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Sample records for assay elisa-like fluorescence

  1. Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    PubMed Central

    Yuan, Han; Tank, Mihir; Alsofyani, Abeer; Shah, Naman; Talati, Nishant; LoBello, Jaclyn C; Kim, Jin Ryoun; Oonuki, Yoji; de la Motte, Carol A; Cowman, Mary K

    2013-01-01

    Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ∼150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20–150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ∼150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases. PMID:23964097

  2. 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. PMID:26677026

  3. LIMITATIONS OF THE FLUORESCENT PROBE VIABILITY ASSAY

    EPA Science Inventory

    Cell viability commonly is determined flow cytometrically by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. FDA is taken up by the viable cell and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF). F fluorescence intensity is...

  4. Fluorescence polarization assays in signal transduction discovery.

    PubMed

    Sportsman, J Richard; Daijo, Janet; Gaudet, Elizabeth A

    2003-05-01

    Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP. PMID:12678698

  5. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

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

    2009-06-29

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

  6. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

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

    2009-06-05

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

  7. Fluorescence Polarization Assays in Small Molecule Screening

    PubMed Central

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  8. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  9. A fluorescence polarization based assay for glucose sensing

    NASA Astrophysics Data System (ADS)

    Cummins, Brian M.; Coté, Gerard L.

    2012-03-01

    A fluorescence polarization (FP) assay was developed to determine concentrations of glucose using concanavalin A (ConA) and fluorescently-labeled dextran. Predictive FP responses to glucose were elicited for different assay configurations using mathematical modeling and displayed herein. Using 4 kDa FITC-dextran, we predicted a change of 0.120 P units from 0 mg/dL glucose to 500 mg/dL. This shows the potential that a homogenous, reproducible FP assay can be engineered to measure glucose concentrations using tetrameric ConA and 4k kDa FITC-dextran.

  10. Rapid fluorescence screening assay for tetracyclines in chicken muscle.

    PubMed

    Schneider, Marilyn J; Lehotay, Steven J

    2004-01-01

    A simple, rapid fluorescence assay was developed for screening tetracyclines in chicken muscle at the U.S. tolerance level (2 mg/kg). The method requires only a homogenization of the tissue in acetonitrile-ammonium hydroxide, centrifugation, addition of Mg+2, and another centrifugation before fluorescence of the supernatant is measured at 505 nm (excitation at 385 nm). Comparison of the fluorescence of control chicken muscle extracts with extracts from muscle fortified with either 2 mg/kg tetracycline, oxytetracycline, or chlortetracycline showed no overlap. A threshold level set at the average fluorescence for a series of fortified 2 mg/kg samples minus 3sigma minimized false-negative responses to provide a successful screening method. The method was tested with blinded samples as controls or samples fortified with tetracycline, oxytetracycline, or chlortetracycline in order to demonstrate its utility. This approach can provide an alternative to microbial screening assays. PMID:15287655

  11. 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

  12. A novel fluorescence-based cellular permeability assay.

    PubMed

    Chandra, Ankur; Barillas, Samuel; Suliman, Ahmed; Angle, Niren

    2007-04-10

    Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated. PMID:16962665

  13. 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. PMID:18596348

  14. Paper-based microfluidic device with upconversion fluorescence assay.

    PubMed

    He, Mengyuan; Liu, Zhihong

    2013-12-17

    A paper-based microfluidic device with upconversion fluorescence assay (named as UC-μPAD) is proposed. The device is fabricated on a normal office printing sheet with a simple plotting method. Upconversion phosphors (UCPs) tagged with specific probes are spotted to the test zones on the μPAD, followed by the introduction of assay targets. Upconversion fluorescence measurements are directly conducted on the test zones after the completion of the probe-to-target reactions, without any post-treatments. The UC-μPAD features very easy fabrication and operation, simple and fast detection, low cost, and high sensitivity. UC-μPAD is a promising prospect for a clinical point-of-care test. PMID:24308347

  15. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    PubMed Central

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  16. Automated fluorescent analysis for drug-induced cytotoxicity assays.

    PubMed

    Funa, K; Dawson, N; Jewett, P B; Agren, H; Ruckdeschel, J C; Bunn, P A; Gazdar, A F

    1986-10-01

    The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug-exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P less than 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency. PMID:3019545

  17. A fluorescence polarization assay for cyclic nucleotide phosphodiesterases.

    PubMed

    Huang, Wei; Zhang, Yan; Sportsman, J Richard

    2002-06-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3'-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented. PMID:12097184

  18. Smartphone-enabled filterless fluorescence assay utilizing the pyrene excimer

    NASA Astrophysics Data System (ADS)

    Goertz, John P.; White, Ian M.

    2015-03-01

    Fluorescence microscopy offers a number of advantages for cell- and biomarker-based diagnostics with regards to ease of use and interpretation, sensitivity, and specificity. However, its use in low-resource settings is often hindered by the need for bulky microscopes with expensive excitation and filter setups. While many advances have been made towards utilizing smartphones as microscopes, there remains a reliance on complex attachments to facilitate fluorescence microscopy. Here, we report progress towards a filter-less fluorescent assay utilizing ultraviolet light, an unmodified smartphone, and pyrene-labeled aptamers. The pyrene monomer is excited at a wavelength of 350 nm and emits at approximately 390 nm; when two pyrene molecules are brought into close proximity, however, they form an excimer which emits at approximately 490 nm. We have engineered pyrene-conjugated DNA sequences such that the fluorophores, normally in monomeric configuration, are brought into proximity upon binding of the DNA to its target. The large Stokes shift between excitation and emission of the excimer allows us to detect such biorecognition events with an unfiltered smartphone camera, enabling the use of this assay in low-resource settings where portability and easeof- use are paramount.

  19. 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.

  20. Fluorescence assays for F-pili and their application.

    PubMed

    Daehnel, Katrin; Harris, Robin; Maddera, Lucinda; Silverman, Philip

    2005-11-01

    Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell-cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0.1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions. PMID:16272377

  1. Cell-Based Lipid Flippase Assay Employing Fluorescent Lipid Derivatives.

    PubMed

    Jensen, Maria S; Costa, Sara; Günther-Pomorski, Thomas; López-Marqués, Rosa L

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid flippase activities in the plasma membrane of cells, using yeast as an example. PMID:26695048

  2. A Sensitive and Versatile Fluorescent Activity Assay for ABHD12.

    PubMed

    Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

    2016-01-01

    Despite great progress in identifying and deorphanizing members of the human metabolic serine hydrolase (mSH) family, the fundamental role of numerous enzymes in this large protein class has remained unclear. One recently found mSH is α/β-hydrolase domain containing 12 (ABHD12) enzyme, whose natural substrate in vivo appears to be the lysophospholipid lysophosphatidylserine (LPS). In vitro, ABHD12 together with monoacylglycerol lipase (MAGL) and ABHD6 hydrolyzes also monoacylglycerols (MAGs) such as the primary endocannabinoid 2-arachidonoyl glycerol (2-AG). Traditional approaches for determining 2-AG hydrolase activity are rather laborious, and often utilize unnatural substrates. Here, we describe a sensitive fluorescent assay of ABHD12 activity in a 96-well-plate format that allows simultaneous testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD12 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred MAG substrate of this enzyme. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. This methodology has helped to identify the first class of inhibitors showing selectivity for ABHD12 over the other mSHs. PMID:27245904

  3. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

    PubMed Central

    McCall, Jennifer R.; Elliott, Elizabeth A.; Bourdelais, Andrea J.

    2014-01-01

    Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine. PMID:25251033

  6. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    PubMed

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  7. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  8. Calcofluor fluorescence assay for wort beta-glucan in a microplate format

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The widely-used fluorescent (Calcofluor) flow injection analysis method for determining the concentrations of beta-glucans in Congress worts from barley malts is adapted to microplate format. Adaptation of the Calcofluor assay to use widely available fluorescent microplate readers makes the assay m...

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

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

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

  10. Novel 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.

  11. Fluorescence-based assays for RGS box function.

    PubMed

    Willard, Francis S; Kimple, Randall J; Kimple, Adam J; Johnston, Christopher A; Siderovski, David P

    2004-01-01

    Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride. PMID:15313559

  12. Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats.

    PubMed

    Miick, Siobhan M; Jalali, Shila; Dwyer, Brian P; Havens, John; Thomas, Donald; Jimenez, Manuel A; Simpson, Mathew T; Zile, Betsy; Huss, Karen L; Campbell, Robert M

    2005-06-01

    A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats. PMID:15964934

  13. 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

  14. Development of a quantitative fluorescence-based ligand-binding assay.

    PubMed

    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

  15. 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...

  16. Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores.

    PubMed

    Schneider, Ralf; Gohla, Anne; Simard, Jeffrey R; Yadav, Dharmendra B; Fang, Zhizhou; van Otterlo, Willem A L; Rauh, Daniel

    2013-06-01

    In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays. PMID:23672540

  17. A triple-color fluorescent probe for multiple nuclease assays.

    PubMed

    Xu, Qinfeng; Zhang, Yihong; Zhang, Chun-yang

    2015-06-01

    We develop a triple-color fluorescent probe which may function as a lab-on-a-DNA-molecule for simultaneous detection of multiple exonucleases/restriction endonucleases. This triple-color fluorescent probe can be further applied for the discrimination of seven exonucleases and four cell lines as well as the screening of various nuclease inhibitors. PMID:25940190

  18. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    SciTech Connect

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-02-20

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

    PubMed

    Lo, Mei-Chu; Aulabaugh, Ann; Jin, Guixian; Cowling, Rebecca; Bard, Jonathan; Malamas, Michael; Ellestad, George

    2004-09-01

    The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed. PMID:15301960

  1. Phytoplankton photosynthetic characteristics from fluorescence induction assays of individual cells

    SciTech Connect

    Olson, R.J.; Chekalyuk, A.M.; Sosik, H.M.

    1996-09-01

    Saturating-flash fluorescence techniques, which can provide information about the physiological state of phytoplankton, at present measure bulk water samples and so provide {open_quotes}averaged{close_quotes} values for all the fluorescent particles present. In analyzing natural samples, however, more detailed information about the distribution of photosynthetic characteristics among different cell types and(or) individual cells is desirable. Therefore we developed two methods for applying a {open_quotes}pump-during-probe{close_quotes} technique on a cell-by-cell basis. We used either an epifluorescence microscope or a flow cytometer to make time-resolved measurements of the increase in chlorophyll fluorescence induced by a rectangular excitation pulse of 100-{mu}s duration. We used a biophysical model of fluorescence induction to obtain information about the quantum yield of photochemistry in photosystem 2 (PS2) and the functional absorption cross-section for PS2. For several species (including the smallest phytoplankton, Prochlorococcus, which are 0.7 {mu}m in diameter), the maximum quantum yield of photochemistry in PS2 obtained by averaging data from many individual cells agreed well with estimates derived from bulk measurements of DCMU enhancement of Chl fluorescence. 40 refs., 9 figs.

  2. 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...

  3. 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...

  4. Quenched near-infrared fluorescent peptide substrate for HIV-1 protease assay

    NASA Astrophysics Data System (ADS)

    Peng, Xinzhan; Draney, Daniel R.; Volcheck, William M.

    2006-02-01

    The HIV-1 protease enzyme is an excellent target for drug therapy of HIV infection/AIDS. To measure the protease activity and screen for potent protease inhibitors, homogeneous protease assays based on quenched fluorescent peptide substrates have been widely used as a high-throughput screening methods. The major problem in these assays is the compound interference or assay artifacts from colored or insoluble materials in the assay, e.g. assay components, screening library compounds, etc. We report in this paper a near-infrared fluorescence resonance energy transfer (NIRFRET) based HIV-1 protease assay that can dramatically reduce or completely eliminate these assay artifacts by using a novel near-IR donor-quencher pair and long wavelength excitation (780 nm) and detection (820+/-10 nm). In this assay, a HIV-1 protease peptide substrate is conjugated with a near-IR fluorescent donor (IRDye TM 800CW), and a novel near-IR non-fluorescent quencher (QC1) on opposite sides of the proteolytic cleavage site. The quencher, QC1, has extremely good spectral overlap of its absorption spectrum with the donor emission spectrum to ensure the efficient quenching of the donor's fluorescence. In the HIV-1 protease assay, this NIR-FRET system shows a large dynamic range, high signal to noise ratio, excellent Z'-factors, a wide range of DMSO tolerance, and no compound interference. This system provides a sensitive, robust assay for high-throughput screening (HTS) and can be readily adapted to other therapeutically significant protease targets.

  5. Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

    PubMed Central

    McCall, Jennifer R.; Jacocks, Henry M.; Baden, Daniel G.; Bourdelais, Andrea J.

    2012-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®-PbTx-2 conjugate was found to displace [3H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various

  6. Influence of fluorescent tag on the motility properties of kinesin-1 in single-molecule assays.

    PubMed

    Norris, Stephen R; Núñez, Marcos F; Verhey, Kristen J

    2015-03-10

    Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SNAP and HALO ligands. We found that although both green and red SNAP ligands provide sufficient fluorescent signal, only the tetramethyl rhodamine (TMR) HALO ligand provides sufficient signal for detection in these assays. This study will serve as a valuable reference for choosing fluorescent labels for single-molecule motility assays. PMID:25762325

  7. Assay of Flippase Activity in Proteoliposomes Using Fluorescent Lipid Derivatives.

    PubMed

    Marek, Magdalena; Günther-Pomorski, Thomas

    2016-01-01

    Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level. PMID:26695033

  8. DNA detection assay based on fluorescence quenching of rhodamine B by gold nanoparticles: The optical mechanisms

    NASA Astrophysics Data System (ADS)

    Pylaev, T. E.; Volkova, E. K.; Kochubey, V. I.; Bogatyrev, V. A.; Khlebtsov, N. G.

    2013-12-01

    The different ability of single- and double-stranded oligonucleotides to stabilize gold nanoparticles (GNPs) in solution has recently been used to design several label-free hybridization assays on the basis of optical changes associated with GNP aggregation. DNA hybridization can be detected through changes in dye fluorescence quenching by GNPs. Here we examine the mechanisms behind a fluorescent DNA assay for model systems containing DNA oligonucleotides, 15-nm GNPs, and Rhodamine B (RB). There was a direct correlation between complete disappearance of fluorescence and complete adsorption of all RB molecules on nonaggregated GNPs, as revealed by an analysis of the colloids' supernatant liquids. We show that both the inner filter effect and the quenching of the dye owing to its adsorption on GNPs contribute to the observed changes in fluorescence intensity. Therefore, both factors should be properly adjusted to optimize the assay sensitivity. In particular, the low detection limit of the fluorescent DNA assay lies in the range 30-100 pM, which is close to the data reported previously for colorimetric and dynamic light scattering DNA assays.

  9. Mutagenicity assays on fluorescent whitening agents using microorganisms.

    PubMed

    Kilbey, B J; Zetterberg, L G

    1975-01-01

    Six fluorescent whitening agents (FWAs) have been re-examined for their activity as inducers of cytoplasmic petite mutants and mitotic gene conversion in diploid yeast Saccharomyces cerebisiae and reversion from auxotrophy to prototrophy in Neurospora crassa, Escherichia coli and Salmonella typhimurium. The results provide no indication that the FWAs examinded produce mutagenic changes or any other alterations in the gene material. In a recent re-examination with Salmonella using the method of Ames et al., the four examined compounds failed to elicit a mutagenic response in the presence of rat liver postmitochondrial supernatant and cofactors. PMID:132347

  10. Homogeneous time resolved fluorescence assay to measure histamine release.

    PubMed

    Claret, Emmanuel J; Ouled-Diaf, Josy; Seguin, Patrick

    2003-12-01

    Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20 micro l does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods. PMID:14683484

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  12. Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag

    PubMed Central

    Gesner, Marianne; Maiti, Mekhala; Grant, Robert; Cavrois, Marielle

    2016-01-01

    The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

  13. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids.

    PubMed

    Wang, Yuhong; Nugen, Sam R

    2013-10-01

    The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used. PMID:23525961

  14. 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.

  15. Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

    PubMed

    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

  16. 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

  17. Assessment of Nuclear Resonance Fluorescence for Spent Nuclear Fuel Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Ambers, Scott

    2011-06-30

    In nuclear resonance fluorescence (NRF) measurements, resonances are excited by an external photon beam leading to the emission of gamma rays with specific energies that are characteristic of the emitting isotope. NRF promises the unique capability of directly quantifying a specific isotope without the need for unfolding the combined responses of several fissile isotopes as is required in other measurement techniques. We have analyzed the potential of NRF as a non-destructive analysis technique for quantitative measurements of Pu isotopes in spent nuclear fuel (SNF). Given the low concentrations of 239Pu in SNF and its small integrated NRF cross sections, the main challenge in achieving precise and accurate measurements lies in accruing sufficient counting statistics in a reasonable measurement time. Using analytical modeling, and simulations with the radiation transport code MCNPX that has been experimentally tested recently, the backscatter and transmission methods were quantitatively studied for differing photon sources and radiation detector types. Resonant photon count rates and measurement times were estimated for a range of photon source and detection parameters, which were used to determine photon source and gamma-ray detector requirements. The results indicate that systems based on a bremsstrahlung source and present detector technology are not practical for high-precision measurements of 239Pu in SNF. Measurements that achieve the desired uncertainties within hour-long measurements will either require stronger resonances, which may be expressed by other Pu isotopes, or require quasi-monoenergetic photon sources with intensities that are approximately two orders of magnitude higher than those currently being designed or proposed.This work is part of a larger effort sponsored by the Next Generation Safeguards Initiative to develop an integrated instrument, comprised of individual NDA techniques with complementary features, that is fully capable of

  18. Protecting Quantum Dot Fluorescence from Quenching to Achieve a Reliable Automated Multiplex Fluorescence In Situ Hybridization Assay.

    PubMed

    Zhang, Wenjun; Hubbard, Antony; Pang, Lizhen; Parkinson, Leslie Baca; Brunhoeber, Patrick; Wang, Yixin; Tang, Lei

    2015-09-01

    Quantum dots (QD) are novel inorganic fluorochromes that are ultra-bright, photo-stable, and available in multiple, highly-resolvable colors. QDs represent an ideal detection material for in situ hybridization (ISH) because they may provide unprecedented resolution and strong signal intensities that are not attainable with traditional fluorophores. Unfortunately, lack of reliability has been an impediment to widespread adoption of QD-based fluorescence in situ hybridization (QD FISH) technology. By optimizing QD-to-target accessibility, we have developed a QD FISH staining procedure that dramatically improves the reliability of an automated ERG/PTEN QD FISH assay (91% 1st pass rate). Here, we report improvements to the assay that protects QD fluorescence from quenching due to trace amounts of heavy metals and minimizes QD background signals. When using this method, highly-consistent staining was observed with the ERG/PTEN QD FISH assay in prostate tissue. Successful staining of several other clinically-relevant genetic markers was also possible. We further demonstrated improved reliability for determining HER2 gene status in breast cancer, identifying anaplastic lymphoma kinase (ALK) gene break-apart in non-small cell lung cancer, and detecting human papillomavirus 16 (HPV16) in cervical intraepithelial neoplasia. The enhanced QD FISH assay allows for examining complicated genetic aberrances without use of enzymatic amplification. Our optimized methods now demonstrate reliability sufficient for QD FISH technology to be a diagnostic tool in a clinical setting. PMID:26485928

  19. Osteoconductivity of Complex Biomaterials Assayed by Fluorescent-Engineered Osteoblast-like Cells.

    PubMed

    Manfrini, Marco; Mazzoni, Elisa; Barbanti-Brodano, Giovanni; Nocini, Pierfrancesco; D'agostino, Antonio; Trombelli, Leonardo; Tognon, Mauro

    2015-04-01

    Biomaterials employed for the bone regeneration can be assayed for specific features such as osteoconductivity and gene expression. In this study, the composite HA/collagen/chondroitin-sulfate biomaterial was investigated using an engineered human cell line, named Saos-eGFP. This cell line, a green fluorescent engineered human osteoblast-like cell, was employed as a cellular model for the in vitro study of biomaterial characteristics. The cytotoxicity was indirectly evaluated by fluorescence detection, osteoconductivity was assayed both by fluorescence and electron microscope analysis as well as cell morphology, whereas the RT-PCR technique was employed to assay gene expression. Saos-eGFP cells viability detection after 24 and 96 h of incubation showed that biomaterial enables the adhesion and proliferation of seeded cells as well as that of the plastic surface, the control. Fluorescence and scanning electron microscopy (SEM) analyses indicated that Saos-eGFP cells were homogeneously distributed on the HA granule surfaces, exhibiting cytoplasmic bridges, and were localized on the collagen-chondroitin sulfate extra-cellular matrix. An expression analysis of specific genes encoding for differentiation markers, showed that biomaterial assayed did not alter the osteogenic pathway of the Saos-eGFP cell line. Our assays confirm the cytocompatibility of this biomaterial, suggesting an osteoconductive capacity mediated by its chemical contents. We showed that the Saos-eGFP cellular model is suitable for in vitro biomaterial assays, and more specifically for assessing osteoconductivity. This result suggests that the cytocompatibility and osteoconductive features of the biomaterial assayed as bone substitute, could have a positive downstream effect on implant osteo-integration. PMID:25388843

  20. Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay.

    PubMed

    Son, Dohyun; Kim, Chung Sub; Lee, Kang Ro; Park, Hyun-Ju

    2016-05-01

    A fluorescence-based cellular assay system was established to identify potential epigenetic modulator ligands. This assay method is to detect the de-repression of an EGFP reporter in cancer cells by the treatment of HDAC (histone deacetylase) or DNMT (DNA methyltransferase) inhibitor. Using this system, we conducted a preliminary screening of in-house natural product library containing extracts and pure compounds, and identified several active compounds. Among them, novel quinic acid derivatives were recognized as excellent HDAC inhibitors by both enzymatic and cell-based HDAC assays. PMID:26996372

  1. DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAY

    EPA Science Inventory

    A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposur...

  2. New artificial fluoro-cofactor of hydride transfer with novel fluorescence assay for redox biocatalysis.

    PubMed

    Zhang, Lei; Yuan, Jun; Xu, Yufang; Zhang, Y-H Percival; Qian, Xuhong

    2016-05-11

    A new artificial fluoro-cofactor was developed for the replacement of natural cofactors NAD(P), exhibiting a high hydride transfer ability. More importantly, we established a new and fast screening method for the evaluation of the properties of artificial cofactors based on the fluorescence assay and visible color change. PMID:27100122

  3. Evaluation of Sustained BMP-2 Release Profiles Using a Novel Fluorescence-Based Retention Assay

    PubMed Central

    Jang, Jun-Hyeog

    2015-01-01

    The purpose of this study was to develop and characterize a novel fluorescence-based retention assay for the evaluation of the release profile of bone morphogenetic protein-2 (BMP-2) released from bone graft carrier. In this study, we evaluated the binding, release kinetics, and delivery efficacies of BMP-2 incorporated into hydroxyapatite (HA) bone grafts. The evaluation of the release profile of BMP-2 from HA bone grafts using a fluorescence-based retention assay revealed initial burst releases from the HA bone grafts followed by long sustained releases up to 14 weeks. The sustained biological activity of the released BMP-2 from HA bone grafts over the full 14-week period supports a long sustained mechanism via fluorescence-based retention assay. Thus, the results from this study show that BMP-2 could be incorporated into HA bone grafts for sustained release over a prolonged period of time with retention of bioactivity and our fluorescence-based retention assay, which is principally detecting the retention profile of BMP-2 in HA bone grafts, is more accurate than conventionally collecting the released BMP-2 for evaluation of BMP-2 release profiles. PMID:25901352

  4. beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.

    PubMed

    Gong, Haibiao; Zhang, Bin; Little, Garrick; Kovar, Joy; Chen, Huaxian; Xie, Wen; Schutz-Geschwender, Amy; Olive, D Michael

    2009-03-01

    beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use. PMID:19103143

  5. Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection.

    PubMed

    Peterson, Eric M; Manhart, Michael W; Harris, Joel M

    2016-06-21

    Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability

  6. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

    PubMed Central

    Chehab, F F; Kan, Y W

    1989-01-01

    We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation. Images PMID:2594760

  7. Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay

    PubMed Central

    Kim, Jinsu; Adhikari, Meena; Dhamane, Sagar; Hagström, Anna E. V.; Kourentzi, Katerina; Strych, Ulrich; Willson, Richard C.; Conrad, Jacinta C.

    2015-01-01

    We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently-labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin-biotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses. PMID:25581289

  8. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    PubMed

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  9. A fluorescence-based assay for human type II phospholipase A2.

    PubMed

    Blanchard, S G; Harris, C O; Parks, D J

    1994-11-01

    A fluorescence assay for quantitation of human Type II Phospholipase A2 activity is described. Hydrolysis of 1-Acyl-2-(N-4-nitrobenzo-2-oxo-1,3-diazole)aminododecanoyl Phosphatidylethanolamine is accompanied by an increase in fluorescence intensity that is linearly proportional to enzyme activity. Substrate is prepared in the absence of detergents as a sonicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slower under identical assay conditions. A plot of initial rate versus substrate concentration could be fit to a simple Michaelis-Menten relationship with Km = 13 microM. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and allows estimation of enzyme activity without separation of substrate from product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue culture plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A2 based on (a) high sensitivity and (b) the use of a commercially available substrate. PMID:7864369

  10. Rapid algal toxicity assay using variable chlorophyll fluorescence for Chlorella kessleri (chlorophyta).

    PubMed

    Kvíderová, Jana

    2010-12-01

    Three methods of algal assays--the standard assay, microassay, and the proposed fluorescence assay--are compared from the point of view of reliability of EC50 detection, the minimum required time for the detection, sensitivity of individual measurement, i.e. at which cell density the particular assay can be used for EC50 estimation, and the time stability of the EC50 values. The assays were performed with green alga Chlorella kessleri strain LARG/1 growing in potassium dichromate solution in Z-medium ranging from 0.01 to 100 mg Cr L⁻¹. The inoculation cell density was set according to the standards to 10⁴ cells mL⁻¹ and according to spectrophotometer/plate reader detection limit. The average EC50 ranged from 0.096 to 0.649 mg Cr L⁻¹ and there were no significant differences in EC50 between the assay type and the inoculation methods with the exception of the significant difference between EC(c)50₇₂ (EC50 established from biomass measured as chlorophyll a concentration after 72 h of cultivation) in the standard assay and EC(r)50 (EC50 derived from growth rate) in the microassay in the standard inoculation experiment due to low variability of their values. The EC(f)50 (EC50 derived from variable fluorescence measurement) values correspond to EC50 values derived from the growth rates. Fluorescence measurement revealed the toxic effect of the chromium after 24 h of exposure at cell density of 5 x 10⁴ cells mL⁻¹, less by half than other used assay methods. The positive correlation of EC(f)50 and time was found in the standard inoculation experiment but opposite effect was observed at the spectrophotometric one. PMID:19551890

  11. Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.

    PubMed

    Ferrer, Marc; Zuck, Paul; Kolodin, Garrett; Mao, Shi Shan; Peltier, Richard R; Bailey, Carolyn; Gardell, Stephen J; Strulovici, Berta; Inglese, James

    2003-06-01

    An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening. PMID:12729605

  12. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. PMID:21277981

  13. [Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay].

    PubMed

    Feng, Weizong; Lin, Junhan; Cai, Shaoli; Zou, Youtu; Chen, Guoren; Huang, Ping; Lin, Yajing; Wang, Bingbing; Lin, Lin

    2011-04-01

    We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement. PMID:21847993

  14. A fluorescence-based assay for Core 1 β3galactosyltransferase (T-synthase) activity.

    PubMed

    Ju, Tongzhong; Cummings, Richard D

    2013-01-01

    Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6-10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays. PMID:23765650

  15. Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

    PubMed Central

    2015-01-01

    The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs. PMID:25226846

  16. Fluorescent vesicles for signal amplification in reverse phase protein microarray assays.

    PubMed

    Bally, Marta; Syed, Shahida; Binkert, Andreas; Kauffmann, Ekkehard; Ehrat, Markus; Vörös, Janos

    2011-09-15

    Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning. PMID:21669176

  17. A Vinblastine Fluorescent Probe for Pregnane X Receptor in a Time-Resolved Fluorescence Resonance Energy Transfer Assay

    PubMed Central

    Lin, Wenwei; Chen, Taosheng

    2013-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and non-radioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand-PXR interactions. Here we report the characterization of BODIPY FL Vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a Kd value of 673 nM. We provide evidence that BODIPY FL Vinblastine is a unique chemical entity different from either vinblastine or the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene in its function as a high-affinity human PXR ligand. We describe a BODIPY FL Vinblastine-based human PXR Time-Resolved Fluorescence Resonance Energy Transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL Vinblastine–based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR. PMID:24044991

  18. Azadioxatriangulenium (ADOTA+): A long fluorescence lifetime fluorophore for large biomolecule binding assay

    PubMed Central

    Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

    2013-01-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes. PMID:24058730

  19. Enhanced fluorescence anisotropy assay for human cardiac troponin I and T detection.

    PubMed

    Qiao, Yanling; Tang, Hongmin; Munske, Gerhard R; Dutta, Prashanta; Ivory, Cornelius F; Dong, Wen-Ji

    2011-11-01

    Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5-40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15-500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing. PMID:21647606

  20. Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection

    PubMed Central

    Qiao, Yanling; Tang, Hongmin; Munske, Gerhard R.; Dutta, Prashanta; Ivory, Cornelius F.

    2012-01-01

    Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5–40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15–500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing. PMID:21647606

  1. Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases.

    PubMed

    Rojas, Rafael J; Kimple, Randall J; Rossman, Kent L; Siderovski, David P; Sondek, John

    2003-06-01

    Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases. PMID:12769685

  2. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    PubMed

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

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection. PMID:27283884

  3. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.

    PubMed

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-06-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop-loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop-loop interactions in hammerhead ribozymes. PMID:26939886

  4. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  5. A Microfluidic Microbeads Fluorescence Assay with Quantum Dots-Bead-DNA Probe.

    PubMed

    Ankireddy, S R; Kim, Jongsung

    2016-03-01

    A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA. PMID:27455729

  6. Quantitative Fluorescence Assays Using a Self-Powered Paper-Based Microfluidic Device and a Camera-Equipped Cellular Phone.

    PubMed

    Thom, Nicole K; Lewis, Gregory G; Yeung, Kimy; Phillips, Scott T

    2014-01-01

    Fluorescence assays often require specialized equipment and, therefore, are not easily implemented in resource-limited environments. Herein we describe a point-of-care assay strategy in which fluorescence in the visible region is used as a readout, while a camera-equipped cellular phone is used to capture the fluorescent response and quantify the assay. The fluorescence assay is made possible using a paper-based microfluidic device that contains an internal fluidic battery, a surface-mount LED, a 2-mm section of a clear straw as a cuvette, and an appropriately-designed small molecule reagent that transforms from weakly fluorescent to highly fluorescent when exposed to a specific enzyme biomarker. The resulting visible fluorescence is digitized by photographing the assay region using a camera-equipped cellular phone. The digital images are then quantified using image processing software to provide sensitive as well as quantitative results. In a model 30 min assay, the enzyme β-D-galactosidase was measured quantitatively down to 700 pM levels. This Communication describes the design of these types of assays in paper-based microfluidic devices and characterizes the key parameters that affect the sensitivity and reproducibility of the technique. PMID:24490035

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

    PubMed

    Vuojola, Johanna; Riuttamäki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero

    2012-05-01

    Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC(50) values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an internally quenched substrate molecule diminishes the background resulting from radiatively excited acceptor molecules. The reduction of autofluorescence and radiative background result in high signal-to-background ratios (ratios of approximately 100 were obtained). By further utilizing assay miniaturization and signal enhancement in a white microtitration plate, a significant reduction in the reagent consumption can be achieved rendering the assay applicable for high-throughput screening. PMID:22502613

  8. Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate.

    PubMed

    Gong, Haibiao; Little, Garrick; Cradduck, Mark; Draney, Daniel R; Padhye, Nisha; Olive, D Michael

    2011-05-15

    Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit. PMID:21482307

  9. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.

    PubMed

    Borg-von Zepelin, M; Wagner, T

    1995-01-01

    We describe an assay based on photometric analysis for the measurement of adherence of Candida species to epithelial target cells (Vero cell line). Adherent Candida cells were detected by staining the cells with the fluorescent dye Calcofluor white (CFW), which binds to chitin and glucan in the yeasts. The tests were performed on microtest plates, which were analysed automatically by fluorescence plate readers. The assay is based on the following steps: (i) coating of the microtest plates with target cells (e.g. Vero cells); (ii) infection with Candida: (iii) staining of Candida with CFW; (iv) rinsing to remove non-adherent Candida cells and unbound dye; (v) detection of adherent fluorescent Candida cells. The test was able to detect 4 x 10(4) cells ml-1. The standard deviation was +/- 8%. Day-to-day variation was +/- 10% at most. The adherence of strains of different Candida species was assayed by a standard procedure. The results confirmed the order of adherence, with C. albicans ranking first, followed by C. tropicalis, C. parapsilosis and C. glabrata. PMID:8569807

  10. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    PubMed

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  11. Real-time quantification of fatty acid uptake using a novel fluorescence assay.

    PubMed

    Liao, Jinfang; Sportsman, Richard; Harris, Jeff; Stahl, Andreas

    2005-03-01

    Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery. PMID:15547301

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

    PubMed

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

    2016-06-15

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

  13. A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells.

    PubMed

    Gasser, Jean-Philippe; Hehl, Michaela; Millward, Thomas A

    2009-01-01

    A simple, "mix-and-measure" microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z' values of 0.6-0.7. PMID:18835236

  14. 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. PMID:26179334

  15. A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

    PubMed Central

    Eggleston, A K; Rahim, N A; Kowalczykowski, S C

    1996-01-01

    We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID:8614617

  16. A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

    PubMed

    Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

    2012-06-01

    N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. PMID:22410281

  17. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

    PubMed

    Farino, Zachary J; Morgenstern, Travis J; Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A; Freyberg, Zachary

    2016-01-01

    Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

  18. A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis

    PubMed Central

    Jenkins, Ronald J.; Dotson, Garry D.

    2012-01-01

    UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of Lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among Gram-negative bacteria which synthesize lipopolysaccharide, and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP) dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Herein, we exploit the single free-thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol specific labeling reagent, ThioGlo. The assay is continuously monitored as a change in fluorescence at λex = 379 nm and λem = 513 nm using a microtiter plate reader. This assay marks the first continuous and non-radioactive assay for either acyltransferase. PMID:22381368

  19. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

    PubMed Central

    Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E.; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A.; Freyberg, Zachary

    2016-01-01

    Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

  20. Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase

    PubMed Central

    Bulfer, Stacie L.; McQuade, Thomas J.; Larsen, Martha J.; Trievel, Raymond C.

    2011-01-01

    Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism. PMID:21073853

  1. New In Vitro Phenotypic Assay for Epilepsy: Fluorescent Measurement of Synchronized Neuronal Calcium Oscillations

    PubMed Central

    Pacico, Nathalie; Mingorance-Le Meur, Ana

    2014-01-01

    Research in the epilepsy field is moving from a primary focus on controlling seizures to addressing disease pathophysiology. This requires the adoption of resource- and time-consuming animal models of chronic epilepsy which are no longer able to sustain the testing of even moderate numbers of compounds. Therefore, new in vitro functional assays of epilepsy are needed that are able to provide a medium throughput while still preserving sufficient biological context to allow for the identification of compounds with new modes of action. Here we describe a robust and simple fluorescence-based calcium assay to measure epileptiform network activity using rat primary cortical cultures in a 96-well format. The assay measures synchronized intracellular calcium oscillations occurring in the population of primary neurons and is amenable to medium throughput screening. We have adapted this assay format to the low magnesium and the 4-aminopyridine epilepsy models and confirmed the contribution of voltage-gated ion channels and AMPA, NMDA and GABA receptors to epileptiform activity in both models. We have also evaluated its translatability using a panel of antiepileptic drugs with a variety of modes of action. Given its throughput and translatability, the calcium oscillations assay bridges the gap between simplified target-based screenings and compound testing in animal models of epilepsy. This phenotypic assay also has the potential to be used directly as a functional screen to help identify novel antiepileptic compounds with new modes of action, as well as pathways with previously unknown contribution to disease pathophysiology. PMID:24416277

  2. Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease.

    PubMed

    Embers, Monica E; Hasenkampf, Nicole R; Barnes, Mary B; Didier, Elizabeth S; Philipp, Mario T; Tardo, Amanda C

    2016-04-01

    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 fromBorrelia 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 fiveB. burgdorferiantigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection ofB. burgdorferiantigen-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

  3. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    PubMed

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

    2016-04-01

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

  4. Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence.

    PubMed

    Yang, Xiaoming; Zhuo, Yan; Zhu, Shanshan; Luo, Yawen; Feng, Yuanjiao; Xu, Yan

    2015-02-15

    Herein, we have successfully built up connections between nanoparticles and nanoclusters, and further constructed a surface-enhanced fluorescence (SEF) strategy based on the two types of nanomaterials for selectively assaying carcinoembryonic antigen (CEA). Specifically, silver nanoclusters provided the original fluorescence signal, while gold nanoparticles modified with DNA served as the fluorescence enhancer simultaneously. On the basis of this proposed nano-system, the two nanomaterials were linked by CEA-aptamer, thus facilitating SEF occurring. Nevertheless, more competitive interactions between CEA and CEA-aptamer emerged once CEA added, leading to SEF failed and their fluorescence decreased. Significantly, this creative method was further applied to detect CEA, and showed the linear relationship between the fluorescence intensity and CEA concentrations in the range of 0.01-1 ng mL(-1) with a detection limit of 3 pg mL(-1) at a signal-to-noise ratio of 3, demonstrating its sensitivity and promising towards multiple applications. On the whole, this approach we established may broaden potential ways of combining nanoparticles and nanoclusters for detecting trace targets in bioanalytical fields. PMID:25259877

  5. Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor.

    PubMed

    Xiao, Sai Jin; Hu, Ping Ping; Li, Yuan Fang; Huang, Cheng Zhi; Huang, Tao; Xiao, Geng Fu

    2009-10-15

    An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP(C)) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3'-terminal, and a fluophor modified at 5'-terminal. It was found that the guanine bases at the 3'-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5'-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP(C) in the range from 1.1 to 44.7 microg/mL with a limit of detection of 0.3 microg/mL (3sigma). PMID:19635360

  6. Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders.

    PubMed

    Lu, Hao; Kopcho, Lisa; Ghosh, Kaushik; Witmer, Mark; Parker, Michael; Gupta, Sumit; Paul, Marilyn; Krishnamurthy, Prasad; Laksmaiah, Basanth; Xie, Dianlin; Tredup, Jeffrey; Zhang, Litao; Abell, Lynn M

    2016-05-15

    Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies. PMID:26874021

  7. A fluorescence turn on assay for alkaline phosphatase based on the Cu(2+) catalyzed Fenton-like reaction.

    PubMed

    Zhang, Qingfeng; Zhang, Cuiyun; Shahzad, Sohail Anjum; Yu, Cong

    2016-09-01

    A fluorescence turn-on assay was established for ALP (alkaline phosphatase) based on Cu(2+) catalyzed Fenton-like reaction and Graphene Oxide (GO). GO was utilized to quench the fluorescence of fluorescein (FAM) labeled single strand DNA (F-DNA). ALP can remove the phosphate group in sodium ascorbyl phosphate (SAP), and convert it into reducing ascorbate. Highly reactive hydroxyl radicals (·OH) were generated in the presence of ascorbate and Cu(2+) through the Fenton-like reaction. The reactive radicals generated in situ caused the cleavage of F-DNA into small fragments. When GO was added, the fluorescence emission of the sample without ALP was quenched and fluorescence emission recovered in the presence of ALP. The intensity of the recovered fluorescence was directly related to the concentration of ALP in the assay solution, and a sensitive and selective facile ALP assay is therefore established. PMID:27343614

  8. Establishment of a New Cell-Based Assay To Measure the Activity of Sweeteners in Fluorescent Food Extracts

    PubMed Central

    2011-01-01

    Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca2+ concentration using Ca2+-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca2+-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca2+ indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. PMID:21981007

  9. Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

    PubMed

    Toda, Yasuka; Okada, Shinji; Misaka, Takumi

    2011-11-23

    Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca(2+) concentration using Ca(2+)-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca(2+)-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca(2+) indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. PMID:21981007

  10. Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

    PubMed Central

    Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barcelo, Nicolas; Lo, Wai; Wronska, Danuta; Kakkilaya, Srinivas; Rao, Aravinda; Bhat, Shalia T.; Sinha, Ruchi; Omar, Sabah; Moro, Manuel; Gilman, Robert H.; Harris, Nick

    2015-01-01

    Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively. PMID:26333092

  11. A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

    PubMed Central

    van der Wel, Tom; Janssen, Freek J.; Baggelaar, Marc P.; Deng, Hui; den Dulk, Hans; Overkleeft, Herman S.; van der Stelt, Mario

    2015-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors. PMID:25684760

  12. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    PubMed Central

    2010-01-01

    Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology. PMID:20840797

  13. A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

    SciTech Connect

    Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. Black-Right-Pointing-Pointer We have established an activity assay for ubiquitin C-terminal hydrolases. Black-Right-Pointing-Pointer This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub{sup F45W}-Xaa) and di-ubiquitin chains (Ub{sup F45W}-diUb). After removal of the intact substrate by Ni{sup 2+}-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub{sup F45W} product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

  14. An Affinity-Based Fluorescence Polarization Assay for Protein Tyrosine Phosphatases

    PubMed Central

    Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin

    2007-01-01

    Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity. PMID:17532513

  15. Measurement of glycated haemoglobin in whole blood by a novel fluorescence quenching assay.

    PubMed

    Blincko, S; Anzetse, J; Edwards, R

    2000-07-01

    We describe a method for the specific measurement of glycated Hb (GHb) by fluorescence quenching. Whole blood is added to lysing solution, then the lysate is mixed with eosin-boronic acid solution and reacted for at least 5 min at room temperature. The quenching of the fluorescence of the eosin-boronic acid solution is proportional to the concentration of GHb present. Total Hb concentration was measured by absorbance and the GHb expressed as a percentage of the total Hb. Comparison with a commercial high-performance liquid chromatography (HPLC) system for HbA1c showed: %GHb=1.30 (SD 0.04) %HbA1c + 1.36 (SD 0.30), S(y/x) 0.803, n=95, r=0.965 (SD=standard deviation). Intra-assay coefficients of variation were <2.5% (for GHb concentrations in the range 6-20%) and inter-assay coefficients of variation were <4.1% (10 assays on six samples with GHb concentrations in the range 6-20%). Linearity of response was demonstrated by dilution. The effect of adding exogenous glucose, bilirubin and triglycerides was tested on samples with low, medium and high GHb concentrations. No significant interference was found. Variation of haematocrit over the range 0.4-0.6 also had no significant effect on percentage GHb. Preliminary results with samples containing variant Hb (HbAS and HbAC) indicated good agreement with HPLC for these samples also. PMID:10902866

  16. A Rapid Fluorescence Assay for Danofloxacin in Beef Muscle. Effect of Muscle Type on Limit of Quantitation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple, rapid fluorescence screening assay was applied to the analysis of beef muscle for danofloxacin at the U.S. tolerance level of 200 ng/g. Muscle samples were homogenized in acetic acid/acetonitrile, the resultant mixture centrifuged, and fluorescence of the supernatants was then measured. ...

  17. Sensitive detection of p53 antibodies in a homogeneous fluorescence assay format

    NASA Astrophysics Data System (ADS)

    Neuweiler, Hannes; Schulz, Andreas; Wolfrum, Juergen M.; Sauer, Markus

    2002-06-01

    Circulating p53 autoantibodies are found to be a universal and highly specific tumor marker for malignant diseases. Hence, sereological screening for p53 autoantibodies at low concentration levels has become increasingly relevant for early-stage and follow-up of tumor diagnostics. We developed a new method for the highly sensitive detection of p53 antibodies in a homogeneous fluorescence assay format. Short, linear peptide derived form antibody recognition sequences so human p53 were labeled with an oxazine dye. Hydrophobic interactions constrain a conformation, where the dye interacts selectively with a tryptophan residue in the peptide sequence. Subsequently, the fluorescence of the dye is quenched efficiently due to electron transfer from the indole derivative to the dye in the excited state. Specific antibody recognition induces a conformational change in the peptide structure, repealing the dye-tryptophan interaction. Consequently, a fluorescence increase upon antibody binding signals the binding event. The long-wavelength absorption and emission characteristics of the probe and the use of a red pulsed diode laser as excitation source in a confocal fluorescence microscopic set-up allows ultra sensitive antibody detection at the single-molecule level. The effectiveness of the probes are highlighted by the detection of individual p53 autoantibodies directly in serum dilutions of cancer patients.

  18. New applications of fluorescence polarization for enzyme assays and in genomics

    NASA Astrophysics Data System (ADS)

    Nikiforov, Theo; Coffin, Jill; Jeong, Sang; Simeonov, Anton; Bi, Xiahui

    2001-05-01

    We have developed new, fluorescence polarization-based approaches for performing enzyme assays in homogeneous solutions and for detecting the hybridization of peptide nucleic acids to DNA targets. In the first method, fluorescein-labeled peptides serving as protein kinase sustrates are thiophosphorylated in the presence of the ATP analog ATPγS. A sulfer-reactive biotin derivative is then added to the mixture and allowed to react with the thiophosphorylated peptide. The formation of a fluorescein-labeled, biotinylated product can be detected by measuring the fluorescence polarization signal of fluorescein upon addition of streptavidin. In the second method, fluorescein-labeled peptides are subjected to enzymatic phosphorylation, desphosphorlation, or proteollytic cleavage by protein kinases, phosphatases, and proteases. The differential binding of the enzymatic substrates and products to cationic polymers such as polyaraginine can be conveniently measured by fluorescence polarization. Finally, we have discovered that the process of hybridization of peptide nucleic acid probes (PNAs) to their target DNA molecules can be followed by measuring the fluorescence polarization of a fluorophore attached to the PNA probes. These measurements can be done either in the presence or absence of polylysine in solution. Examples of the application of this method for single nucleotide polymorphism (SNP) typing are presented.

  19. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    PubMed Central

    2011-01-01

    A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting. PMID:22011343

  20. Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells.

    PubMed

    Blaszczak, Ewa; Prigent, Claude; Rabut, Gwenaël

    2016-01-01

    Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism. PMID:27613039

  1. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  2. GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

    PubMed Central

    Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

    2014-01-01

    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

  3. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

    PubMed

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  4. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors

    PubMed Central

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A.

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  5. Quantitative ELISA-Like Immunohistochemistry of Fibroblast Growth Factor 23 in Diagnosis of Tumor-Induced Osteomalacia and Clinical Characteristics of the Disease

    PubMed Central

    Hu, Fangke; Jiang, Chengying; Zhang, Qiang; Shi, Huaiyin; Wei, Lixin

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disorder and fibroblast growth factor 23 (FGF23) plays a key role in its pathogenesis. This study was conducted to describe a novel FGF23 detecting procedure and describe clinical features of the disease. Fourteen TIO cases were retrieved and FGF23 expression was measured by quantitative ELISA-like immunohistochemistry using formalin-fixed and paraffin-embedded tissues. As summarized from 14 TIO cases, clinical features of TIO were long-standing history of osteomalacia, hypophosphatemia, and urinary phosphate wasting. The associated tumors were mostly benign phosphaturic mesenchymal tumors mixed connective tissue variant (PMTMCT) which could be located anywhere on the body, and most of them could be localized by conventional examinations and octreotide scanning. By quantitative ELISA-like immunohistochemistry, all the 14 TIO cases had high FGF23 expression (median 0.69, 25%–75% interquartile 0.57–1.10, compared with 26 non-TIO tumors of median 0.07, 25%–75% interquartile 0.05–0.11, p < 0.001). The quantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect the high FGF23 expression in formalin-fixed and paraffin-embedded biopsies or specimens. Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of TIO and PMTMCT, respectively. PMID:27034530

  6. Quantitative ELISA-Like Immunohistochemistry of Fibroblast Growth Factor 23 in Diagnosis of Tumor-Induced Osteomalacia and Clinical Characteristics of the Disease.

    PubMed

    Hu, Fangke; Jiang, Chengying; Zhang, Qiang; Shi, Huaiyin; Wei, Lixin; Wang, Yan

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disorder and fibroblast growth factor 23 (FGF23) plays a key role in its pathogenesis. This study was conducted to describe a novel FGF23 detecting procedure and describe clinical features of the disease. Fourteen TIO cases were retrieved and FGF23 expression was measured by quantitative ELISA-like immunohistochemistry using formalin-fixed and paraffin-embedded tissues. As summarized from 14 TIO cases, clinical features of TIO were long-standing history of osteomalacia, hypophosphatemia, and urinary phosphate wasting. The associated tumors were mostly benign phosphaturic mesenchymal tumors mixed connective tissue variant (PMTMCT) which could be located anywhere on the body, and most of them could be localized by conventional examinations and octreotide scanning. By quantitative ELISA-like immunohistochemistry, all the 14 TIO cases had high FGF23 expression (median 0.69, 25%-75% interquartile 0.57-1.10, compared with 26 non-TIO tumors of median 0.07, 25%-75% interquartile 0.05-0.11, p < 0.001). The quantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect the high FGF23 expression in formalin-fixed and paraffin-embedded biopsies or specimens. Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of TIO and PMTMCT, respectively. PMID:27034530

  7. Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

    PubMed Central

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  8. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    PubMed

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  9. A direct fluorescence-based assay for RGS domain GTPase accelerating activity.

    PubMed

    Willard, Francis S; Kimple, Adam J; Johnston, Christopher A; Siderovski, David P

    2005-05-15

    Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Galpha subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Galpha subunits. In this article, we describe the use of the ribose-conjugated fluorescent guanine nucleotide analog BODIPYFL-GTP as a spectroscopic probe to measure intrinsic and RGS protein-catalyzed nucleotide hydrolysis by Galphao. BODIPYFL-GTP bound to Galphao exhibits a 200% increase in fluorescence quantum yield. Hydrolysis of BODIPYFL-GTP to BODIPYFL-GDP reduces the quantum yield to 27% above its unbound value. We demonstrate that BODIPYFL-GTP can be used as a rapid real-time probe for measuring RGS domain-catalyzed GTP hydrolysis by Galphao. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Galphao and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds. PMID:15840508

  10. DNA-mediated supercharged fluorescent protein/graphene oxide interaction for label-free fluorescence assay of base excision repair enzyme activity.

    PubMed

    Wang, Zhen; Li, Yong; Li, Lijun; Li, Daiqi; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

    2015-09-01

    The interaction between supercharged green fluorescent protein (ScGFP) and graphene oxide (GO) as well as the resulting quenching effect of GO on ScGFP were investigated. Based on this unique quenching effect and the DNA-mediated ScGFP/GO interaction, a label-free fluorescence method has been established for homogeneously assaying the activity and inhibition of base excision repair enzyme. PMID:26208330

  11. A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk.

    PubMed

    Varriale, Antonio; Pennacchio, Anna; Pinto, Gabriella; Oliviero, Giorgia; D'Errico, Stefano; Majoli, Adelia; Scala, Andrea; Capo, Alessandro; Pennacchio, Angela; Di Giovanni, Stefano; Staiano, Maria; D'Auria, Sabato

    2015-10-21

    Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17β-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17β-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17β-estradiol derivative. The successful detection of 17β-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety. PMID:26434254

  12. Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease.

    PubMed

    Liu, Xue-Guo; Xing, Xiao-Jing; Li, Bo; Guo, Yong-Ming; Zhang, Ye-Zhen; Yang, Yan; Zhang, Lian-Feng

    2016-07-15

    A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3'-end (F-DNA) and its complementary sequence modified with a 5'-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5'-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field. PMID:27015149

  13. Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors.

    PubMed Central

    Luciani, N; de Rocquigny, H; Turcaud, S; Romieu, A; Roques, B P

    2001-01-01

    The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors. PMID:11389689

  14. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  15. Optimization of a polyglutamine aggregation inhibitor peptide (QBP1) using a thioflavin T fluorescence assay.

    PubMed

    Hamuro, Lora; Zhang, Guangtao; Tucker, Timothy J; Self, Christopher; Strittmatter, Warren J; Burke, James R

    2007-10-01

    Polyglutamine protein aggregates are a hallmark of several neurodegenerative diseases, including Huntington's disease, and increasing evidence suggests that reducing or inhibiting aggregation produces a therapeutic benefit in animal models of disease. Part of the challenge in designing compounds that interfere with protein aggregation is having a sensitive and consistent in vitro assay that allows for efficient screening and lead optimization. Here we describe a simplified polyglutamine assay that uses a soluble, pathological-length polyglutamine construct (62 glutamines [Q62]) fused to glutathione-S-transferase (GST) and measure aggregate formation with fluorescence generated by thioflavin T binding. Controlled release of Q62 from GST using proteolytic cleavage resulted in time-dependent aggregate formation that was not observed for a non-pathological-length GST-Q19 construct. Cleavage of the polyglutamine domain from GST increased the rate of Q62 aggregation from days to hours, significantly decreasing the time for compound analysis. Controlled aggregate formation combined with the fluorescence sensitivity of the dye thioflavin T allowed us to screen a series of peptide analogs for lead optimization of a previously identified peptide aggregation inhibitor, QBP1. QBP1 analogs showed the greatest inhibitory potency when added prior to Q62 aggregate initiation, suggesting that the mechanism of inhibition was interference with early formed aggregates that were not detectable by ultraviolet or dye binding. The assay detected activities that differed by three orders of magnitudes with Z' = 0.56, which is suitable for high-throughput screening and allowed us to do lead optimization of QBP1 analogs for pharmacophore model building. PMID:17939755

  16. EGFR fluorescence in situ hybridisation assay: guidelines for application to non-small-cell lung cancer.

    PubMed

    Varella-Garcia, M; Diebold, J; Eberhard, D A; Geenen, K; Hirschmann, A; Kockx, M; Nagelmeier, I; Rüschoff, J; Schmitt, M; Arbogast, S; Cappuzzo, F

    2009-11-01

    There is a need for predictive biomarkers that identify non-small-cell lung cancer (NSCLC) patients most likely to respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment. There are numerous potential candidates, although none has been proven in prospective clinical trials. The EGFR gene copy number evaluated by fluorescence in situ hybridisation (FISH) has been highlighted as one of the most effective markers for sensitivity to EGFR TKIs in large phase III, randomised placebo-controlled trials and has been used in clinical settings to assist physicians in defining the therapeutic regimen. The EGFR FISH assay has technical challenges and it is critical that detailed guidelines are provided to help clinical laboratories in performing and interpreting the test. Excellent assay reproducibility and portability rates among laboratories are crucial to guarantee that accurate clinical decisions can be made for patients with NSCLC. This article discusses the consensus outcomes of a global workshop convened to discuss key technical issues and standardise reading strategies for the EGFR FISH assay of NSCLC tumour tissue. PMID:19861557

  17. A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases.

    PubMed

    Gaudet, Elizabeth A; Huang, Kuo-Sen; Zhang, Yan; Huang, Wei; Mark, David; Sportsman, J Richard

    2003-04-01

    Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery. PMID:12844437

  18. A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

    PubMed

    Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

    2014-11-11

    Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM. PMID:25233044

  19. Modified bimolecular fluorescence complementation assay to study the inhibition of transcription complex formation by JAZ proteins.

    PubMed

    Qi, Tiancong; Song, Susheng; Xie, Daoxin

    2013-01-01

    The jasmonate (JA) ZIM-domain (JAZ) proteins of Arabidopsis thaliana repress JA signaling and negatively regulate the JA responses. Recently, JAZ proteins have been found to inhibit the transcriptional function of several transcription factors, among which the basic helix-loop-helix (bHLH) (GLABRA3 [GL3], ENHANCER OF GLABRA3 [EGL3], and TRANSPARENT TESTA8 [TT8]) and R2R3-MYB (GL1 and MYB75) that can interact with each other to form bHLH-MYB complexes and further control gene expression. The bimolecular fluorescence complementation (BiFC) assay is a widely used technique to study protein-protein interactions in living cells. Here we describe a modified BiFC experimental procedure to study the inhibition of the formation of the bHLH (GL3)-MYB (GL1) complex by JAZ proteins. PMID:23615997

  20. Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

    PubMed

    Shekarchi, I C; Sever, J L; Nerurkar, L; Fuccillo, D

    1985-01-01

    The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available. PMID:2981902

  1. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay

    PubMed Central

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung

    2014-01-01

    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm2. A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times. PMID:25332741

  2. A Fluorescence-Based Assay for Proteinuria Screening in Larval Zebrafish (Danio rerio).

    PubMed

    Hanke, Nils; King, Benjamin L; Vaske, Bernhard; Haller, Hermann; Schiffer, Mario

    2015-10-01

    Analysis of genes compromising the glomerular filtration barrier in rodent models using transgenic or knockdown approaches is time- and resource-consuming and often leads to unsatisfactory results. Therefore, it would be beneficial to have a selection tool indicating that your gene of interest is in fact associated with proteinuria. Zebrafish (Danio rerio) is a rapid screening tool to study effects in glomerular filtration barrier integrity after genetic manipulation. We use either injection of high-molecular-weight dextrans or a transgenic fluorescent fish line [Tg(l-fabp:DBP:EGFP)] expressing a vitamin D-binding protein fused with eGFP for indirect detection of proteinuria. A loss of high-molecular-weight proteins from the circulation of the fish into the urine can be identified by monitoring fluorescence intensity in the zebrafish eye. Paired with an optimized analysis method, this assay provides an effective screening solution to detect filtration barrier damage with proteinuria before moving to a mammalian system. PMID:26125680

  3. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay.

    PubMed

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung

    2014-09-01

    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm(2). A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times. PMID:25332741

  4. Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

    PubMed

    Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

    2016-01-01

    We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent. PMID:26960620

  5. A fluorescence-based high throughput assay for the determination of small molecule–human serum albumin protein binding

    PubMed Central

    McCallum, Megan M.; Pawlak, Alan J.; Shadrick, William R.; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David J.; Arnold, Leggy A.

    2014-01-01

    Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the LOPAC small molecule library of 1280 compounds identifying known high protein binders. The small molecule competition of HSA–Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3–24 μM. The compound affinity towards HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. PMID:24390461

  6. Multi-residue fluorescent microspheres immunochromatographic assay for simultaneous determination of macrolides in raw milk.

    PubMed

    Li, Xiangmei; Shen, Jianzhong; Wang, Qi; Gao, Shuxia; Pei, Xingyao; Jiang, Haiyang; Wen, Kai

    2015-12-01

    A rapid, reliable, sensitive, and quantitative multi-residue fluorescent microspheres immunochromatographic assay (FMCA) was developed for simultaneous detection of four macrolides in raw milk. The IC50 value of the optimized FMCA was 1.36, 1.22, 1.01, and 1.39 ng/mL for erythromycin (ERY), spiramycin (SPI), tilmicosin (TIM), and tylosin (TYL), respectively. The limits of detection (LODs) for the four macrolides was 0.13 ng/mL. The recoveries of ERY, SPI, TIM, and TYL from spiked raw milk ranged from 91.8-109.2, 89.6-114.4, 84.8-111.6, and 85.8-115.2%, respectively, with coefficients of variation (CVs) of 5.4-11.3, 7.9-15.7, 6.2-13.7, and 3.2-14.9%, respectively. The whole testing process was completed within 20 min. The antibody-mixed labeled method was successfully applied to the FMCA, which greatly simplified the operation steps and saved a lot of time. Compared with the immunogold chromatographic assay (IGCA), the FMCA is more sensitive and stable and has less antibody consumption. A parallel analysis in blind raw milk samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS); the results showed good correlation (r(2) = 0.99) between the two methods. Therefore, the developed multi-residue FMCA is reliable and can be easily applied to other antibiotics or other contaminants. PMID:26497839

  7. Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes.

    PubMed

    Lehmusvuori, Ari; Tapio, Antti-Heikki; Mäki-Teeri, Petra; Rantakokko-Jalava, Kaisu; Wang, Qi; Takalo, Harri; Soukka, Tero

    2013-05-01

    We have developed a duplex polymerase chain reaction (PCR) assay based on switchable lanthanide chelate complementation probes. In the complementation probe technology, two nonfluorescent oligonucleotide probes, one labeled with a lanthanide ion carrier chelate and another with a light absorbing antenna ligand, form a fluorescent complex by self-assembly of the reporter molecules when the two probes are hybridized in adjacent positions to the target DNA. Here we report the synthesis of a new terbium(III) (Tb(III)) ion carrier chelate and a new light-absorbing antenna ligand for Tb(III) and the development of a duplex Chlamydia trachomatis (Ct) PCR assay. For the detection of Ct in urine samples, a specific sequence in Ct cryptic plasmid was amplified and detected using europium(III) (Eu(III)) complementation probes. An internal amplification control was amplified in each reaction and detected using Tb(III) complementation probes to verify the Ct negative results. Ct bacteria were concentrated from urine samples with a rapid and simple centrifugation-based sample preparation method. Good diagnostic accuracy (99-100%) was achieved, and also Ct positive reactions yielded a very high Eu(III) signal-to-background ratio (maximum of 244). High performance of the complementation probes is advantageous when sample may contain impurities after a simple sample preparation. PMID:23353013

  8. Small molecule aptamer assays based on fluorescence anisotropy signal-enhancer oligonucleotides.

    PubMed

    Perrier, Sandrine; Bouilloud, Prisca; De Oliveira Coelho, Gisella; Henry, Mickael; Peyrin, Eric

    2016-08-15

    Herein, we design novel fluorescence anisotropy (FA) aptamer sensing platforms dedicated to small molecule detection. The assay strategy relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer mediated by an unlabelled, partially complementary oligonucleotide. The signal-enhancer oligonucleotide (SEO) essentially served as a free probe fraction revealer. By targeting specific regions of the signalling functional nucleic acid, the SEO binding to the unbound aptamer triggered perturbations of both the internal DNA flexibility and the localized dye environment upon the free probe to duplex structure transition. This potentiating effect determined increased FA variations between the duplex and target bound states of the aptameric probe. FA assay responses were obtained with both pre-structured (adenosine) and unstructured (tyrosinamide) aptamers and with dyes of different photochemical properties (fluorescein and texas red). The multiplexed analysis ability was further demonstrated through the simultaneous multicolour detection of the two small targets. The FA method appears to be especially simple, sensitive and widely applicable. PMID:27085946

  9. Fluorescence-Based Transport Assays Revisited in a Human Renal Proximal Tubule Cell Line.

    PubMed

    Caetano-Pinto, Pedro; Janssen, Manoe J; Gijzen, Linda; Verscheijden, Laurens; Wilmer, Martijn J G; Masereeuw, Rosalinde

    2016-03-01

    Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in

  10. Modeling and Application of a Rapid Fluorescence-Based Assay for Biotoxicity in Anaerobic Digestion.

    PubMed

    Chen, Jian Lin; Steele, Terry W J; Stuckey, David C

    2015-11-17

    The sensitivity of anaerobic digestion metabolism to a wide range of solutes makes it important to be able to monitor toxicants in the feed to anaerobic digesters to optimize their operation. In this study, a rapid fluorescence measurement technique based on resazurin reduction using a microplate reader was developed and applied for the detection of toxicants and/or inhibitors to digesters. A kinetic model was developed to describe the process of resazurin reduced to resorufin, and eventually to dihydroresorufin under anaerobic conditions. By modeling the assay results of resazurin (0.05, 0.1, 0.2, and 0.4 mM) reduction by a pure facultative anaerobic strain, Enterococcus faecalis, and fresh mixed anaerobic sludge, with or without 10 mg L(-1) spiked pentachlorophenol (PCP), we found it was clear that the pseudo-first-order rate constant for the reduction of resazurin to resorufin, k1, was a good measure of "toxicity". With lower biomass density and the optimal resazurin addition (0.1 mM), the toxicity of 10 mg L(-1) PCP for E. faecalis and fresh anaerobic sludge was detected in 10 min. By using this model, the toxicity differences among seven chlorophenols to E. faecalis and fresh mixed anaerobic sludge were elucidated within 30 min. The toxicity differences determined by this assay were comparable to toxicity sequences of various chlorophenols reported in the literature. These results suggest that the assay developed in this study not only can quickly detect toxicants for anaerobic digestion but also can efficiently detect the toxicity differences among a variety of similar toxicants. PMID:26457928

  11. Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis.

    PubMed

    Sachdeva, Poonam; Patel, Achchhe Lal; Sachdev, Divya; Ali, Mashook; Mittal, Aruna; Saluja, Daman

    2009-07-01

    To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India. PMID:19502371

  12. Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

    PubMed Central

    Hollender, Courtney A.; Liu, Zhongchi

    2010-01-01

    Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells. PMID:20567209

  13. Bimolecular fluorescence complementation (BiFC) assay for protein-protein interaction in onion cells using the helios gene gun.

    PubMed

    Hollender, Courtney A; Liu, Zhongchi

    2010-01-01

    Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay(1) allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)(2) and the visualization of their interactions mediated by BiFC in onion epidermal cells. PMID:20567209

  14. Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

    ERIC Educational Resources Information Center

    Hicks, Katherine A.

    2016-01-01

    Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

  15. Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Tuvshintulga, Bumduuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-08-30

    The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (-)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. PMID:27523944

  16. Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients.

    PubMed

    Martins, João R M; Passerotti, Carlo C; Maciel, Rui M B; Sampaio, Lucia O; Dietrich, Carl P; Nader, Helena B

    2003-08-01

    A practical fluorescence-based assay method for determination of hyaluronan (HA) was developed. Plates were coated with hyaluronan-binding proteins (HABP) obtained from bovine cartilage and successively incubated with samples containing standard solutions of hyaluronan or serum from normal and cyrrhotic patients, biotin-conjugated HABP, and europium-labeled streptavidin. After release of europium from streptavidin with enhancement solution the final fluorescence is measured in a fluorometer. The method is specific for HA even in the presence of substantial amounts of other glycosaminoglycans (chondroitin, dermatan sulfate, and heparan sulfate, and heparin) or proteins. It is possible to quantify HA between 0.2 and 500.0 microg/L. Analyses of HA concentration in 545 normal subjects and 40 cirrhotic patients gave average values of 14.5 and 542.0 microg/L, respectively. It was also shown that older subjects (> or =51 years old) have more HA (28.0 microg/L) than younger subjects (12.0 to 14.0 microg/L). This new sandwich technique has shown high precision and sensitivity similar to those of a recently described fluorescence-based assay method, being able to measure HA in amounts as small as 0.2 microg/L. In addition, this noncompetitive assay avoids preincubation, consumes less time (<5 h) than the previous competitive fluorescence-based assay (>72 h), and avoids the use of radioactive materials. PMID:12842108

  17. A simple aptamer-based fluorescent assay for the detection of Aflatoxin B1 in infant rice cereal.

    PubMed

    Chen, Lu; Wen, Fang; Li, Ming; Guo, Xiaodong; Li, Songli; Zheng, Nan; Wang, Jiaqi

    2017-01-15

    A fluorescent assay for the rapid, sensitive and specific detection of Aflatoxin B1 (AFB1) was developed in this study. Initially, a DNA/DNA duplex was formed between a fluorescein-labeled AFB1 aptamer and its partially complementary DNA strand containing a quencher moiety, resulting in fluorescence quenching due to the close proximity of fluorophore and quencher. Upon the addition of AFB1, an aptamer/AFB1 complex was generated to release the quencher-modified DNA strand, thus recovered the fluorescence of fluorescein and enabled quantitative detection for AFB1 by monitoring fluorescence enhancement. Under optimized conditions, this assay exhibited a linear response to AFB1 in the range of 5-100ng/mL with a detection limit down to 1.6ng/mL. Trials of this assay in infant rice cereal with satisfactory recovery in the range of 93.0%-106.8%, demonstrate that the new assay could be a potential sensing platform for AFB1 determination in food. PMID:27542489

  18. A fast and indirect fluorescent antibody assay for the vibrio in large yellow croaker Pseudosciaena crocea (Richardson)

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Su, Yongquan; Yan, Qingpi

    2003-03-01

    A fast and indirect fluorescent antibody assay for the Vibrio alginolyticus and V. parahaemolyticus infecting the large yellow croaker has been developed. The specific antisera for the two strains of vibrio were prepared with New Zealand rabbit and the antiserum and cross-reactive efficacy was tested by coagulation in tube. It showed that the goat anti-rabbit IgG had been labeled by fluorescence isothiocyanate (FITC). The results showed that positive reactions were 100% for the large yellow croaker Pseudosciaena crocea with typical symptom of vibrio infection, while the positive reaction to the pathogen in healthy yellow croakers reached 40%, but seemed negative for aquaculture water. The results demonstrated that this fast and indirect fluorescent antibody assay can be used not only to test the vibrio pathogen in diseased yellow croaker but also in infected animals with no symptom.

  19. Velocity of movement of actin filaments in in vitro motility assay. Measured by fluorescence correlation spectroscopy.

    PubMed Central

    Borejdo, J; Burlacu, S

    1992-01-01

    We have measured the velocity of actin filaments in in vitro motility assay by fluorescence correlation spectroscopy. In this method, one measures fluctuations in the number of filaments in an open sample volume. The number of filaments was calculated from measurements of fluorescence of rhodamine-phalloidin bound to F-actin. Sample volume was defined by a diaphragm placed in front of the photomultiplier. Fluctuations arise when actin filaments enter and leave the sample volume due to translations driven by mechanochemical interactions with myosin heads which are immobilized on a glass surface. The average velocity of the translation of filaments determined by the correlation method, (Vc), was equal to the diameter of the diaphragm divided by the half-time of the relaxation of fluctuations. The average number of moving filaments determined by correlation method, (Nc), was inversely proportional to the relative fluctuations. By the fluctuation method it was possible to determine the average velocity of over 800 moving filaments in less than 4 min. There was good agreement between (Vc) and (Nc) and the average velocity and the average number of moving filaments determined manually. To be able to apply correlation measurements to an experimental problem, neither (Vc) nor (Nc) must depend on the position of observation of filaments. We first confirmed that this was indeed the case. We then applied the method to investigate the dependence of motility on the ATPase activity of myosin heads. ATPase activity was varied by mixing intact heads with heads which were labeled with different thiol reagents. It was found that the motion was drastically influenced by the reagent used for modification. When the reagent was N-ethyl-maleimide, 1.5% modification was sufficient to completely inhibit the motion. When the reagent was 5-iodoacetamidofluorescein, motion declined hyperbolically with the fraction of modified heads. Images FIGURE 2 FIGURE 4 FIGURE 11 PMID:1534696

  20. Monitoring water supplies for weaponized bacteria and bacterial toxins using rapid fluorescence-based viability and affinity assays

    NASA Astrophysics Data System (ADS)

    Van Tassell, Roger L.; Evans, Mishell

    2004-03-01

    The rapid detection of weaponized bacteria and toxins is a major problem during a biological attack. Although sensitive detection formats exist for many biowarfare agents, they often require advanced training and complex procedures. Luna has developed simple, rapid means for determining the presence of pathogens and bacterial toxins in water supplies using fluorescence-based assays that can be adapted for field use. The batteries of rapid assays are designed for i) determining cell viability and bacterial loads by exploiting metabolic markers (e.g., acid-production, redox potentials, etc) and ii) detecting bacterial toxins using fluorescent, polymerized affinity liposomes (fluorosomes). The viability assays were characterized using E. coli, S. aureus and the anthrax simulant, B. globigii. The viability assays detected bacterial loads of ~ 104 CFU/ml and with simple filtration ~ 100CFU/ml could be detected. The affinity fluorosomes were characterized using cholera toxin (CT). Affinity liposomes displaying GM1 and anti-CT antibodies could detect CT at <μg/ml levels. Stability studies showed that affinity vesicles could be stored for weeks at 4°C or freeze-dried with no significant loss of binding capacity. Using an in-house fiber optic fluorescence system, Luna characterized the binding of affinity fluorosomes to respective targets and determined the responses of bacterial loads in the fluorescent viability assays. Using this two-tiered approach, Luna demonstrated that water susceptible to sabotage could be easily monitored and confirmed for specific agents using simple, general and specific fluorescence-based detection schemes based on metabolism and ligand-target interactions.

  1. Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

    PubMed Central

    Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

    2014-01-01

    Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

  2. Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.

    PubMed

    Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

    2014-01-01

    Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1 orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

  3. Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume.

    PubMed

    Billich, Andreas; Bilban, Melitta; Meisner, Nicole-Claudia; Nussbaumer, Peter; Neubauer, Andreas; Jäger, Stefan; Auer, Manfred

    2004-02-01

    Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening. PMID:15090207

  4. A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

    PubMed Central

    Mendoza, Oscar; Gueddouda, Nassima Meriem; Boulé, Jean-Baptiste; Bourdoncle, Anne; Mergny, Jean-Louis

    2015-01-01

    Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures. PMID:25765657

  5. Quantum Dot-Bead-DNA Probe-Based Hybridization Fluorescence Assays on Microfluidic Chips.

    PubMed

    Ankireddy, Seshadri Reddy; Kim, Jongsung

    2015-10-01

    The development of chip-based, quantum dot (QD)-bead-DNA conjugate probes for hybridization detection is a prime research focus in the field of microfluidics. QD-Bead-DNA probe-based hybridization detection methods are often called "bead-based assays," and their success is substantially influenced by the dispensing and manipulation capabilities of microfluidic technology. Met was identified as a prognostic marker in different cancers including lung, renal, liver, head and neck, stomach, and breast. In this report, the cancer causing Met gene was detected with QDs attached to polystyrene microbeads. We constructed a microfluidic platform using a flexible PDMS polymer. The chip consists of two channels, with two inlets and two outlets. The two channels were integrated with QD-bead-DNA probes for simultaneous detection of wild type target DNA and mutant DNA, containing three nucleotide changes compared to the wild type sequence. The fluorescence quenching ability of QDs within the channels of microfluidic chips were compared for both DNAs. PMID:26726440

  6. Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy.

    PubMed

    Liu, Miaomiao; Xie, Jun; Zheng, Hailing; Zhou, Yang; Wang, Bing; Hu, Zhiwen

    2015-01-01

    The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories. PMID:26656824

  7. A homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion.

    PubMed

    Smeulders, Liesbet; Bunkens, Lieve; Vereycken, Inge; Van Acker, Koen; Holemans, Pascale; Gustin, Emmanuel; Van Loock, Marnix; Dams, Géry

    2013-01-01

    The human immunodeficiency virus type 1 (HIV-1) initiates infection through sequential interactions with CD4 and chemokine coreceptors unmasking the gp41 subunit of the viral envelope protein. Consequently, the N-terminal heptad repeats of gp41 form a trimeric coiled-coil groove in which the C-terminal heptad repeats collapse, generating a stable six-helix bundle. This brings the viral and cell membrane in close proximity enabling fusion and the release of viral genome in the cytosol of the host cell. In this chapter, we describe a homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion, based on the ability of soluble peptides, derived from the N- and C-terminal domains of gp41, to form a stable six-helix bundle in vitro. Labeling of the peptides with allophycocyanin and the lanthanide europium results in a Föster resonance energy transfer (FRET) signal upon formation of the six-helix bundle. Compounds interfering with the six-helix bundle formation inhibit the HIV-1 fusion process and suppress the FRET signal. PMID:23821256

  8. Photonic Crystal Enhancement of a Homogeneous Fluorescent Assay using Submicron Fluid Channels Fabricated by E-jet Patterning

    PubMed Central

    Tan, Yafang; Sutanto, Erick; Alleyne, Andrew G.; Cunningham, Brian T.

    2016-01-01

    We demonstrate the enhancement of a liquid-based homogenous fluorescence assay using the resonant electric fields from a photonic crystal (PC) surface. Because evanescent fields are confined to the liquid volume nearest to the photonic crystal, we developed a simple approach for integrating a PC fabricated on a silicon substrate within a fluid channel with submicron height, using electrohydrodynamic jet (e-jet) printing of a light-curable epoxy adhesive to define the fluid channel pattern. The PC is excited by a custom-designed compact instrument that illuminates the PC with collimated light that precisely matches the resonant coupling condition when the PC is covered with aqueous media. Using a molecular beacon nucleic acid fluorescence resonant energy transfer (FRET) probe for a specific miRNA sequence, we demonstrate an 8x enhancement of the fluorescence emission signal, compared to performing the same assay without exciting resonance in the PC detecting a miRNA sequence at a concentration of 62nM from a liquid volume of only ~20 nl. The approach may be utilized for any liquid-based fluorescence assay for applications in point-of-care diagnostics, environmental monitoring, or pathogen detection. PMID:24376013

  9. Photonic crystal enhancement of a homogeneous fluorescent assay using submicron fluid channels fabricated by E-jet patterning.

    PubMed

    Tan, Yafang; Sutanto, Erick; Alleyne, Andrew G; Cunningham, Brian T

    2014-04-01

    We demonstrate the enhancement of a liquid-based homogenous fluorescence assay using the resonant electric fields from a photonic crystal (PC) surface. Because evanescent fields are confined to the liquid volume nearest to the photonic crystal, we developed a simple approach for integrating a PC fabricated on a silicon substrate within a fluid channel with submicron height, using electrohydrodynamic jet (e-jet) printing of a light-curable epoxy adhesive to define the fluid channel pattern. The PC is excited by a custom-designed compact instrument that illuminates the PC with collimated light that precisely matches the resonant coupling condition when the PC is covered with aqueous media. Using a molecular beacon nucleic acid fluorescence resonant energy transfer (FRET) probe for a specific miRNA sequence, we demonstrate an 8× enhancement of the fluorescence emission signal, compared to performing the same assay without exciting resonance in the PC detecting a miRNA sequence at a concentration of 62 nM from a liquid volume of only ∼20 nL. The approach may be utilized for any liquid-based fluorescence assay for applications in point-of-care diagnostics, environmental monitoring, or pathogen detection. PMID:24376013

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

    PubMed Central

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

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

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

    PubMed

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

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

  12. An electrochemical ELISA-like immunosensor for miRNAs detection based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes.

    PubMed

    Tran, H V; Piro, B; Reisberg, S; Huy Nguyen, L; Dung Nguyen, T; Duc, H T; Pham, M C

    2014-12-15

    We design an electrochemical immunosensor for miRNA detection, based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes. An original immunological approach is followed, using antibodies directed to DNA.RNA hybrids. An electrochemical ELISA-like amplification strategy was set up using a secondary antibody conjugated to horseradish peroxidase (HRP). Hydroquinone is oxidized into benzoquinone by the HRP/H2O2 catalytic system. In turn, benzoquinone is electroreduced into hydroquinone at the electrode. The catalytic reduction current is related to HRP amount immobilized on the surface, which itself is related to miRNA.DNA surface density on the electrode. This architecture, compared to classical optical detection, lowers the detection limit down to 10 fM. Two miRNAs were studied: miR-141 (a prostate biomarker) and miR-29b-1 (a lung cancer biomarker). PMID:24973539

  13. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    PubMed Central

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA-3′) can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future. PMID:25634469

  14. A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

    PubMed

    Nagarajan, Archana; Bodhicharla, Rakesh; Winter, Jody; Anbalagan, Charumathi; Morgan, Kevin; Searle, Mark; Nazir, Aamir; Adenle, Ademola; Fineberg, April; Brady, Declan; Vere, Kelly; Richens, Jo; O'Shea, Paul; Bell, David; de-Pomerai, David

    2015-01-01

    The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo. PMID:26295817

  15. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    NASA Astrophysics Data System (ADS)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  16. A sensitive "turn-on" fluorescent assay for quantification of ceftriaxone based on l-tryptophan-Pd(II) complex fluorophore.

    PubMed

    Qiao, Man; Jiang, Junze; Yang, Jidong; Liu, Shaopu; Liu, Zhongfang; Hu, Xiaoli

    2016-05-15

    Based on l-tryptophan-Pd(II) system, a sensitive and selective fluorimetric assay for the quantification of ceftriaxone (CTRX) had been developed. The experimental results showed that in pH 4.0 Britton-Robinson (BR) buffer medium, the fluorescence of l-tryptophan (l-Trp) (λex/λem=276nm/352nm) could be efficiently quenched by Pd(II). When CTRX was added to the mixed solution of the l-tryptophan and Pd(II), the fluorescence of l-Trp recovered. The reaction mechanism and the reasons for the fluorescence recovery were also discussed. Pd(II) reacted with l-Trp to form a 1:1 chelate complex, and then, after CTRX was added in l-Try-Pd(II) system, the ligand exchange reaction occurred between l-Trp and CTRX, which resulted in the fluorescence recovery. Under the optimized experimental conditions, the recovered fluorescence intensities at 352nm showed excellent linear relationship with the concentration of CTRX over the range of 6.0×10(-8)-2.4×10(-)(6)molL(-1) (0.040-1.59μgmL(-1)). The correlation coefficient (R) was 0.9997 and the detection limit was 1.8×10(-)(8)molL(-1) (11.9ngmL(-1)). Furthermore, the assay had been applied to determine trace amount of CTRX human urine samples with satisfactory results. PMID:26963730

  17. A sensitive "turn-on" fluorescent assay for quantification of ceftriaxone based on L-tryptophan-Pd(II) complex fluorophore

    NASA Astrophysics Data System (ADS)

    Qiao, Man; Jiang, Junze; Yang, Jidong; Liu, Shaopu; Liu, Zhongfang; Hu, Xiaoli

    2016-05-01

    Based on L-tryptophan-Pd(II) system, a sensitive and selective fluorimetric assay for the quantification of ceftriaxone (CTRX) had been developed. The experimental results showed that in pH 4.0 Britton-Robinson (BR) buffer medium, the fluorescence of L-tryptophan (L-Trp) (λex/λem = 276 nm/352 nm) could be efficiently quenched by Pd(II). When CTRX was added to the mixed solution of the L-tryptophan and Pd(II), the fluorescence of L-Trp recovered. The reaction mechanism and the reasons for the fluorescence recovery were also discussed. Pd(II) reacted with L-Trp to form a 1:1 chelate complex, and then, after CTRX was added in L-Try-Pd(II) system, the ligand exchange reaction occurred between L-Trp and CTRX, which resulted in the fluorescence recovery. Under the optimized experimental conditions, the recovered fluorescence intensities at 352 nm showed excellent linear relationship with the concentration of CTRX over the range of 6.0 × 10- 8-2.4 × 10-6 mol L- 1 (0.040-1.59 μg mL- 1). The correlation coefficient (R) was 0.9997 and the detection limit was 1.8 × 10-8 mol L- 1 (11.9 ng mL- 1). Furthermore, the assay had been applied to determine trace amount of CTRX human urine samples with satisfactory results.

  18. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    PubMed

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  19. Fluorescence turn-on and colorimetric dual readout assay of glutathione over cysteine based on the fluorescence inner-filter effect of oxidized TMB on TMPyP.

    PubMed

    Jiang, Xiangyu; Geng, Fenghua; Wang, Yongxiang; Liu, Jinhua; Qu, Peng; Xu, Maotian

    2016-07-15

    Quantitative fluorescence turn-on and colorimetric detection of glutathione (GSH) with rapid speed, low cost have attained much attention. Herein, we developed a sensitive fluorescence turn-on and colorimetric sensor for GSH based on the inner-filter effect (IFE), which is the first time to select oxTMB and TMPyP as the IFE absorber and fluorophore pair, respectively. The absorption band of oxTMB matches well with the emission band of TMPyP in the IFE-based fluorescent assay. In the absence of GSH, the absorption peak of oxTMB at 652nm significantly overlaps with the emission of TMPyP, resulting in the efficient IFE and inhibition of the fluorescence of TMPyP. In the presence of GSH, the absorption intensity at 652nm decreases, generating the recovery of the fluorescence of TMPyP. Therefore, this approach is demonstrated to be a novel candidate for detection of GSH, with high sensitivity and selectivity. The linear dynamic range for the concentrations of GSH is between 0.1μM to 20μM along with a limit of detection (LOD) of about 30nM (calculated LOD as 3σ/slope). Finally, this novel sensor was successfully applied for GSH detection in fetal calf serum, and satisfactory recovery was achieved. PMID:26971272

  20. A homogenous fluorescence quenching based assay for specific and sensitive detection of influenza virus A hemagglutinin antigen.

    PubMed

    Chen, Longyan; Neethirajan, Suresh

    2015-01-01

    Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs). The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs), and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs). When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human) and H5 (avian). The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures. PMID:25884789

  1. A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen

    PubMed Central

    Chen, Longyan; Neethirajan, Suresh

    2015-01-01

    Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs). The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs), and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs). When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human) and H5 (avian). The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures. PMID:25884789

  2. Development of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection.

    PubMed

    Kumagai, Kazuo; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

    2014-02-15

    Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z'-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. PMID:24299989

  3. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

    PubMed

    Fu, Ying; Lu, Danqin; Lin, Bin; Sun, Qianqian; Liu, Kai; Xu, Lili; Zhang, Shengping; Hu, Chen; Wang, Chuangui; Xu, Zhiai; Zhang, Wen

    2013-11-21

    Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells. PMID:24098881

  4. A rapid fluorescence "switch-on" assay for glutathione detection by using carbon dots-MnO2 nanocomposites.

    PubMed

    Cai, Qi-Yong; Li, Jie; Ge, Jia; Zhang, Lin; Hu, Ya-Lei; Li, Zhao-Hui; Qu, Ling-Bo

    2015-10-15

    Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence "switch-on" assay was developed to detect trace amount of GSH based on carbon dots-MnO2 nanocomposites, which was fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots-MnO2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO2 nanosheets to Mn(2+) ions and subsequently release carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples. PMID:25957074

  5. In vitro assay for drug-induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis.

    PubMed

    Fujimura, Hisako; Murakami, Naoko; Kurabe, Michie; Toriumi, Wataru

    2009-05-01

    To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down-regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up-regulated by peroxisome proliferator-activated receptor-alpha agonists: CFR and VPA. From these findings, we concluded that the cell-based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis. PMID:19224547

  6. Rapid and quantitative detection of 4(5)-methylimidazole in caramel colours: A novel fluorescent-based immunochromatographic assay.

    PubMed

    Wu, Xinlan; Huang, Minghui; Yu, Shujuan; Kong, Fansheng

    2016-01-01

    A novel fluorescence-based immunochromatographic assay (ICA) for rapid detecting 4(5)-methylimidazole (4-MI) is presented in this study. In our work, the conjugates of fluorescent microspheres (FMs) and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85% to 102.31%, with the coefficient of variation (n = 3) below 9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of 4-MI in food safety control. PMID:26213047

  7. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    SciTech Connect

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  8. Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays

    PubMed Central

    Chaudhery, Vikram; Huang, Cheng-Sheng; Pokhriyal, Anusha; Polans, James; Cunningham, Brian T.

    2011-01-01

    By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer. PMID:22109210

  9. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

    PubMed Central

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  10. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    PubMed

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  11. Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization.

    PubMed

    Copple, Susan S; Giles, S Rashelle; Jaskowski, Troy D; Gardiner, Anna E; Wilson, Andrew M; Hill, Harry R

    2012-05-01

    We evaluated 5 commercially available HEp-2 antinuclear antibody (ANA) indirect fluorescent antibody (IFA) assays using patient serum samples from 45 patients with rheumatoid arthritis, 50 with systemic lupus erythematosus (SLE), 35 with scleroderma, 20 with Sjögren syndrome, 10 with polymyositis, and 100 healthy control subjects. In addition, 12 defined serum samples from the Centers for Disease Control and Prevention and 100 patient serum samples sent to ARUP Laboratories (Salt Lake City, UT) for ANA IFA testing were also examined (n = 372). Standardization among the HEp-2 IFA assays occurred when they exhibited the same titer ± 1 doubling dilution. Agreement of the 5 assays was 78%. Within the specific groups of serum samples, agreement ranged from 44% in scleroderma serum samples to 93% in healthy control subjects, with 72% agreement in the SLE group. Variations in slide and substrate quality were also noted (ie, clarity, consistency of fluorescence, cell size, number and quality of mitotic cells). Along with subjectivity of interpretation, HEp-2 IFA assays are also vulnerable to standardization issues similar to other methods for ANA screening. PMID:22523223

  12. High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

    PubMed

    Lokesh, G L; Rachamallu, Aparna; Kumar, G D Kishore; Natarajan, Amarnath

    2006-05-01

    The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening. PMID:16500609

  13. Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.

    PubMed

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M

    2011-08-01

    The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods. PMID:21692532

  14. An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

    PubMed

    Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing; Marko, John F; Matouschek, Andreas

    2016-09-15

    The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. PMID:27296635

  15. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    SciTech Connect

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer An endothelial cell apoptosis assay using FRET-based biosensor was developed. Black-Right-Pointing-Pointer The fluorescence of the cells changed from green to blue during apoptosis. Black-Right-Pointing-Pointer This method was developed into a high-throughput assay in 96-well plates. Black-Right-Pointing-Pointer This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z Prime factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  16. A reliable non-separation fluorescence quenching assay for total glycated serum protein: a simple alternative to nitroblue tetrazolium reduction.

    PubMed

    Blincko, S; Colbert, D; John, W G; Edwards, R

    2000-05-01

    A simple non-separation assay for the measurement of total glycated serum protein is described. It was found that the fluorescence intensity of a solution of a fluorescein-boronic acid derivative was quenched in proportion to the amount of serum added. This led to the development of an assay in which 10 microL of serum is added to 4 mL of a solution of the fluorescein-boronic acid derivative and the fluorescence intensity is measured after 15 min. The results, as measured by drop in fluorescence intensity, calibrated by a single standard, were compared with the results for nitroblue tetrazolium (NBT) reduction of fructosamine and showed good correlation (r=0.936, n=114). The intra-assay precision (seven samples each measured 10 times) was less than 2.1% (concentration range 190-660 micromol/L); inter-assay precision for seven samples in 10 assays was less than 2.5% (over the same concentration range). Dilution of serum that had a high concentration of total glycated protein showed the assay to be linear. Serum samples (with low, medium and high total glycated protein concentrations) showed less than 2.1% difference from base results with added glucose (up to 60 mmol/L), less than 9.7% difference with added bilirubin (up to 250 micromol/L) and less than 6.9% with added triglycerides (up to 50 mmol/L). Addition of haemoglobin (up to 0.9 g/dL) with high glycation (11.7% HbA1c) to plasma (298 micromol/L total glycated protein) showed less than 10% difference from the base result. Assays performed over a range of temperatures (12-34 degrees C) showed no significant differences in the results. The assay gives similar results to the currently used NTB method but with significantly less susceptibility to interferences. As such the method should be a useful aid in the management of diabetes. PMID:10817254

  17. Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway.

    PubMed

    Hecht, Nir; Gur, Eyal

    2015-09-15

    The Pup-proteasome system (PPS) carries out regulated tagging and degradation of proteins in bacterial species belonging to the phyla Actinobacteria and Nitrospira. In the pathogen Mycobacterium tuberculosis, where this proteolytic pathway was initially discovered, PPS enzymes are essential for full virulence and persistence in the mammalian host. As such, PPS enzymes are potential targets for development of antituberculosis therapeutics. Such development often requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. Here, we describe the development of an in vitro activity assay for Dop, the first enzyme in the PPS. Based on fluorescence anisotropy measurements, this assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. We demonstrate how this assay can also be reliably and conveniently used for detailed kinetic measurements of Dop activity. As such, this assay is of value for basic research into Dop and the PPS. Finally, we show that the assay developed here primarily for the mycobacterial Dop can be readily employed with other Dop enzymes, using the same simple protocol. PMID:26095396

  18. Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes.

    PubMed

    Shigano, Miyuki; Takashima, Rie; Takasawa, Hironao; Hamada, Shuichi

    2016-04-01

    The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly those that require metabolic activation. For this assay, hepatocytes (HEPs) can be isolated by collagenase treatment but without requirement for in situ liver perfusion. Consequently, the liver MN assay can be integrated into a general repeated-dose (RD) toxicity study. The method is also applicable to liver MN assays involving partial hepatectomy or the use of juvenile rats. Here, we propose an improved method for staining HEPs prepared from formalin-fixed liver tissues for MN assays, without collagenase treatment. HEP suspensions are prepared by treating the tissues with concentrated KOH and a fluorescent dye, SYBR(®) Gold (SYGO), is used for staining. Visualization of the MN in SYGO-stained HEPs is clearer than with Wright-Giemsa staining. We compared the induction of MN as measured with our new method versus the conventional method using collagenase dispersion. Our method not only enables the integration of the liver MN assay into a general RD toxicity study but also allows it to be conducted retrospectively. PMID:27085473

  19. How to Illustrate Ligand-Protein Binding in a Class Experiment: An Elementary Fluorescent Assay.

    ERIC Educational Resources Information Center

    Marty, Alain; And Others

    1986-01-01

    Describes an experiment (taking approximately five hours) which illustrates the binding of a small molecule to a protein. By using an appropriate fluorescent ligand and a given protein, the fluorescent probe technique is applied to measure the number of bonding sites, and number of site classes, and their association constants. (JN)

  20. Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

    PubMed

    Rahier, Renaud; Noiriel, Alexandre; Abousalham, Abdelkarim

    2016-01-01

    Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale. PMID:26636829

  1. 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. PMID:22459802

  2. Aptamer-based fluorescent screening assay for acetamiprid via inner filter effect of gold nanoparticles on the fluorescence of CdTe quantum dots.

    PubMed

    Guo, Jiajia; Li, Ying; Wang, Luokai; Xu, Jingyue; Huang, Yanjun; Luo, Yeli; Shen, Fei; Sun, Chunyan; Meng, Rizeng

    2016-01-01

    This paper reports a novel aptamer-based fluorescent detection method for small molecules represented by acetamiprid based on the specific binding of aptamers with acetamiprid, and the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (CdTe QDs). When CdTe QDs were mixed with AuNPs, the fluorescence of CdTe QDs was significantly quenched via IFE. The IFE efficiency could be readily modulated by the absorption and the aggregation state of AuNPs. The presence of salt could easily induce the aggregation of AuNPs, resulting in the fluorescence recovery of the quenched QDs. Acetamiprid-binding aptamer (ABA) could adsorb on the negatively charged AuNPs through the coordination interaction to protect AuNPs from salt-induced aggregation, so the fluorescence of CdTe QDs would be quenched by the IFE of AuNPs. However, the specific binding of ABA with acetamiprid could release the ABA from the surfaces of AuNPs and decrease the salt tolerance of AuNPs, so the IFE-decreased fluorescence of CdTe QDs was regained with the presence of acetamiprid, and the fluorescence enhancement efficiency was driven by the concentration of acetamiprid. Based on this principle, the aptamer-based fluorescent method for acetamiprid has been established and optimized. The assay exhibited excellent selectivity towards acetamiprid over its analogues and other pesticides which may coexist with acetamiprid. Under the optimum experiment conditions, the established method could be applied for the determination of acetamiprid with a wide linear range from 0.05 to 1.0 μM, and a low detection limit of 7.29 nM (3σ). Furthermore, this IFE-based method has been successfully utilized to detect acetamiprid in six types of vegetables, and the results were in full agreement with those from HPLC and LC-MS. The proposed method displays remarkable advantages of high sensitivity, rapid analysis, excellent selectivity, and would be suitable for the practical application

  3. Selective and sensitive determination of cypermethrin in fish via enzyme-linked immunosorbent assay-like method based on molecularly imprinted artificial antibody-quantum dot optosensing materials.

    PubMed

    Xiao, Ting-Ting; Shi, Xi-Zhi; Jiao, Hai-Feng; Sun, Ai-Li; Ding, Hao; Zhang, Rong-Rrong; Pan, Dao-Dong; Li, De-Xiang; Chen, Jiong

    2016-01-15

    Molecularly imprinted silica layers appended to quantum dots (MIP-QDs) with customized selective artificial recognition sites were fabricated in this study by optimizing the ratio of the functional monomer to the template. Scanning electron microscopy, transmission electron microscopy, Brunauer–emmett–teller, Fourier transform infrared spectroscopy, and selectivity assay analyses were also performed. Results demonstrated that the selective fluorescence quenching properties of MIP-QDs toward cypermethrin (CYP) are due to strong interactions between these molecules. An enzyme-linked immunosorbent assay (ELISA)-like method based on the MIP-QDs was established under optimal conditions. The fluorescence quenching observed from this method showed a linear relationship with CYP concentration over the range of 0.05–60.0 mg/kg with a correlation coefficient of 0.9838. Good recovery (82.7–92.4%) and a relative standard deviation of less than 10.1% were obtained from fish samples spiked with three levels of CYP. This method also demonstrated a low detection limit of 1.2 μg/kg. The ELISA-like method based on MIP-QDs can be successfully employed to detect residual of CYP in fish samples. PMID:26283587

  4. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    PubMed

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  5. A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction.

    PubMed

    Kimple, Adam J; Yasgar, Adam; Hughes, Mark; Jadhav, Ajit; Willard, Francis S; Muller, Robin E; Austin, Christopher P; Inglese, James; Ibeanu, Gordon C; Siderovski, David P; Simeonov, Anton

    2008-06-01

    The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively. PMID:18537560

  6. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

    PubMed Central

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  7. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

    PubMed

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  8. Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

    PubMed

    Dajer, A; Luna-Martínez, E; Zapata, D; Villegas, S; Gutiérrez, E; Peña, G; Gurría, F; Nielsen, K; Gall, D

    1999-05-14

    A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions. PMID:10343334

  9. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

    PubMed

    Kruger, Wade; Gilbert, Daniel; Hawthorne, Rebecca; Hryciw, Deanne H; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W

    2005-06-01

    There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. PMID:15862914

  10. A fluorescence photobleaching assay of gap junction-mediated communication between human cells.

    PubMed

    Wade, M H; Trosko, J E; Schindler, M

    1986-04-25

    Gap junction-mediated communication between contiguous cells has been implicated in the regulation of cell proliferation and differentiation. This report describes a new technique to measure cell-cell communication, gap fluorescence redistribution after photobleaching, which is based on the diffusion-dependent return of 6-carboxyfluorescein-mediated fluorescence in a photobleached cell that is in contact with other fluorescently labeled cells. Fluorescence recovery rates are interpreted as dye transport across gap junctions. Results of experiments on normal human fibroblasts and human teratocarcinoma cells show that this technique can measure rapid dye transfer and detect inhibition of communication (between teratocarcinoma cells) by the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and the pesticide dieldrin. PMID:3961495

  11. Development of fluorescent substrates and assays for the key autophagy-related cysteine protease enzyme, ATG4B.

    PubMed

    Nguyen, Thanh G; Honson, Nicolette S; Arns, Steven; Davis, Tara L; Dhe-Paganon, Sirano; Kovacic, Suzana; Kumar, Nag S; Pfeifer, Tom A; Young, Robert N

    2014-04-01

    The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway. PMID:24735444

  12. Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo

    PubMed Central

    Lai, Hsien-Tsung; Chiang, Cheng-Ming

    2016-01-01

    Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1–158 amino acid residues, called Venus-N, and its C-terminal 159–239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is

  13. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    NASA Astrophysics Data System (ADS)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  14. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

    PubMed Central

    Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

    2012-01-01

    A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

  15. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

    PubMed Central

    2016-01-01

    Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  16. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors.

    PubMed

    Cisneros, José A; Robertson, Michael J; Valhondo, Margarita; Jorgensen, William L

    2016-07-13

    Human macrophage migration inhibitory factor (MIF) is both a keto-enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein-ligand hydrogen bonding, aryl-aryl, and cation-π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  17. A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition.

    PubMed

    Wang, Lei; Yan, Xu; Su, Xingguang

    2016-09-01

    In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5'-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1-2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease. PMID:27543031

  18. Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format.

    PubMed

    Auld, Douglas S; Johnson, Ronald L; Zhang, Ya-qin; Veith, Henrike; Jadhav, Ajit; Yasgar, Adam; Simeonov, Anton; Zheng, Wei; Martinez, Elisabeth D; Westwick, John K; Austin, Christopher P; Inglese, James

    2006-01-01

    Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput. PMID:17110211

  19. A Fluorescence Immunochromatographic Assay Using Europium (III) Chelate Microparticles for Rapid, Quantitative and Sensitive Detection of Creatine Kinase MB.

    PubMed

    Lai, Xiao-Hong; Liang, Rong-Liang; Liu, Tian-Cai; Dong, Zhi-Ning; Wu, Ying-Song; Li, Lin-Hai

    2016-05-01

    The isoenzyme creatine kinase MB is very important for diagnosis of acute myocardial infarction (AMI). Some CK-MB immunoassays are sensitive, accurate and available for clinical application, but they are expensive and time-consuming procedures. Furthermore, conventional fluorescence immunochromatographic assays (FL-ICAs) have suffered from background fluorescence interference and low analytical sensitivity. A rapid and simple FL-ICA with Eu (III) chelate polystyrene microparticles was developed to determine CK-MB in 50uL serum samples using a portable test strip reader by measuring the fluorescence peak heights of the test line (HT) and the control line (HC) in 12 min. The assay was reliable with a good correlation coefficient between HT/HC ratio and CK-MB concentration in samples. A linear range was 0.85-100.29 ng/mL for CK-MB, and the LOD was 0.029 ng/mL. The intra- and inter-assay coefficients of variation (CV) were both <10 % and the average recoveries were from 90.17 % -112.63 % for CK-MB. The system performed well in interference experiments. Furthermore, a highly significant correlation (r = 0.9794, P < 0.001) between this method and the commercially available bioMérieux mini VIDAS system were attained for measuring 120 CK-MB samples. These results indicated that the Eu (III) chelate microparticles-based FL-ICA is simple, fast, highly sensitive, reliable, and reproducible for point-of-care testing of CK-MB concentrations in serum. Graphical Abstract ᅟ. PMID:27034063

  20. 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. PMID:25809520

  1. A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays

    PubMed Central

    Lee, Linda G.; Nordman, Eric S.; Johnson, Martin D.; Oldham, Mark F.

    2013-01-01

    We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE) to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA) as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG) as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting. PMID:25586412

  2. Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

    PubMed Central

    Dillingham, Mark S.; Tibbles, Katherine L.; Hunter, Jackie L.; Bell, Jason C.; Kowalczykowski, Stephen C.; Webb, Martin R.

    2008-01-01

    The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases. PMID:18599625

  3. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    PubMed

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels. PMID:24830141

  4. Determination of ochratoxin A in food: comparison of a stable isotope dilution assay, liquid chromatography-fluorescence detection and an enzyme-linked immunosorbent assay.

    PubMed

    Lindenmeier, Michael; Schieberle, Peter; Rychlik, Michael

    2011-05-01

    Quantitative results for the mycotoxin ochratoxin A (OTA), obtained by a stable isotope dilution assay (SIDA) were compared with two commonly used analytical methods for OTA quantitation. For this, different types of food, such as wheat, coffee, sultanas, and blood sausages, were analyzed. Because results obtained by the SIDA method were closest to the certified contents of an OTA reference material, data obtained by this method were considered as reference data. For liquid chromatography-fluorescence detection, a clean-up by solid phase extraction on silica was found to be necessary, and a correction for recovery had to be performed to match the data from the SIDA experiments. The enzyme-linked immunosorbent assay (ELISA) strongly overestimated the OTA content in coffee and nutmeg therefore an extract clean-up by immunoaffinity chromatography had to be used to match the SIDA results. Following this sample preparation, ELISA gave correct qualitative and semiquantitative results, and proved to be a suitable screening method. SIDA was also established as a valuable tool to quantify OTA in meat products, when using a clean-up procedure developed recently for blood samples. PMID:23605702

  5. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    PubMed

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity. PMID:16834534

  6. A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay.

    PubMed

    Carmona, Adriana K; Schwager, Sylva L; Juliano, Maria A; Juliano, Luiz; Sturrock, Edward D

    2006-01-01

    Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader. PMID:17487185

  7. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples.

    PubMed

    Otero, Paz; Alfonso, Amparo; Alfonso, Carmen; Aráoz, Rómulo; Molgó, Jordi; Vieytes, Mercedes R; Botana, Luis M

    2011-09-01

    In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication. PMID:21801889

  8. Fluorescent single-stranded DNA-based assay for detecting unchelated Gadolinium(III) ions in aqueous solution.

    PubMed

    Edogun, Osafanmwen; Nguyen, Nghia Huu; Halim, Marlin

    2016-06-01

    The main concern pertaining to the safety of Gadolinium(III)-based contrast agents (GBCAs) is the toxicity caused by the unchelated ion, which may be inadvertently present in the solution due most commonly to excess unreacted starting material or dissociation of the complexes. Detecting the aqueous free ion during the synthesis and preparation of GBCA solutions is therefore instrumental in ensuring the safety of the agents. This paper reports the development of a sensitive fluorogenic sensor for aqueous unchelated Gadolinium(III) (Gd(III)). Our design utilizes single-stranded oligodeoxynucleotides with a specific sequence of 44 bases as the targeting moiety. The fluorescence-based assay may be run at ambient pH with very small amounts of samples in 384-well plates. The sensor is able to detect nanomolar concentration of Gd(III), and is relatively unresponsive toward a range of biologically relevant ions and the chelated Gd(III). Although some cross-reactivity with other trivalent lanthanide ions, such as Europium(III) and Terbium(III), is observed, these are not commonly found in biological systems and contrast agents. This convenient and rapid method may be useful in ascertaining a high purity of GBCA solutions. Graphical abstract Fluorescent aptamer-based assay for detecting unchelated Ln(III) ions in aqueous solution. PMID:27071762

  9. Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay.

    PubMed

    Guo, Sujuan; Pridham, Kevin J; Sheng, Zhi

    2016-01-01

    Autophagy is a catabolic process whereby cellular components are degraded to fuel cells for longer survival during stress. Hence, autophagy plays a vital role in determining cell fate and is central for homeostasis and pathogenesis of many human diseases including chronic myeloid leukemia (CML). It has been well established that autophagy is important for the leukemogenesis as well as drug resistance in CML. Thus, autophagy is an intriguing therapeutic target. However, current approaches that detect autophagy lack reliability and often fail to provide quantitative measurements. To overcome this hurdle and facilitate the development of autophagy-related therapies, we have recently developed an autophagy assay termed as the Cyto-ID fluorescence spectrophotometric assay. This method uses a cationic fluorescence dye, Cyto-ID, which specifically labels autophagic compartments and is detected by a spectrophotometer to permit a large-scale and quantitative analysis. As such, it allows rapid, reliable, and quantitative detection of autophagy and estimation of autophagy flux. In this chapter, we further provide technical details of this method and step-by-step protocols for measuring autophagy or autophagy flux in CML cell lines as well as primary hematopoietic cells. PMID:27581142

  10. A fluorescent assay for γ-glutamyltranspeptidase via aggregation induced emission and its applications in real samples.

    PubMed

    Hou, Xianfeng; Zeng, Fang; Wu, Shuizhu

    2016-11-15

    γ-Glutamyl transpeptidase (GGT) plays crucial roles in some physiological processes. Herein a turn-on fluorescent probe for γ-glutamyl transpeptidase (GGT) assay based on aggregation-induced-emission (AIE) effect and the enzyme-induced transformation of hydrophilicity to hydrophobicity has been developed by functionalizing tetraphenylethylene (TPE) derivative with two γ-glutamyl amide groups, which simultaneously work as recognition units and hydrophilic groups. When the γ-glutamyl amide groups are cleaved through GGT enzymatic reaction, the hydrophobic reaction product readily aggregate and correspondingly strong blue fluorescence can be observed, as a result of activated AIE process. By virtue of the probe's good solubility in totally aqueous solution, high sensitivity and excellent photostability, the probe can be employed to detect GGT level in human serum samples. Furthermore, the probe can be used for imaging endogenous GGT in living A2780 cells. Hence, the probe holds great promise for acting as a convenient one-step straightforward assay for GGT detection in diagnostic-related applications, and also it could provide a useful approach for conducting pathological analysis for diseases involving GGT. PMID:27183282

  11. Dopamine assay based on an aggregation-induced reversed inner filter effect of gold nanoparticles on the fluorescence of graphene quantum dots.

    PubMed

    Lin, Feng-E; Gui, Chuang; Wen, Wei; Bao, Ting; Zhang, Xiuhua; Wang, Shengfu

    2016-09-01

    We describe a fluorescent dopamine assay that is based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of graphene quantum dots (GQDs). The green fluorescence of GQDs is remarkably inhibited in the presence of citrate-stabilized AuNPs via IFE. Upon the addition of dopamine (DA), aggregation of the AuNPs occurs which is associated with a color change from red to blue. The IFE can no longer occur and the fluorescence of GQDs is recovered. Under the optimum conditions, a linear correlation exists between fluorescence intensity and the concentration of DA in the range from 20nM to 200nM with a detection limit of 15nM (at 3σ/s). The assay is rapid, inexpensive and highly sensitive. PMID:27343608

  12. Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

    PubMed

    Branchini, Bruce

    2016-01-01

    We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues. PMID:27424898

  13. Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

    PubMed

    Akbar, S Md; Sreeramulu, K; Sharma, Hari C

    2016-06-01

    Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery. PMID:26905428

  14. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    PubMed Central

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2014-01-01

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. PMID:25555152

  15. Boronate Affinity Fluorescent Nanoparticles for Förster Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules.

    PubMed

    Wang, Shuangshou; Ye, Jin; Li, Xinglin; Liu, Zhen

    2016-05-17

    Förster resonance energy transfer (FRET) has been essential for many applications, in which an appropriate donor-acceptor pair is the key. Traditional dye-to-dye combinations remain the working horses but are rather nonspecifically susceptive to environmental factors (such as ionic strength, pH, oxygen, etc.). Besides, to obtain desired selectivity, functionalization of the donor or acceptor is essential but usually tedious. Herein, we present fluorescent poly(m-aminophenylboronic acid) nanoparticles (poly(mAPBA) NPs) synthesized via a simple procedure and demonstrate a FRET scheme with suppressed environmental effects for the selective sensing of cis-diol biomolecules. The NPs exhibited stable fluorescence properties, resistance to environmental factors, and a Förster distance comparable size, making them ideal donor for FRET applications. By using poly(mAPBA) NPs and adenosine 5'-monophosphate modified graphene oxide (AMP-GO) as a donor and an acceptor, respectively, an environmental effects-suppressed boronate affinity-mediated FRET system was established. The fluorescence of poly(mAPBA) NPs was quenched by AMP-GO while it was restored when a competing cis-diol compounds was present. The FRET system exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. Quantitative inhibition assay of glucose in human serum was demonstrated. As many cis-diol compounds such as sugars and glycoproteins are biologically and clinically significant, the FRET scheme presented herein could find more promising applications. PMID:27089186

  16. Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis

    PubMed Central

    Peng, Jingfu; Yu, Xiaoli; Cui, Zhenling; Xue, Wenfei; Luo, Ziyi; Wen, Zilu; Liu, Minghua; Jiang, Danqing; Zheng, Heping; Wu, Hai; Zhang, Shulin; Li, Yao

    2016-01-01

    Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH). Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay. Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate. Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates. PMID:27199947

  17. [Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA].

    PubMed

    Xue, Li-Jun; Wang, Yong-Zhi; Ren, Hao; Tong, Yi-Min; Zhao, Ping; Zhu, Shi-Ying; Qi, Zhong-Tian

    2006-09-01

    The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future. PMID:17037203

  18. Fluorescence assay for monitoring Zn-deficient superoxide dismutase in vitro

    NASA Astrophysics Data System (ADS)

    Martyshkin, D. V.; Mirov, S. B.; Zhuang, Y.-X.; Crow, J. P.; Ermilov, V.; Beckman, J. S.

    2003-11-01

    A method has been developed for selective detection of the zinc-deficient form of Cu, Zn superoxide dismutase (SOD1) in vitro. Zinc-deficient SOD1 mutants have been implicated in the death of motor neurons leading in amyotrophic lateral sclerosis (ALS or Lou Gerhig's disease). Thus, this method may have applicability for detecting zinc-deficient SOD1 mutants in human ALS patients samples as well as in a transgenic mouse model of ALS and in cultured motor neurons. We determined previously that structural analogs of 1,10 phenanthroline, which react specifically with Cu(I), react with the active Cu(I) of SOD1 when zinc is absent, but not when zinc is also bound, as evidenced by the fact that the reaction is inhibited by pretreatment of the enzyme with zinc. We report herein that bathocuproine, or its water-soluble derivative bathocuproine disulfonate, react with zinc-deficient SOD1 to form a complex which fluoresces at 734 nm when excited at 482 nm. Fluorescent intensity is concentration dependent, thus we propose to use fluorescent confocal microscopy to measure intracellular levels of zinc-deficient SOD1 in situ.

  19. A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization.

    PubMed

    Yue, Qiaoli; Shen, Tongfei; Wang, Lei; Xu, Shuling; Li, Haibo; Xue, Qingwang; Zhang, Yuanfu; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2014-06-15

    A new aptamer biosensor was presented for the detection of thrombin in this work, which was based on fluorescence polarization (FP) using silica nanoparticles as enhancement probe. The silica nanoparticles covered by streptavidin were tagged with a thrombin aptamer (5'-biotin-GGTTGGTGTGGTTGG-3'), which was bound to the surface of silica nanoparticle through the specific interaction between streptavidin and biotin. In the presence of thrombin, it induced the aptamer to form quadruplex structure. When the other thrombin aptamer labeled with fluorescein (5'-FAM-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') was added to the above system, a sandwich structure can form at the surface of silica nanoparticles. The fluorescence polarization was therefore enhanced and quantification between fluorescence polarization signal and concentration of thrombin was built. The sensor provided a linear range from 0.6 to 100 nM for thrombin with a detection limit of 0.20 nM (3.29 SB/m, according to the recent recommendation of IUPAC) in a homogeneous media. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.26 nM), demonstrating high anti-interference of the sensor in a complex biological sample matrix. And the sensor can be used to monitor spiked concentration of thrombin level in real human plasma with satisfactory results obtained. PMID:24508546

  20. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

    EPA Science Inventory

    One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

  1. The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.

    PubMed

    Jensen, Jesper Langholm; Jacobsen, Jonas; Moss, Marcia L; Rasmussen, Fred; Qvist, Karsten Bruun; Larsen, Sine; van den Brink, Johannes M

    2015-05-01

    Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method. PMID:25726113

  2. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.

    PubMed

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  3. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging

    PubMed Central

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  4. HPLC with fluorescence detection assay of perampanel, a novel AMPA receptor antagonist, in human plasma for clinical pharmacokinetic studies.

    PubMed

    Mano, Yuji; Takenaka, Osamu; Kusano, Kazutomi

    2015-10-01

    Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. PMID:25828925

  5. A Dual-Mode Single-Molecule Fluorescence Assay for the Detection of Expanded CGG Repeats in Fragile X Syndrome

    PubMed Central

    Cannon, Brian; Pan, Cynthia; Chen, Liangjing; Hadd, Andrew G.

    2012-01-01

    Fragile X syndrome is the leading cause of inherited mental impairment and is associated with expansions of CGG repeats within the FMR1 gene. To detect expanded CGG repeats, we developed a dual-mode single-molecule fluorescence assay that allows acquisition of two parallel, independent measures of repeat number based on (1) the number of Cy3-labeled probes bound to the repeat region and (2) the physical length of the electric field-linearized repeat region, obtained from the relative position of a single Cy5 dye near the end of the repeat region. Using target strands derived from cell-line DNA with defined numbers of CGG repeats, we show that this assay can rapidly and simultaneously measure the repeats of a collection of individual sample strands within a single field of view. With a low occurrence of false positives, the assay differentiated normal CGG repeat lengths (CGGN, N = 23) and expanded CGG repeat lengths (CGGN, N = 118), representing a premutation disease state. Further, mixtures of these DNAs gave results that correlated with their relative populations. This strategy may be useful for identifying heterozygosity or for screening collections of individuals, and it is readily adaptable for screening other repeat disorders. PMID:22311273

  6. An Improved Method for Estimating Antibody Titers in Microneutralization Assay Using Green Fluorescent Protein

    PubMed Central

    Yang, Hongmei; Baker, Steven F.; González, Mario E.; Topham, David J.; Martínez-Sobrido, Luis; Zand, Martin; Holden-Wiltse, Jeanne; Wu, Hulin

    2015-01-01

    Viruses that express reporter genes upon infection have been recently used to evaluate neutralizing antibody responses, where a lack of reporter expression indicates specific virus inhibition. The traditional model-based methods using standard outcome of percent neutralization could be applied to the data from the assays to estimate antibody titers. However, the data produced is sometimes irregular, which can yield meaningless outcomes of percent neutralization that do not fit the typical curves for immunoassays, making automated or semi-high throughput antibody titer estimation unreliable. We developed a type of new outcomes model, which is biologically meaningful and fits typical immunoassay curves well. Our simulation study indicates that the new response approach outperforms the traditional response approach regardless of the data variability. The proposed new response approach can be used in similar assays for other disease models. PMID:26010892

  7. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents.

    PubMed

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-02-24

    In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z' factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening. PMID:22290227

  8. Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

    PubMed

    Kwon, Keehwan; Nagarajan, Rajesh; Stivers, James T

    2004-11-30

    Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover. PMID:15554707

  9. Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

    PubMed

    Kováčová, Kristína; Farkaš, Vladimír

    2016-04-01

    Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated. PMID:26754421

  10. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    SciTech Connect

    Kent, Stephen

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.