes a strong candidate to be used in hormone replacement therapy during menopause. PMID:24134316

  10. Use of a Standardized MxA Protein Measurement-Based Assay for Validation of Assays for the Assessment of Neutralizing Antibodies Against Interferon-β

    PubMed Central

    Subramanyam, Meena; Goelz, Susan; Goyal, Jaya; Jethwa, Vijay; Jones, Wendy; Files, James G.; Kramer, Daniel; Bird, Chris; Dilger, Paula; Tovey, Michael; Lallemand, Christophe; Thorpe, Robin

    2013-01-01

    Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-β in multiple sclerosis (MS) patients on IFN-β therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-β products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-β products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-β NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-β1a rather than IFN-β1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-β1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described. PMID:23848523

  11. Development of a quantitative fluorescence-based ligand-binding assay

    PubMed Central

    Breen, Conor J.; Raverdeau, Mathilde; Voorheis, H. Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  12. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  13. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    PubMed

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  14. A high-throughput, modified ALS activity assay for Cyperus difformis and Schoenoplectus mucronatus seedlings.

    PubMed

    Pedroso, Rafael M; Al-Khatib, Kassim; Hanson, Bradley D; Fischer, Albert J

    2017-01-01

    Cyperus difformis L. (CYPDI) and Schoenoplectus mucronatus (L.) Palla (SCHMU) are major weeds of California (CA) rice, where resistance to acetolactate synthase (ALS)-inhibitors was identified in several CYPDI and SCHMU populations that have also evolved resistance to photosystem II (PSII)-inhibiting herbicides. The mechanism of ALS resistance in these populations remains to be clarified but this information is crucial in a weed management program, especially in a scenario where resistance to multiple herbicides has been identified. ALS activity assays are commonly used to diagnose resistance to ALS-inhibitors, but protocols currently available are burdensome for the study of CYPDI and SCHMU, as they require large amounts of plant material from young seedlings and have low yields. Our objective was to investigate the ALS resistance mechanism in suspected ALS-resistant (R) CYPDI and SCHMU biotypes using a modified ALS activity assay that requires less plant material. ALS enzymes from suspected R biotypes were at least 10,000-fold less sensitive to bensulfuron-methyl than susceptible (S) cohorts, indicating ALS resistance that is likely due to an altered target-site. Protein concentration (mgg(-1) tissue) did not differ between R and S biotypes within each species, suggesting that R biotypes do not over produce ALS enzymes. CYPDI biotypes had up to 4-fold more protein per mg of tissue than SCHMU biotypes, but up to 7-fold more acetoin per mg(-1) protein was quantified in SCHMU, suggesting greater ALS catalytic ability in SCHMU biotypes, regardless of their herbicide resistance status. Our optimized protocol to measure ALS activity allowed for up to a 3-fold increase in the number of assays performed per g of leaf tissue. The modified assay may be useful for measuring ALS activity in other weed species that also produce small amount of foliage in early growth stages when protein in tissue is most abundant.

  15. Development and Validation of a Luminescence-based, Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

    PubMed Central

    Lalli, Cristiana; Guidi, Alessandra; Gennari, Nadia; Altamura, Sergio; Bresciani, Alberto; Ruberti, Giovina

    2015-01-01

    Background Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. Methodology/Principal Findings The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. Conclusions The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies. PMID:25635836

  16. Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    PubMed

    Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

    2015-11-01

    Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed.

  17. Flourescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

  18. Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

  19. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    PubMed Central

    Elnager, Abuzar; Hassan, Rosline; Idris, Zamzuri; Mustafa, Zulkifli; Wan-Arfah, Nadiah; Sulaiman, S. A.; Gan, Siew Hua; Abdullah, Wan Zaidah

    2015-01-01

    Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted. PMID:25664321

  20. From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

    PubMed Central

    Kraemer, Stephan; Vaught, Jonathan D.; Bock, Christopher; Gold, Larry; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Saccomano, Nicholas A.; Wilcox, Sheri K.; Zichi, Dom; Sanders, Glenn M.

    2011-01-01

    Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community. PMID:22022604

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

    PubMed Central

    Takatsuki, Hanae; Satoh, Katsuya; Sano, Kazunori; Fuse, Takayuki; Nakagaki, Takehiro; Mori, Tsuyoshi; Ishibashi, Daisuke; Mihara, Ban; Takao, Masaki; Iwasaki, Yasushi; Yoshida, Mari; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission. PMID:26070208

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

    NASA Astrophysics Data System (ADS)

    Fitriastuti, Dhina; Jumina, Priatmoko

    2017-03-01

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

  3. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma

    PubMed Central

    Goiffon, Reece J.; Martinez, Sara C.; Piwnica-Worms, David

    2015-01-01

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l−1 MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders. PMID:25666092

  4. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    SciTech Connect

    Schultz, R.M.; Nanda, S.; Altom, M.G.

    1984-01-01

    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  5. Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases.

    PubMed

    Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Baldwin, Enoch P; Fraser, Christopher S

    2014-07-01

    Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.

  6. Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation.

    PubMed

    Hosomi, Hiroko; Fukami, Tatsuki; Iwamura, Atsushi; Nakajima, Miki; Yokoi, Tsuyoshi

    2011-08-01

    Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6β-hydroxylase activity (670 pmol · min(-1) · mg(-1)). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the drug-induced cytotoxicity associated with CYP3A4.

  7. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

    PubMed

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

    2017-02-01

    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.

  8. In vitro cell-based assays for evaluation of antioxidant potential of plant-derived products.

    PubMed

    Nascimento da Silva, Luís Cláudio; Bezerra Filho, Clovis Macêdo; Paula, Raiana Apolinário de; Silva E Silva, Cristiane Santos; Oliveira de Souza, Larissa Isabela; Silva, Márcia Vanusa da; Correia, Maria Tereza Dos Santos; Figueiredo, Regina Célia Bressan Queiroz de

    2016-08-01

    Several plant-derived compounds have been screened by antioxidant assays, but many of these results are questionable, since they do not evaluate the pharmacologic parameters. In fact, the development of better antioxidants stills a great challenge. In vitro cell-based assays have been employed to assess the antioxidant effect of various compounds at subcellular level. Cell-based assays can also reveal compounds able to enhance the antioxidant pathways, but without direct radical scavenging action (which could not be detected by traditional assays). These methodologies are general of easy implementation and reproducible making them suitable for the early stages of drug discovery. Hydrogen peroxide, a nonradical derivative of oxygen, can be employed as an oxidative agent in these assays due its biochemical properties (presence of all biological systems, solubility) and capacity to induce cell death. Truthfully, if their limitations are understood (such as difference on cell metabolism when in in vitro conditions), these cell-based assays can provide useful information about the pathways involved in the protective effects of phytochemicals against cell death induced by oxidative stress, which can be exploited to develop new therapeutic approaches.

  9. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned.

    PubMed

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

    2008-06-01

    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.

  10. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay

    PubMed Central

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

    2017-01-01

    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. PMID:28270973

  11. A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus.

    PubMed

    Fabbrini, Monica; Sammicheli, Chiara; Margarit, Immaculada; Maione, Domenico; Grandi, Guido; Giuliani, Marzia Monica; Mori, Elena; Nuti, Sandra

    2012-04-30

    Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.

  12. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

    PubMed

    Esposito, Anthony M; Cheung, Pamela; Swartz, Talia H; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D; Basler, Christopher F; Felsenfeld, Dan P; Chen, Benjamin K

    2016-03-01

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

  13. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    PubMed Central

    Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

    2016-01-01

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. PMID:26803470

  14. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    PubMed

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

  15. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

    PubMed Central

    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

    2016-01-01

    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

  16. A ring barrier-based migration assay to assess cell migration in vitro.

    PubMed

    Das, Asha M; Eggermont, Alexander M M; ten Hagen, Timo L M

    2015-06-01

    Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier-based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cells of interest are seeded around this barrier, and after the formation of a peripheral monolayer the barrier is removed and migration into the cell-free area is monitored. This assay is highly reproducible and convenient to perform, and it allows the deduction of several parameters of migration, including total and effective migration, velocity and cell polarization. An advantage of this assay over the conventional scratch assay is that the cells move over an unaltered and virgin surface, and thus the effect of matrix components on cell migration can be studied. In addition, the cells are not harmed at the onset of the assay. Through computer automation, four individual barrier assays can be monitored at the same time. The procedure can be used in a 12-well standard plate allowing higher throughput, or it can be modified to perform invasion assays. The basic procedure takes 2-3 d to complete.

  17. EVALUATION OF NEW 2,2-DIMETHYL-5,5-DIPROPOXYBENZIDINE- AND 3,3-DIPROPOXYBENZIDINE-BASED DIRECT DYE ANALOGS FOR MUTAGENIC ACTIVITY BY USE OF THE SALMONELLA/MAMMALIAN MUTAGENICITY ASSAY

    EPA Science Inventory

    A series of new metallized and unmetallized direct dyes based on benzidine analogs, 2,2'dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenicity in Salmonella typhimurium strains TA98 and TA100. All of the dyes examined were judged non-mutagen...

  18. Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.

    PubMed

    Ramesh Kumar, D; Sanjuktha, M; Rajan, J J S; Ananda Bharathi, R; Santiago, T C; Alavandi, S V; Poornima, M

    2014-09-01

    Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.

  19. Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

    PubMed

    Jenkins, David Paul; Yu, Weifeng; Brown, Brandon M; Løjkner, Lars Damgaard; Wulff, Heike

    2013-01-01

    The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

  20. "Singing in the Tube"--audiovisual assay of plant oil repellent activity against mosquitoes (Culex pipiens).

    PubMed

    Adams, Temitope F; Wongchai, Chatchawal; Chaidee, Anchalee; Pfeiffer, Wolfgang

    2016-01-01

    Plant essential oils have been suggested as a promising alternative to the established mosquito repellent DEET (N,N-diethyl-meta-toluamide). Searching for an assay with generally available equipment, we designed a new audiovisual assay of repellent activity against mosquitoes "Singing in the Tube," testing single mosquitoes in Drosophila cultivation tubes. Statistics with regression analysis should compensate for limitations of simple hardware. The assay was established with female Culex pipiens mosquitoes in 60 experiments, 120-h audio recording, and 2580 estimations of the distance between mosquito sitting position and the chemical. Correlations between parameters of sitting position, flight activity pattern, and flight tone spectrum were analyzed. Regression analysis of psycho-acoustic data of audio files (dB[A]) used a squared and modified sinus function determining wing beat frequency WBF ± SD (357 ± 47 Hz). Application of logistic regression defined the repelling velocity constant. The repelling velocity constant showed a decreasing order of efficiency of plant essential oils: rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus globulus), lavender (Lavandula angustifolia), citronella (Cymbopogon nardus), tea tree (Melaleuca alternifolia), clove (Syzygium aromaticum), lemon (Citrus limon), patchouli (Pogostemon cablin), DEET, cedar wood (Cedrus atlantica). In conclusion, we suggest (1) disease vector control (e.g., impregnation of bed nets) by eight plant essential oils with repelling velocity superior to DEET, (2) simple mosquito repellency testing in Drosophila cultivation tubes, (3) automated approaches and room surveillance by generally available audio equipment (dB[A]: ISO standard 226), and (4) quantification of repellent activity by parameters of the audiovisual assay defined by correlation and regression analyses.

  1. Recent developments in cell-based assays and stem cell technologies for Botulinum neurotoxin research and drug discovery

    PubMed Central

    Kiris, Erkan; Kota, Krishna P.; Burnett, James C.; Soloveva, Veronica; Kane, Christopher D.; Bavari, Sina

    2015-01-01

    Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons, and stem-cell derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors. PMID:24450833

  2. Prompt activation of telomerase by chemical carcinogens in rats detected with a modified TRAP assay.

    PubMed

    Miura, M; Karasaki, Y; Abe, T; Higashi, K; Ikemura, K; Gotoh, S

    1998-05-08

    The maintenance of telomere length is crucial for survival of cells. Telomerase is an RNA-containing reverse transcriptase, which is responsible for elongation of shortened telomeres. Telomerase reactivation has been suggested to be involved in malignant progressions. To study on the involvement of telomerase activation in in vivo carcinogenesis, we first modified the original TRAP assay by changing the primer designs and the labeling method of PCR products to an end-labeling method. Second, we investigated the activation of telomerase in different organs after treatments of rats with various chemical carcinogens. Very early after the beginning of the treatment, telomerase activity in the liver, kidney, and lung was increased. In most cases, telomerase activation occurred in the primary or favorite target organs. The present results suggest that telomerase activation occurs promptly when animals are exposed to chemical carcinogens, which may contribute to in vivo chemical carcinogenesis.

  3. A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate.

    PubMed

    Martínez-Martínez, Irene; Montoro-García, Silvia; Lozada-Ramírez, José Daniel; Sánchez-Ferrer, Alvaro; García-Carmona, Francisco

    2007-10-15

    A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

  4. The Fibrin slide assay for detecting urokinase activity in human fetal kidney cells

    NASA Technical Reports Server (NTRS)

    Sedor, K.

    1985-01-01

    The Fibrin Slide Technique of Hau C. Kwaan and Tage Astrup is discussed. This relatively simple assay involves two steps: the formation of an artificial clot and then the addition of an enzyme (UKOKINASE) to dissolve the clot. The actual dissolving away of the clot is detected by the appearance of holes (lysis zones) in the stained clot. The procedure of Kwaan and Astrup is repeated, along with modifications and suggestions for improvements based on experience with the technique.

  5. Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay

    PubMed Central

    Amakura, Yoshiaki; Tsutsumi, Tomoaki; Yoshimura, Morio; Nakamura, Masafumi; Handa, Hiroshi; Matsuda, Rieko; Teshima, Reiko; Watanabe, Takahiro

    2016-01-01

    The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay. PMID:28231110

  6. A spectrophotometric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase activity.

    PubMed

    Bernal, Cristobal; Mendez, Eva; Terencio, José; Boronat, Albert; Imperial, Santiago

    2005-05-15

    We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.

  7. Long term response of a Concanavalin-A based fluorescence glucose sensing assay

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    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.

  8. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  9. Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease

    PubMed Central

    Hasenkampf, Nicole R.; Barnes, Mary B.; Didier, Elizabeth S.; Philipp, Mario T.; Tardo, Amanda C.

    2016-01-01

    The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. PMID:26843487

  10. In Vitro Assay for the Rap GTPase-Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin.

    PubMed

    Pascoe, Heath G; Wang, Yuxiao; Zhang, Xuewu

    2017-01-01

    Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

  11. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    PubMed

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  12. Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

    PubMed

    Blackford, John A; Brimacombe, Kyle R; Dougherty, Edward J; Pradhan, Madhumita; Shen, Min; Li, Zhuyin; Auld, Douglas S; Chow, Carson C; Austin, Christopher P; Simons, S Stoney

    2014-07-01

    Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

  13. Activation of chemical promutagens by Selenastrum capricornutum in the plant cell/microbe coincubation assay

    SciTech Connect

    Gentile, J.M.; Lippert, M.; Johnson, P.; Shafer, T. )

    1990-05-01

    The critical balance of organisms living in aquatic environments is influenced by the presence and relationship of plants to those environments. However, even though plants occupy a fundamental trophic level within aquatic ecosystems, few studies have focused upon the effect of xenobiotics on aquatic plants, and even fewer studies have dealt with xenobiotic metabolism by aquatic plants. It is well established that plants can metabolize chemicals into mutagens. The impact of these unique plant-activated chemical mutagens on ecosystems, food chains and, ultimately, human health is an important question that will require intensive and integrative investigation. The plant cell/microbe coincubation assay is particularly advantageous for use with unicellular algae. The conditions of this assay are such that chemical metabolism and subsequent mutagen detection can be followed in intact algal cells under simulated field conditions. The purpose of this research was to demonstrate that a unicellular algal species could be used effectively in the plant cell/microbe coincubation assay to activate model chemical mutagens.

  14. A high content assay for biosensor validation and for examining stimuli that affect biosensor activity

    PubMed Central

    Slattery, Scott D.; Hahn, Klaus M.

    2015-01-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor’s maximally activated and inactivated state, and examine response to specific proteins. This involves considerable labor and expense, as expression conditions must be optimized to saturate the biosensor with the regulator, and multiple replicates and controls are required. We describe here a protocol for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays a