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Sample records for 35sgtpgammas binding assay

  1. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  2. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  3. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  4. FACS binding assay for analysing GDNF interactions.

    PubMed

    Quintino, Luís; Baudet, Aurélie; Larsson, Jonas; Lundberg, Cecilia

    2013-08-15

    Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

  5. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  6. Improved flow cytometer measurement of binding assays

    DOEpatents

    Saunders, G.C.

    1984-05-30

    The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

  7. DNA Origami Seesaws as Comparative Binding Assay

    PubMed Central

    Nickels, Philipp C.; Høiberg, Hans C.; Simmel, Stephanie S.; Holzmeister, Phil; Tinnefeld, Philip

    2016-01-01

    Abstract The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami‐based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw‐like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays. PMID:27038073

  8. Integrated Summary Report: Validation of Two Binding Assays ...

    EPA Pesticide Factsheets

    This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 89

  9. Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay.

    PubMed

    McKnight, Ruel E; Gleason, Aaron B; Keyes, James A; Sahabi, Sadia

    2007-02-15

    A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.

  10. Binding of TH-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    SciTech Connect

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.

    1985-09-01

    Binding of TH-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method.

  11. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  12. Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay.

    PubMed

    Romaniuk, Paul J

    2016-01-01

    Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the measurement of the effects of mutations in either the WT1 zinc finger domain or the putative binding sites within a promoter element or cellular RNA.

  13. Hormone-binding assay using living bacteria expressing eukaryotic receptors.

    PubMed

    Romanov, Georgy A; Lomin, Sergey N

    2009-01-01

    Studies on hormone-receptor interaction include, as a rule, isolation and extensive purification of the receptor protein or a particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we proposed a live cell-based assay using transgenic bacteria expressing single eukaryotic receptors. We describe here 3H-cytokinin binding to corresponding plant receptors as an example. The method includes procedures of bacteria growing, incubation with labeled hormone, separation of bound from unbound ligand, determination of radioactivity in bacterial precipitates, and mathematical analysis of primary data. The established simple protocol for specific labeling hormone-binding sites in intact bacteria allows determination of the main parameters of the ligand-receptor interaction.

  14. 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

  15. 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

  16. 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

  17. Binding assays with artificial tethered membranes using surface plasmon resonance.

    PubMed

    Wiltschi, Birgit; Knoll, Wolfgang; Sinner, Eva-Kathrin

    2006-06-01

    Surface sensitive optical techniques based on surface plasmon resonance have become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces. We use surface plasmon resonance spectroscopy (SPS) in combination with surface plasmon enhanced fluorescence spectroscopy (SPFS) for the characterization of interaction processes associated with biomembranes. The biological membrane is mimicked by a tethered membrane consisting of a planar lipid bilayer attached to a gold surface via a hydrophilic anchor peptide. The interaction between membrane-bound hydrophobic compounds and free hydrophilic molecules is monitored in real-time and with high sensitivity and selectivity by combined SPS/SPFS. In this review we shortly discuss the principles of surface plasmon resonance and its utilization in SPS and SPFS. A detailed description of the required instrumentation for combined SPS and SPFS is presented. Furthermore, we outline the design of a binding assay with a tethered bilayer and the procedure of the artificial membrane system built-up is delineated. We also present examples that demonstrate the potential of combined SPS/SPFS assays with artificial tethered membranes. The method provides insight into the interaction of integral membrane proteins with various hydrophilic ligands and the specific recognition of small lipophilic molecules by soluble proteins.

  18. International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

    EPA Science Inventory

    An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

  19. Competitive MS binding assays for dopamine D2 receptors employing spiperone as a native marker.

    PubMed

    Niessen, Karin V; Höfner, Georg; Wanner, Klaus T

    2005-10-01

    A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays.

  20. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation.

    PubMed

    Ye, Zhengqi; Zetterberg, Craig; Gao, Hong

    2017-03-14

    Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

  1. Flourescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

  2. Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

  3. Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry.

    PubMed

    Wuhrer, Manfred; van Remoortere, Alexandra; Balog, Crina I A; Deelder, André M; Hokke, Cornelis H

    2010-11-15

    Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.

  4. Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay.

    PubMed

    Vater, C A; Reid, K; Bartle, L M; Goldmacher, V S

    1995-01-01

    A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.

  5. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    PubMed

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

  6. 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

  7. Characterization of protein-membrane binding interactions via a microplate assay employing whole liposome immobilization.

    PubMed

    Smith, Matthew D; Best, Michael D

    2011-01-01

    Protein-cell membrane binding interactions control numerous vital biological processes, many of which can go awry during disease onset. However, the study of these events is complicated by the complexity of the membrane bilayer. These efforts would benefit from a rapid and easily accessible method for characterizing protein-membrane recognition events. Herein, we describe a microplate-based method for the detection of protein-membrane binding that employs whole liposome immobilization using a biotin anchor. First, control studies are detailed to test for nonspecific liposome immobilization (fluorescence assay; see Subheading 3.2), and to ensure that liposomes remain intact on the microplate surface (dye leakage assay; see Subheading 3.3). Finally, a protein-membrane binding detection assay is described through the example of protein kinase Cα binding to surface-immobilized whole liposomes (see Subheading 3.4).

  8. Monoclonal antibody binding-site diversity assessment with a cell-based clustering assay.

    PubMed

    Liao-Chan, Sindy; Zachwieja, Joseph; Gomez, Steven; Duey, Dana; Lippincott, John; Theunissen, Jan-Willem

    2014-03-01

    The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.

  9. A robust assay to measure DNA topology-dependent protein binding affinity.

    PubMed

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.

  10. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    PubMed Central

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  11. Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

    PubMed

    Tahir, S K; Kovar, P; Rosenberg, S H; Ng, S C

    2000-07-01

    We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

  12. A polyplex qPCR-based binding assay for protein-DNA interactions.

    PubMed

    Moreau, Morgane J J; Schaeffer, Patrick M

    2012-09-21

    The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.

  13. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  14. Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay.

    PubMed

    Döppenschmitt, S; Langguth, P; Regårdh, C G; Andersson, T B; Hilgendorf, C; Spahn-Langguth, H

    1999-01-01

    Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to difficulties in the interpretation of results. An assay for quantification of drug affinity to the transporter is presented on the basis of the radioligand-binding assay principle. This has the advantage of directly quantifying the interaction between drugs and P-gp. Because of the reversible and competitive interaction of numerous substrates with P-gp, a radioligand-binding assay was developed by taking [3H]verapamil and [3H]vinblastine as radioligands and the human intestinal Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clear induction of the expression of P-gp was demonstrated in the Caco-2 cells grown in the presence of vinblastine, as well as in the transfected cells, although to a lesser extent. Both radioligands were shown to bind to P-gp. Verapamil was the radioligand of choice for further investigations due to its lower nonspecific binding to the transporter preparation. Kinetics as well as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the data derived from saturation as well as from competition experiments, in accordance with previous findings on two exsorption sites for P-gp. The binding properties of [3H]verapamil and [3H]vinblastine to

  15. Radioligand binding assays in the drug discovery process: potential pitfalls of high throughput screenings.

    PubMed

    Noël, F; Mendonça-Silva, D L; Quintas, L E

    2001-02-01

    Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if

  16. Improving time to optimal Staphylococcus aureus treatment using a penicillin-binding protein 2a assay.

    PubMed

    Rao, Sonia N; Wang, Sheila K; Gonzalez Zamora, Jose; Hanson, Amy P; Polisetty, Radhika S; Singh, Kamaljit

    2016-12-01

    The penicillin-binding protein 2a (PBP2a) assay is a quick, accurate and inexpensive test for determining methicillin susceptibility in Staphylococcus aureus. A pre-post-study design was conducted using a PBP2a assay with and without the impact of an antimicrobial stewardship intervention to improve time to optimal therapy for methicillin-susceptible and methicillin-resistant S. aureus isolates. Our results demonstrate significantly improved time to optimal therapy and support the use of a PBP2a assay as part of an programme for all healthcare facilities, especially those with limited resources.

  17. Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores.

    PubMed

    Ikanovic, Milada; Rudzinski, Walter E; Bruno, John G; Allman, Amity; Carrillo, Maria P; Dwarakanath, Sulatha; Bhahdigadi, Suneetha; Rao, Poornima; Kiel, Johnathan L; Andrews, Carrie J

    2007-03-01

    A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.

  18. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    PubMed Central

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with Kd values of 2.50 and 4.00 μM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques. PMID:17038331

  19. Relative Chemical Binding Affinities for Trout and Human Estrogen Receptor Using Different Competitive Binding Assays

    EPA Science Inventory

    Rainbow trout-based assays for estrogenicity are currently being used for development of predictive models based upon quantitative structure activity relationships. A predictive model based on a single species raises the question of whether this information is valid for other spe...

  20. A novel assay to identify the trafficking proteins that bind to specific vesicle populations

    PubMed Central

    Bentley, Marvin; Banker, Gary

    2016-01-01

    Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin–binding domain (FRB)–tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

  1. Application of a silver-binding assay to the determination of protein in cerebrospinal fluid.

    PubMed

    Krystal, G; Lam, V; Schreiber, W E

    1989-05-01

    We evaluated a silver-binding assay for use in measuring total protein in cerebrospinal fluid. The advantage of this procedure over other methods is that, because of its sensitivity, it requires only a 0.5-microL sample. The procedure, which takes approximately 40 min to complete, involves dilution of 0.5-microL samples to 1 mL with distilled water containing sodium dodecyl sulfate, followed by addition of glutaraldehyde and an ammoniacal silver solution. After color development for 30 min, the reaction is terminated with sodium thiosulfate and the absorbance is measured at 420 nm. This assay displayed within-run and day-to-day precision (CV) of 3.1% to 13% over the range of 210 to 1370 mg/L. It showed substantially less protein-to-protein variation than the Coomassie Blue dye-binding procedure when tested with albumin, globulin, and transferrin. It also yielded an accurate estimation of hemoglobin. Moreover, preliminary studies suggested that it was capable of quantifying immunoglobulin light chains and glycoproteins. In a study of 54 human cerebrospinal fluid samples, results of the silver-binding assay corresponded more closely with those obtained with a rate biuret assay (intraclass correlation coefficient = 0.91) than did either the dye-binding or classical Lowry methods.

  2. Relationship between the results of in vitro receptor binding assay to human estrogen receptor alpha and in vivo uterotrophic assay: comparative study with 65 selected chemicals.

    PubMed

    Akahori, Yumi; Nakai, Makoto; Yamasaki, Kanji; Takatsuki, Mineo; Shimohigashi, Yasuyuki; Ohtaki, Masahiro

    2008-02-01

    For screening chemicals possessing endocrine disrupting potencies, the uterotrophic assay has been placed in a higher level in the OECD testing framework than the ER binding assay to detect ER-mediated activities. However, there are no studies that can demonstrate a clear relationship between these assays. In order to clarify the relationship between the in vitro ER binding and in vivo uterotrophic assays and to determine meaningful binding potency from the ER binding assay, we compared the results from these assays for 65 chemicals spanning a variety of chemicals classes. Under the quantitative comparison between logRBAs (relative binding affinities) and logLEDs (lowest effective doses), the log RBA was well correlated with both logLEDs of estrogenic and anti-estrogenic compounds at r(2)=0.67 (n=28) and 0.79 (n=23), respectively. The RBA of 0.00233% was found to be the lowest ER binding potency to elicit estrogenic or anti-estrogenic activities in the uterotrophic assay, accordingly this value is considered as the detection limit of estrogenic or anti-estrogenic activities in the uterotrophic assay. The usage of this value as cutoff provided the best concordance rate (82%). These findings are useful in a tiered approach for identifying chemicals that have potential to induce ER-mediated effects in vivo.

  3. Automate it: ligand-binding assay productivity in a discovery bioanalytical setting.

    PubMed

    Leung, Sheldon S; Dreher, Elizabeth A

    2013-07-01

    In multiple industries, including the biopharmaceutical industry, automation is synonymous with increased productivity. Environments with high-throughput needs commonly employ automation for efficiency. However, in a discovery bioanalytical ligand-binding assay laboratory setting where the focus is not necessarily on sample analysis throughput, but instead on assay development and characterization, is automation applicable? Can automation enhance productivity when tasks are more customized than routine? In this Perspective we review the different categories of automation with ligand-binding assays with these questions in mind. In considering whether automation technology has progressed far enough to result in a positive return in investment in the discovery setting, the resource investment required to operate in this space was contrasted with the gain in productivity. In our opinion, technology advancements in automated technology platforms, and especially personal automation, have allowed these categories to strike the right balance for investment in the discovery laboratory setting.

  4. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay.

    PubMed

    Hendrickson, W; Schleif, R F

    1984-09-25

    DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.

  5. Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay.

    PubMed

    Kim, Jinsu; Vu, Binh; Kourentzi, Katerina; Willson, Richard C; Conrad, Jacinta C

    2017-02-15

    To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters.

  6. A versatile assay for RNA-binding proteins in living cells.

    PubMed

    Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W; Castello, Alfredo

    2014-05-01

    RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

  7. A rapid and simple assay for growth hormone-binding protein activity in human plasma.

    PubMed

    Baumann, G; Shaw, M A; Amburn, K

    1988-12-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

  8. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  9. Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

    PubMed Central

    Cowles, E A; Yunovitz, H; Charles, J F; Gill, S S

    1995-01-01

    The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut. PMID:7618886

  10. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  11. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  12. Data quality in drug discovery: the role of analytical performance in ligand binding assays

    NASA Astrophysics Data System (ADS)

    Wätzig, Hermann; Oltmann-Norden, Imke; Steinicke, Franziska; Alhazmi, Hassan A.; Nachbar, Markus; El-Hady, Deia Abd; Albishri, Hassan M.; Baumann, Knut; Exner, Thomas; Böckler, Frank M.; El Deeb, Sami

    2015-09-01

    Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

  13. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    PubMed

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-04

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  14. A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

    PubMed

    Vainshtein, Inna; Silveria, Scott; Kaul, Poonam; Rouhani, Riaz; Eglen, Richard M; Wang, John

    2002-12-01

    A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta

  15. Development of an albumin copper binding (ACuB) assay to detect ischemia modified albumin.

    PubMed

    Eom, Ji-Eun; Lee, Eunyoung; Jeon, Kyung-Hwa; Sim, Jeongeun; Suh, Minah; Jhon, Gil-Ja; Kwon, Youngjoo

    2014-01-01

    Myocardial ischemia (MI) induces many changes in the body, including pH decrease and electrolyte imbalance. No obvious symptoms of MI appear until irreversible cellular injuries occur. Since early treatment is critical for recovery from ischemia, the development of reliable diagnostic tool is demanded to detect the early ischemic status. Ischemia modified albumin (IMA), formed by cleavage of the last two amino acids of the human serum albumin (HSA) N-terminus, has been considered so far as the most trustworthy and accurate marker for the investigation of ischemia. IMA levels are elevated in plasma within a few minutes of ischemic onset, and may last for up to 6 h. In the present study, we developed a novel assay for the examination of IMA levels to ameliorate the known albumin cobalt binding (ACB) test established previously. We observed a stronger copper ion bound to the HSA N-terminal peptide than cobalt ion by HPLC and ESI-TOF mass spectrometric analyses. The copper ion was employed with lucifer yellow (LY), a copper-specific reagent to develop a new albumin copper binding (ACuB) assay. The parameters capable of affecting the assay results were optimized, and the finally-optimized ACuB assay was validated. The result of the IMA level measurement in normal versus stroke rat serum suggests that the ACuB assay is likely to be a reliable and sensitive method for the detection of ischemic states.

  16. Antibody binding in altered gravity: implications for immunosorbent assay during space flight

    NASA Technical Reports Server (NTRS)

    Maule, Jake; Fogel, Marilyn; Steele, Andrew; Wainwright, Norman; Pierson, Duane L.; McKay, David S.

    2003-01-01

    A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.

  17. Brevenal Is a Natural Inhibitor of Brevetoxin Action in Sodium Channel Receptor Binding Assays

    PubMed Central

    Bourdelais, Andrea J.; Campbell, Susan; Jacocks, Henry; Naar, Jerome; Wright, Jeffery L. C.; Carsi, Jigani; Baden, Daniel G.

    2009-01-01

    Summary 1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures. PMID:15233378

  18. A modified paper-binding procedure for the assay of nucleus-associated protein phosphokinases.

    PubMed

    Goueli, S A; Slungaard, R; Wilson, M J; Ahmed, K

    1980-05-01

    Previously existing paper-binding assay procedures gave results with large variations when employed for the measurement of nucleus-associated protein phosphokinase activities. However, a modified method, utilizing the binding of 32P-labeled phosphoprotein substrates to paper and employing washing procedures in 20% trichloroacetic acid at 60 degrees to 70 degrees C, gave highly reproducible results. This modified procedure was satisfactory with either chromatin or a nonhistone protein fraction derived therefrom as a source of enzyme, and dephosphophosvitin, lysine-rich histones, or casein as phosphoprotein substrates.

  19. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages

    PubMed Central

    Simpson, David J.; Sacher, Jessica C.; Szymanski, Christine M.

    2016-01-01

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages. PMID:26761028

  20. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages.

    PubMed

    Simpson, David J; Sacher, Jessica C; Szymanski, Christine M

    2016-01-11

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages.

  1. An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

    PubMed

    Demissie, Robel D; Kabre, Pauline; Tuntland, Micheal L; Fung, Leslie W-M

    2016-04-01

    Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

  2. A universal homogeneous assay for high-throughput determination of binding kinetics.

    PubMed

    Schiele, Felix; Ayaz, Pelin; Fernández-Montalván, Amaury

    2015-01-01

    There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug-target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR-FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein-protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.

  3. Improved binding of acidic bone matrix proteins to cationized filters during solid phase assays.

    PubMed

    Farach-Carson, M C; Wright, G C; Butler, W T

    1992-01-01

    A number of commercially available matrix filter supports have been designed for the immobilization of proteins following either electrotransfer from sodium dodecyl sulfate (SDS) polyacrylamide gels or direct application during dot blotting assays. These matrices differ with respect to chemical composition, charge, pore size, and degree of hydrophobicity. It follows that the properties of the protein(s) of interest will greatly influence the degree to which they interact with and ultimately bind to various filters. Acidic bone proteins contain diverse post-translational modifications that influence their interactions with solid phase matrices such as those used in immunoblotting (Western or dot blotting) or ion binding (overlay) procedures. This communication describes the results of a study comparing binding of various mixtures of non-collagenous acidic bone matrix phosphoproteins as well as purified osteopontin and osteocalcin to various filters including nitrocellulose and cationized paper or nylon. Based on our findings, we recommend the use of cationized filters for solid phase assays requiring the binding of these acidic macromolecules to background supports.

  4. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  5. Characterization of the novel progestin gestodene by receptor binding studies and transactivation assays.

    PubMed

    Fuhrmann, U; Slater, E P; Fritzemeier, K H

    1995-01-01

    Gestodene is a novel progestin used in oral contraceptives with an increased separation of progestogenic versus androgenic activity and a distinct antimineralocorticoid activity. This specific pharmacological profile of gestodene is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of gestodene to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR) and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for 3-keto-desogestrel and progesterone. The two synthetic progestins displayed identical high affinity to rabbit PR and similar marked binding to rat AR and GR, while progesterone showed high affinity to PR but only low binding to AR and GR. Furthermore, 3-keto-desogestrel exhibited almost no binding to MR, whereas gestodene, similar to progesterone, showed marked affinity to this receptor. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of gestodene on AR-, GR- and MR-mediated induction of transcription. In contrast to progesterone, which showed antiandrogenic activity, gestodene and 3-keto-desogestrel both exhibited androgenic activity. Furthermore, all three progestins exhibited weak GR-mediated antagonistic activity. In contrast to progesterone, which showed almost no glucocorticoid activity, gestodene and 3-keto-desogestrel showed weak glucocorticoid action. In addition, gestodene inhibited the aldosterone-induced reporter gene transcription, similar to progesterone, whereas unlike progesterone, gestodene did not induce reporter gene transcription. 3-Keto-desogestrel showed neither antimineralocorticoid nor mineralocorticoid action.

  6. Cholesterol transport via ABCA1: new insights from solid-phase binding assay.

    PubMed

    Reboul, Emmanuelle; Dyka, Frank M; Quazi, Faraz; Molday, Robert S

    2013-04-01

    It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).

  7. Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors

    PubMed Central

    Montano, Erica N.; Boullier, Agnès; Almazan, Felicidad; Binder, Christoph J.; Witztum, Joseph L.; Hartvigsen, Karsten

    2013-01-01

    Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of OxLDL to macrophages has traditionally been assessed using radiolabeled OxLDL. To allow more efficient and convenient measurements, we developed a nonradioactive binding assay in which biotinylated OxLDL (Bt-OxLDL) is added to macrophages in 96-well microtiter culture plates under various conditions and the extent of binding is determined using solid phase chemiluminescent immunoassay techniques. As examples, we show that Bt-OxLDL displayed high and saturable binding to macrophages in contrast to Bt-LDL, which showed very low binding. In competition assays, unlabeled OxLDL and the anti-OxLDL monoclonal antibody E06 inhibited Bt-OxLDL binding to macrophages in a dose-dependent manner. Specific binding of Bt-OxLDL to ApoE/SR-A/CD36 triple knockout macrophages was reduced by 80% as compared with binding to macrophages from ApoE knockout mice. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells. This assay avoids the use of radioactivity and uses small amounts of materials. It can be used to study binding of OxLDL to macrophages and factors that influence this binding. The techniques described should be readily adaptable to study of other ligands, receptors, and cell types. PMID:23997238

  8. High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

    PubMed Central

    2015-01-01

    We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets. PMID:25233437

  9. A particle agglutination assay for rapid identification of heparin binding to coagulase-negative staphylococci.

    PubMed

    Pascu, C; Hirmo, S; Ljungh, A; Wadström, T

    1996-10-01

    The heparin-binding properties of six different species of coagulase-negative staphylococci were examined by a particle agglutination assay. Heparin (mol. wt 4000-6000), mildly treated with sodium periodate, was covalently coupled to amino-modified latex beads (0.72 micron diameter). The particle agglutination assay was validated by comparing results with the adhesion (percentage binding of adherent cells) of coagulase-negative staphylococcal strains to heparinised microtitration plates. Of 38 different coagulase-negative staphylococcal strains tested, 30 showed agglutination reactivity with heparin-coated latex beads. Strains of different coagulase-negative staphylococcal species agglutinated heparin-coated latex beads to various extents (e.g., cells of Staphylococcus haemolyticus strains reacted more strongly than cells of S. epidermidis strains). The agglutination reaction was significantly inhibited by fucoidan, suramin, lambda-carrageenan and other sulphated compounds, but not by non-sulphated carbohydrate polymers such as hyaluronic acid. Agglutination of staphylococcal cells with heparin-coated latex beads was completely blocked by a cell-surface extract. These results suggest that structures responsible for heparin binding are exposed on the cell surface.

  10. Solid-phase receptor binding assay for /sup 125/I-hCG

    SciTech Connect

    Bortolussi, M.; Selmin, O.; Colombatti, A.

    1987-01-01

    A solid-phase radioligand-receptor assay (RRA) to measure the binding of /sup 125/I-labelled human chorionic gonadotropin (/sup 125/I-hCG) to target cell membranes has been developed. The binding of /sup 125/I-hCG to membranes immobilized on the wells of microtitration plates reached a maximum at about 3 hours at 37 degrees C, was saturable, displayed a high affinity (Ka = 2.4 X 10(9) M-1) and was specifically inhibited by unlabelled hCG. In comparison with RRAs carried out with membranes in suspension, the solid-phase RRA is significantly simpler and much faster to perform as it avoids centrifugation or filtration procedures. The solid-phase RRA was adapted profitably to process large numbers of samples at the same time. It proved particularly useful as a screening assay to detect anti-hCG monoclonal antibodies with high inhibitory activity for binding of /sup 125/I-hCG to its receptors.

  11. ISR: background, evolution and implementation, with specific consideration for ligand-binding assays.

    PubMed

    Findlay, John W A; Kelley, Marian M

    2014-02-01

    ISR was highlighted as a topic of major interest to the US FDA in 2006, having been previously required, then discontinued, by Canadian regulatory authorities. Following an FDA focus on ISR, this topic has also been emphasized by regulatory agencies in Europe, Asia and Latin America. Extensive discussions on proper implementation of programs have taken place in multiple settings, including pharmaceutical companies, regulatory agencies, professional associations and CROs. These efforts have led to recommendations for ISR conduct that are now included in a final guideline on bioanalytical method validation from the European Medicines Agency, a draft validation guidance from the Ministry of Health, Labor and Welfare in Japan and a revised draft validation guidance from the FDA. In this Review we look at the background, evolution and implementation of ISR for all assays, while including some specific considerations on this topic for ligand-binding assays.

  12. Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

    SciTech Connect

    Dawson, R.M.

    1986-12-01

    Benzodiazepine receptors of rat cerebellum were assayed with (/sup 3/H)-labeled flunitrazepam at 37/sup 0/C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37/sup 0/C until just prior to filtration.

  13. A radioligand binding assay to measure anti-thyroperoxidase autoantibodies in mice.

    PubMed

    Hayward, Sarah L; Suzuki, Kunimasa; Elliott, John F

    2007-06-30

    Autoimmune (Hashimoto's) thyroiditis is a chronic inflammatory disease which affects >3% of the population and shows an increasing prevalence with increasing age. Anti-thyroid autoantibodies, particularly against thyroperoxidase (also known as thyroid peroxidase or TPO), occur commonly in humans with autoimmune thyroid disease, and assays for anti-TPO autoantibodies are used in clinical diagnosis. In contrast anti-TPO autoantibodies have not been observed in classical mouse models of autoimmune thyroiditis, except in cases where mice were deliberately immunized with TPO. In the past, detection of anti-TPO autoantibodies in mice has relied on an indirect immunofluorescence assay (iIFA) which screens for thyroid follicle membrane staining in frozen sections of mouse thyroid glands. Since recent transgenic mouse models of autoimmune thyroiditis spontaneously develop anti-TPO autoantibodies, an assay other than serial dilution and iIFA would be useful to detect and quantify these autoantibodies. In this paper we describe such an assay, based on the capacity of autoimmune mouse sera to bind to the extracellular domain of mouse TPO which was produced in a radioactively labeled form using a coupled in vitro transcription/translation system. The same approach, using human TPO, could provide a highly sensitive method to detect anti-TPO autoantibodies in humans.

  14. Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

    PubMed

    Onishi, Reina; Watanabe, Ayahisa; Nakajima, Mado; Sekiguchi, Mitsuaki; Kugimiya, Akira; Kinouchi, Hiroki; Nihashi, Yoichiro; Kamimori, Hiroshi

    2015-01-01

    In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

  15. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    PubMed

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal

    2014-11-01

    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

  16. TR-FRET binding assay targeting unactivated form of Bruton's tyrosine kinase.

    PubMed

    Asami, Tokiko; Kawahata, Wataru; Sawa, Masaaki

    2015-01-01

    Bruton's Tyrosine Kinase (BTK) is one of the crucial kinases for the B cell maturation and mast cell activation, and specific inhibitors of BTK are considered to be attractive targets in drug discovery research. In this Letter, we have designed and synthesized a new fluorescent probe for TR-FRET-based high-throughput screening, to identify compounds that preferentially bind to an inactive conformation of BTK which has a unique structural feature. A set of kinase-focused compound library was screened using this assay method, and compound 31 was successfully identified as a potent inhibitor which preferentially bind to the inactive conformation of BTK. These results suggest that this screening method has a great potential for the discovery of novel selective BTK inhibitors.

  17. A receptor binding assay applied to monitoring the neurotoxicity of parathion to Peromyscus after oral exposure

    USGS Publications Warehouse

    Jett, D.A.; Eldefrawi, A.T.; Eldefrawi, M.E.

    1993-01-01

    Many naturally occurring toxins, as well as pesticides, metals, and other compounds that occur in our environment from anthropogenic activities, stimulate or antagonize neuro-receptors to produce acute and/or chronic toxicities. Recent advances in laboratory instrumentation and the availability of a variety of radiolabeled ligands and type-specific drugs for numerous receptors make it possible to easily screen large numbers of samples and detect changes in sensitivity and density of receptor types and subtypes. A receptor binding assay for examining the chronic dietary toxicity of parathion will be used as a model to describe the methodology.

  18. Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

    PubMed Central

    Axmann, Markus; Schütz, Gerhard J.; Huppa, Johannes B.

    2015-01-01

    T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (µM range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on Förster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment. PMID:26555227

  19. Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

    PubMed

    Stanley, Pamela; Sundaram, Subha

    2014-06-03

    Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed

  20. Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

    SciTech Connect

    Schwarz, S.; Ellen, R.P.; Grove, D.A.

    1987-10-01

    There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.

  1. Integrated Summary Report: Validation of Two Binding Assays Using Human Recombinant Estrogen Receptor Alpha (hrERa)

    EPA Science Inventory

    This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (h...

  2. Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

    ERIC Educational Resources Information Center

    Mascotti, David P.; Waner, Mark J.

    2010-01-01

    A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

  3. A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions.

    PubMed

    Xie, Yongyao; Zhang, Yaling; Zhao, Xiucai; Liu, Yao-Guang; Chen, Letian

    2016-02-15

    Investigation of protein-DNA interactions provides crucial information for understanding the mechanisms of gene regulation. Current methods for studying protein-DNA interactions, such as DNaseI footprinting or gel shift assays, involve labeling DNA with radioactive or fluorescent tags, making these methods costly, laborious, and potentially damaging to the environment. Here, we describe a novel cleaved amplified polymorphic sequence (CAPS)-based binding assay (CBA), which is a label-free method that can simplify the semi-quantitative validation of protein-DNA interactions. The CBA tests the interaction between a protein and its target DNA, based on the CAPS pattern produced due to differences in the accessibility of a restriction endonuclease site (intrinsic or artificial) in amplified DNA in the presence and absence of the protein of interest. Thus, the CBA can produce a semi-quantitative readout of the interaction strength based on the dose of the binding protein. We demonstrate the principle and feasibility of CBA using B3, MADS3 proteins and the corresponding RY or CArG-box containing DNAs.

  4. Evaluation of albumin structural modifications through cobalt-albumin binding (CAB) assay.

    PubMed

    Lee, Eunyoung; Eom, Ji-Eun; Jeon, Kyung-Hwa; Kim, Tae Hee; Kim, Eunnam; Jhon, Gil-Ja; Kwon, Youngjoo

    2014-03-01

    Human serum albumin (HSA) is the most abundant protein in the human body. HSA injections prepared by fractionating human blood have mainly covered the demand for albumin to treat hypoalbuminemia, the state of low concentration of albumin in blood. HSA in solution may exist in various forms such as monomers, oligomers, polymers, or as mixtures, and its conformational change and/or aggregation may occur easily. Considering these characteristics, there is a great chance of modification and polymer formation during the preparation processes of albumin products, especially injections. The albumin cobalt binding (ACB) test reported by Bar-Or et al. was originally designed to detect ischemia modified albumin (IMA), which contains the modified HSA N-terminal sequence by cleavage of the last two amino acids. In this study, we developed a cobalt albumin binding (CAB) assay to correct the flaws of the ACB test with improving the sensitivity and precision. The newly developed CAB assay easily detects albumin configuration alterations and may be able to be used in developing a quality control method for albumin and its pharmaceutical formulations including albumin injections.

  5. Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform.

    PubMed

    Liu, Rong; Hoffpauir, Brian; Chilewski, Shannon D; Gamberdella, Janice; Kavita, Uma; Duo, Jia; Gleason, Carol; Zhang, Yan; Pillutla, Renuka; DeSilva, Binodh; Hamuro, Lora

    2017-01-01

    The Gyrolab™ xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab™ xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was <30% for all six LBAs. High instrument response precision (CV ≤5%) was observed across compact discs (CDs), and methods were validated to use a single standard curve across multiple CDs within a Gyrolab™ xP run. Reduced bioanalysis timelines were achieved compared to standard manual plate-based methods, and methods were successfully transferred across testing labs, paving the way for this platform for use in late-stage clinical development.

  6. 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…

  7. Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy has 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 on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic 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 biomolecule 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 Immunoglobulin 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 of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

  8. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed Central

    Prestwich, G D; Wawrzeńczyk, C

    1985-01-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites. PMID:3860862

  9. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed

    Prestwich, G D; Wawrzeńczyk, C

    1985-08-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.

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

    PubMed

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

    2015-10-01

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

  11. New approach to measure protein binding based on a parallel artificial membrane assay and human serum albumin.

    PubMed

    Lázaro, Elisabet; Lowe, Philip J; Briand, Xavier; Faller, Bernard

    2008-04-10

    We report here a new, label-free approach to measure serum protein binding constants. The assay is able to measure HSA K d values in the milli-molar to micromolar range. The protein is not immobilized on any surface and the assay self-corrects for nonspecific adsorption. No mass balance is required to get accurate binding constants and it is not necessary to wait for equilibrium to extract the binding constant. The assay runs in a 96-well format using commercially available parts and is, therefore, relatively easy to implement and automate. As the chemical membranes used are not water permeable, there is no volume change due to the osmotic pressure and pretreatment (soaking) is not necessary. The concept can potentially be extended to other proteins and could thus serve as a label-free technique for general binding constant measurements.

  12. Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations

    PubMed Central

    Nielson, Carrie M.; Jones, Kerry S.; Chun, Rene F.; Jacobs, Jon M.; Wang, Ying; Hewison, Martin; Adams, John S.; Swanson, Christine M.; Lee, Christine G.; Vanderschueren, Dirk; Pauwels, Steven; Prentice, Ann; Smith, Richard D.; Shi, Tujin; Gao, Yuqian; Schepmoes, Athena A.; Zmuda, Joseph M.; Lapidus, Jodi; Cauley, Jane A.; Schoenmakers, Inez; Orwoll, Eric S.

    2016-01-01

    Context: Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitamin D binding protein (DBP) used to calculate f25OHD. Objectives: Our objective was to assess racial-geographic differences in f25OHD and to understand inconsistencies in racial associations with DBP and calculated f25OHD. Design: This study used a cross-sectional design. Setting: The general community in the United States, United Kingdom, and The Gambia were included in this study. Participants: Men in Osteoporotic Fractures in Men and Medical Research Council studies (N = 1057) were included. Exposures: Total 25OHD concentration, race, and DBP (GC) genotype exposures were included. Outcome Measures: Directly measured f25OHD, DBP assessed by proteomics, monoclonal and polyclonal immunoassays, and calculated f25OHD were the outcome measures. Results: Total 25OHD correlated strongly with directly measured f25OHD (Spearman r = 0.84). Measured by monoclonal assay, mean DBP in African-ancestry subjects was approximately 50% lower than in whites, whereas DBP measured by polyclonal DBP antibodies or proteomic methods was not lower in African-ancestry. Calculated f25OHD (using polyclonal DBP assays) correlated strongly with directly measured f25OHD (r = 0.80–0.83). Free 25OHD, measured or calculated from polyclonal DBP assays, reflected total 25OHD concentration irrespective of race and was lower in African Americans than in US whites. Conclusions: Previously reported racial differences in DBP concentration are likely from monoclonal assay bias, as there was no racial difference in DBP concentration by other methods. This confirms the poor vitamin D status of many African-Americans and the utility of total 25OHD in assessing vitamin D in the general population. PMID:27007693

  13. Role of nonspecific binding: a comparison among flow through and flow over assays in nanoporous material

    NASA Astrophysics Data System (ADS)

    Bettotti, P.; Kumar, N.; Guider, R.; Froner, E.; Scarpa, M.

    2014-02-01

    In this article we describe the fabrication of free standing n-type porous silicon microcavity (MC) and their properties as liquid sensors. We have optimized the etching recipe to keep both large pore size and high quality factor (Q-factor). Thus the fabricated porous layers have pore size in the range of 40 to 110 nm and are thus compatible with mass transport across the porous layer. We found that MC with a Q-factor of 60 can measure down to 1.1*10-5 refractive index variations. Furthermore we analyze the role of non specific binding by comparing flow through versus flow over geometries. We compare these two approaches using different techniques and we show that flow over assay systematically overestimates the sensitivity of the device because of an inefficient rinse of the sample. Our work clearly indicates a limit in the reliability of measurements performed in flow over geometry unless specific controls are taken into account.

  14. A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

    PubMed

    Wang, Jia-Hui; Shao, Xiao-Xia; Hu, Meng-Jun; Wei, Dian; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2017-02-04

    Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

  15. Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.

    PubMed

    Wagner, Meike; Jung, Johannes; Koslowski, Michael; Türeci, Özlem; Tiwari, Vijay K; Sahin, Ugur

    2016-01-01

    DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.

  16. Ligand-binding assays: risk of using a platform supported by a single vendor.

    PubMed

    Yohrling, Jennifer

    2009-06-01

    The use of biological reagents in ligand-binding assays (LBAs) presents inherent challenges when measuring the concentration of large molecules in complex matrices. As a result, there are relatively few platforms that provide the accuracy, precision and robustness needed to determine the concentration of macromolecular therapies and biomarkers, and demonstrate the presence or absence of an immune response. Some bioanalytical laboratories use only one LBA platform to reduce costs, increase efficiency and maintain optimal assay performance. However, the business and regulatory risks of using a single platform supported by only one vendor should be considered. This article summarizes the immunological methods used to support bioanalysis for large molecules that are supported by a single vendor, the benefits of being dedicated to a single platform for bioanalysis used for regulatory filings, the costs associated with restructuring if an immunoassay platform is discontinued and recommendations to mitigate risk when using LBAs in drug development. The experience with the recent discontinuation of the BioVeris™ electrochemiluminescent-based platform is discussed.

  17. Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

    PubMed

    Su, Xin; Li, Zehao; Yan, Xinzhong; Wang, Lei; Zhou, Xu; Wei, Lin; Xiao, Lehui; Yu, Changyuan

    2017-03-21

    Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

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

    SciTech Connect

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

    2006-01-01

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

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

    PubMed

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

    1985-10-01

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

  20. Qualitative analysis of sequence specific binding of flavones to DNA using restriction endonuclease activity assays.

    PubMed

    Duran, Elizabeth; Ramsauer, Victoria P; Ballester, Maria; Torrenegra, Ruben D; Rodriguez, Oscar E; Winkle, Stephen A

    2013-08-01

    Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti-cancer agents. We have examined the binding of two flavones, 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone; FlavA) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site (5') TTTAAA) but not BssHII ((5') GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity.

  1. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    PubMed

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

  2. 3-(4-Hydroxyphenyl)propionic acid: the forgotten detection substrate for ligand-binding assay-based bioanalysis.

    PubMed

    Jordan, Gregor; Stubenrauch, Kay-Gunnar; Heinrich, Julia; Staack, Roland F

    2017-02-01

    Ligand-binding assays are ideal for routine bioanalysis, but we reason that the straightforward replacement of the conventional chromogenic horseradish peroxidase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, of a routinely used preclinical immunoassay to detect hIgG, with the fluorogenic 3-(4-hydroxyphenyl)propionic acid would broaden the narrow dynamic range. The replacement leads to a sensitivity of 0.47 (minimum required dilution [MRD] 10) and 1.02 (MRD 50) ng/ml, and dynamic ranges of 3.3 (MRD 10) and 3.6 (MRD 50) orders of magnitude, and thereby had improved sensitivity and dynamic range compared with other conventional colorimetric ELISAs, other ligand-binding assay technologies or LC-MS assays. Improvements in sensitivity and dynamic range were achieved for the sera of horse, mice and monkeys without assay optimization.

  3. Ligand binding assay critical reagents and their stability: recommendations and best practices from the Global Bioanalysis Consortium Harmonization Team.

    PubMed

    King, Lindsay E; Farley, Esme; Imazato, Mami; Keefe, Jeannine; Khan, Masood; Ma, Mark; Pihl, K Susanne; Sriraman, Priya

    2014-05-01

    The L4 Global Harmonization Team on reagents and their stability focused on the management of critical reagents for pharmacokinetic, immunogenicity, and biomarker ligand binding assays. Regulatory guidance recognizes that reagents are important for ligand binding assays but do not address numerous aspects of critical reagent life cycle management. Reagents can be obtained from external vendors or developed internally, but regardless of their source, there are numerous considerations for their reliable long-term use. The authors have identified current best practices and provided recommendations for critical reagent lot changes, stability management, and documentation.

  4. Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility.

    PubMed

    Williams, Alistair J K; Somerville, Michelle; Rokni, Saba; Bonifacio, Ezio; Yu, Liping; Eisenbarth, George; Akolkar, Beena; Steffes, Michael; Bingley, Polly J

    2009-12-31

    Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1% azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41% (range, 78 to -33%, p<0.001). The effect on binding was epitope specific; median IA-2A binding by 14 sera with antibodies to the protein tyrosine phosphatase region of IA-2 was reduced by 48% (range, 11 to 78%, p<0.001), while binding by 4 sera with antibodies specific to only the juxtamembrane region of IA-2 showed no change (median increase 16% (range 6 to 33%, p=0.125). When the Tween-20 concentration was reduced from 1% to 0.15% the median reduction in IA-2A binding with azide by the 18 sera was only 10% (range, -12 to 41%, p<0.001). Tween-20 also exerted an independent effect, since median IA-2A binding increased by 23% (range 3% to 86%, p<0.001) when Tween-20 concentration was reduced from 1% to 0.15% in the absence of azide. We conclude that common assay reagents such as azide and Tween-20 can strongly influence IA-2A binding in an epitope-related manner, and their use may explain some of the differences between laboratories in IA-2A measurement.

  5. Quantification of IgG on erythrocytes of patients and normals by a radio-ligand-binding assay.

    PubMed

    Giles, C M; Davies, K A; Loizou, S; Moulds, J J; Walport, M J

    1991-12-01

    A monoclonal IgG anti-human IgG, 1B12, was used in a radio-ligand-binding assay to quantify IgG on erythrocytes of patients and normals. The assay detected a range of 10-700 IgG molecules. Good correlation was achieved between the number of molecules and the strength of agglutination in antiglobulin tests performed in capillary tubes. The assay was capable of detecting subagglutinating immune bound IgG on erythrocytes from patients with systemic lupus erythematosus (SLE).

  6. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase.

    PubMed

    Clark, Daniel N; Jones, Scott A; Hu, Jianming

    2017-01-01

    The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

  7. Microbial methodological artifacts in (/sup 3/H)glutamate receptor binding assays

    SciTech Connect

    Yoneda, Y.; Ogita, K.

    1989-03-01

    Incubation of radiolabeled L-glutamic acid, a putative central excitatory neurotransmitter, in 50 mM Tris-acetate buffer (pH 7.4) at 30 degrees C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of L-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 nM. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified (/sup 3/H)glutamate at 30/degree/C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100 degrees C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 micron immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of (/sup 3/H)glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids.

  8. Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

    PubMed

    Jani, Darshana; Allinson, John; Berisha, Flora; Cowan, Kyra J; Devanarayan, Viswanath; Gleason, Carol; Jeromin, Andreas; Keller, Steve; Khan, Masood U; Nowatzke, Bill; Rhyne, Paul; Stephen, Laurie

    2016-01-01

    Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

  9. Magnetic levitation as a platform for competitive protein-ligand binding assays.

    PubMed

    Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

    2012-07-17

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

  10. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  11. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  12. Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

    EPA Science Inventory

    The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

  13. Characterization of ligand binding to the σ(1) receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay.

    PubMed

    Brune, Stefanie; Schepmann, Dirk; Lehmkuhl, Kirstin; Frehland, Bastian; Wünsch, Bernhard

    2012-08-01

    The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 μM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

  14. Tb3+-Cleavage Assays Reveal Specific Mg2+ Binding Sites Necessary to Pre-fold the btuB Riboswitch for AdoCbl Binding

    PubMed Central

    Choudhary, Pallavi K.; Gallo, Sofia; Sigel, Roland K. O.

    2017-01-01

    Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement. PMID:28377919

  15. [Radiocompetitive assay of sulfamido-3-chloro-4-benzoic acid with carbonic anhydrase as binding reagent (author's transl)].

    PubMed

    Khiat, M; Bali, J P; Guibert, M S; Chanal, J L; Marignan, R

    1978-01-16

    The control of patients treated by diuretic sulfonamides can be carried out by a radiocompetitive assay using their binding properties to carbonic anhydrase (CA). In this paper we have studied the assay of sulfamido-3-chloro-4-benzoic acid (SD3) using dialysis equilibrium as separation procedure. With (CA) 2 X 10(-6) M and 14C-SD3 0.5 X 10(-6) M (specific activity: 2 muCi/mg), can be detected 0.5 X 10(-6) M of (SD3) in the assay medium. 6.5 mg protein present in serum lower the assay sensitivity twenty times, owing to an elevated value of the affinity constant, Ka, of albumin-(SD3) complex (10(3) mol-1). On the other hand, the molecules with sulfamidobenzoic group cannot be differentiated in this procedure.

  16. Optimized modification of gold nanoparticles with a self-assembled monolayer for suppression of nonspecific binding in DNA assays

    NASA Astrophysics Data System (ADS)

    Esashika, Keiko; Saiki, Toshiharu

    2016-10-01

    Homogeneous DNA assays using gold nanoparticles (AuNPs) require the reduction of nonspecific binding between AuNPs to improve sensitivity in detecting the target molecule. In this study, we employed alkanethiol self-assembled monolayers (SAMs) for modifying the AuNP surface to attain both good dispersability and high hybridization efficiency. The alkanethiol SAMs enhance the repulsive interaction between AuNPs, reducing nonspecific binding and promoting the extension of surface-immobilized ssDNA into the solvent, thus enhancing the hybridization process. Introduction of oligoethylene glycol into the alkanethiol prevented nonspecific binding caused by the entanglement of alkane chains. Finally, the conditions were optimized by controlling the surface charge density through the introduction of a COOH group at the alkanethiol terminus, resulting in the complete blocking of nonspecific binding and the maintenance of high hybridization efficiency.

  17. Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

    PubMed

    Meleza, Cesar; Thomasson, Bobbie; Ramachandran, Chidambaram; O'Neill, Jason W; Michelsen, Klaus; Lo, Mei-Chu

    2016-10-15

    Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.

  18. 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.

  19. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

    EPA Science Inventory

    The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

  20. Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

    NASA Astrophysics Data System (ADS)

    Lin, Hong; Kitova, Elena N.; Klassen, John S.

    2014-01-01

    Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

  1. Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.

    PubMed

    Nikiforov, Theo T; Beechem, Joseph M

    2006-10-01

    We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.

  2. Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay.

    PubMed

    Nguyen, Kiet T; Hu, Xubo; Pei, Dehua

    2004-06-01

    A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.

  3. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  4. Hemizona assay: use of fresh versus salt-stored human oocytes to evaluate sperm binding potential to the zona pellucida.

    PubMed

    Kruger, T F; Oehninger, S; Franken, D R; Hodgen, G D

    1991-06-01

    The hemizona assay (HZA) has a high predictive value for in vitro fertilization (IVF) results. Oocyte quality plays a significant role in the validation of this test. The question was asked whether human salt-stored oocytes (up to 30 days) are damaged and subsequently lose their sperm binding capacity when compared to fresh human oocytes. Equivalent binding in both the salt-stored and the fresh group of oocytes was observed in the hemizonae incubated with normal semen as well as in their matching halves incubated with semen from an infertile man. Based on the results, we conclude that salt-stored oocytes (pH 7.0) give reliable information regarding sperm binding potential under HZA conditions.

  5. Determination of the drug-DNA binding modes using fluorescence-based assays.

    PubMed

    Williams, Alicia K; Dasilva, Sofia Cheliout; Bhatta, Ankit; Rawal, Baibhav; Liu, Melinda; Korobkova, Ekaterina A

    2012-03-15

    Therapeutic drugs and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-DNA interactions. Two nucleic acid dyes were employed in the method: TO-PRO-3 iodide (TP3) and 4',6-diamidino-2-phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA. We evaluated the DNA binding constant, K(b), for each dye. We also performed fluorescence quenching experiments with 11 molecules of various structures and measured a C(50) value for each compound. We determined preferential binding modes for the aforementioned molecules and found that they bound to DNA consistently, as indicated by other studies. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with calf thymus DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.

  6. Assay for Arf GTP-binding Proteins | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Laboratory of Cellular and Molecular Biology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize an antibody-based proteomics assay.

  7. Comparison of rosetting, phagocytosis, and IgG binding assays for detection of IgG on old red cells

    SciTech Connect

    Bassel, P.; Bosman, G.; Kay, M.

    1986-03-01

    Various methods have been used for detecting or inferring the presence of IgG on senescent red cells. In the authors studies, they have used a method for directly measuring IgG on senescent red cells. In our studies, the authors have used a method for directly measuring IgG on cells (e.g. scanning immunoelectron microscopy) along with determining phagocytosis. Thus, phagocytosis is used as a biological assay for determining the biological significance of the IgG on cells. However, the phagocytosis assay as performed in the authors laboratory is tedious, time-consuming, and requires meticulous technique. In contrast, rosetting is a quick, simple assay that does not require special techniques or supplies. Therefore, the authors compared the phagocytosis assay employed by us to rosetting, and correlated each of these with the amount of IgG present on red cells as determined with an /sup 125/I protein A binding assay. Although senescent red cells were phagocytized, they did not form rosettes with K562 cells even at 25 RBC:K562. Further experiments indicated that the rosette assay depended on the RBC:K561 cell ratio and not on the amount of IgG/red cell. Rosette formation (%) at varying RBC:K562 ratios was as follows: 100:1, 81 +/- 12; 50:1, 65 +/- 18; 25:1, 34 +/- 30, 10:1, 20 +/- 33; 5:1, 15 +/- 29; 1: 1, 3 +/- 7 (n = 14). In contrast, phagocytosis of old RBC correlated well with the amount of IgG present on red cells (r = 0.96, 0.94, 0.92 and 0.94 in each of 4 different experiments with n = 16, 19, 14, and 19 respectively). Thus, the phagocytosis assay the authors have used correlates with IgG on red cells; whereas rosette formation does not.

  8. In vitro estrogen receptor binding of PCBs: measured activity and detection of hydroxylated metabolites in a recombinant yeast assay.

    PubMed

    Layton, Alice C; Sanseverino, John; Gregory, Betsy W; Easter, James P; Sayler, Gary S; Schultz, T Wayne

    2002-05-01

    The estrogenic activities of 17beta-estradiol, biphenyl, chlorinated biphenyls, and Aroclor mixtures 1221, 1242, and 1248 were measured with a modified recombinant yeast estrogen assay (i.e., a Saccharomyces cerevisiae-based lac-Z (beta-galactosidase) reporter assay). Modifications of the assay included the use of glass vials instead of plastic microtiter plates and the addition of the medium and yeast before the test substrate. 14C-labeled compounds were used to follow improvements in the assay procedures. 14C-17beta-estradiol recovery from plastic microtiter plates and glass vials using the standard or the modified procedure was approximately 89%. However, 14C-4-CB (4-chlorobiphenyl) recovery was considerably less, ranging from 3% in plastic microtiter plates using the standard procedure to 26% in vials using the modified procedure. These results suggest that the toxicity of strongly hydrophobic chemicals may be underestimated. Using the modified yeast estrogen assay, full agonist activity was observed for 4-CB, 2,4,6-CB, and 2,5-CB while each of the Aroclor mixtures were only partial agonists. The equivalent EC50 values in ppm were in environmentally relevant concentrations for biphenyl (19 ppm), 4-CB (4.5 ppm), 2,5-CB (21 ppm), 2,4,6-CB (0.8 ppm), Aroclor 1221 (2.9 ppm), Aroclor 1242 (0.65 ppm), and Aroclor 1248 (2.3 ppm). Estrogen receptor binding for the individual PCB congeners was 25- to 650-fold less than the reported estrogen binding for the corresponding hydroxylated PCB metabolite. Gas chromatographic/mass spectrometric analysis of yeast extracts indicated that S. cerevisiae hydroxylated the individual PCB congeners in the ppb range. With the exception of biphenyl, the concentration of hydroxylated metabolites obtained from incubation of S. cerevisiae with PCB congeners was consistent with the concentration necessary to elicit a positive estrogen receptor-binding response. This work provides evidence that S. cerevisiae are capable of metabolic

  9. Periplasmic binding protein-based detection of maltose using liposomes: a new class of biorecognition elements in competitive assays.

    PubMed

    Edwards, Katie A; Baeumner, Antje J

    2013-03-05

    A periplasmic binding protein (PBP) was investigated as a novel binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10(-4) M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these proteins' two globular domains upon target binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose binding protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the protein. MBP conjugated fluorescent dye-encapsulating liposomes served to provide recognition and signal amplification in a competitive assay for maltose using amylose magnetic beads in a microtiter plate-based format. The development of appropriate binding buffers and competitive surfaces are described, with general observations expected to extend to PBPs for other analytes. The resulting assay was specific for d-(+)-maltose versus other sugar analogs including d-(+)-raffinose, sucrose, d-trehalose, d-(+)-xylose, d-fructose, 1-thio-β-d-glucose sodium salt, d-(+)-galactose, sorbitol, glycerol, and dextrose. Cross-reactivity with d-lactose and d-(+)-glucose occurred only at concentrations >10(4)-fold greater than d-(+)-maltose. The limit of detection was 78 nM with a dynamic range covering over 3 orders of magnitude. Accurate detection of maltose as an active ingredient in a pharmaceutical preparation was demonstrated. This method offers a significant improvement over existing

  10. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    PubMed

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-03-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed.

  11. 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

  12. A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.

    PubMed

    Aoki, Takashi; Kazama, Hitoshi; Satoh, Marie; Mizuki, Kazuhiro; Watabe, Hiroyuki

    2005-09-01

    When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.

  13. Early postnatal diagnosis of hereditary spherocytosis by combining light microscopy, acidified glycerol lysis test and eosin-5'-maleimide binding assay.

    PubMed

    Andres, Oliver; Eber, Stefan; Speer, Christian P

    2015-12-01

    Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

  14. Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors.

    PubMed

    Uri, Asko; Lust, Marje; Vaasa, Angela; Lavogina, Darja; Viht, Kaido; Enkvist, Erki

    2010-03-01

    Conjugates of adenosine mimics and d-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.

  15. Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs.

    PubMed

    Tian-Yu, Jiamin; Licht, Stuart; Pardee, Gwynn; Bhat, Arun; Cao, Ying; Gao, Wei; Sangalang, Emma; Zaror, Isabel

    2010-04-15

    Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

  16. [Blood prealbumin: comparison of 2 methods of assay and correlation with the retinol binding protein].

    PubMed

    Romette, J; Mallet, B; Di Constanzo, J

    1984-01-01

    Prealbumin was determined by radial immunodiffusion and laser immunonephelometry methods in serum or plasma from 86 adult subjects. Both methods were reliable in physiologic prealbumin range but immunonephelometry only was reliable for lower levels. Physiologic prealbumin level was 346 +/- 67 mg/l in adult males and 319 +/- 48 mg/l in adult females; no difference was noted for retinol binding protein (60 +/- 14 mg/l). When prealbumin and retinol binding protein levels were low, no close correlation was noted in their variations.

  17. Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

    PubMed

    Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

    2017-03-15

    Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity.

  18. An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

    SciTech Connect

    Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F; Pelletier, Dale A; Hurst, Gregory {Greg} B; Doktycz, Mitchel John; Morrell-Falvey, Jennifer L; Billings, Amanda N

    2009-01-01

    Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris

  19. Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay

    PubMed Central

    Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J. Stephen

    2012-01-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of FDA approved drugs, drug-like molecules and natural products extracts we identified several lead compounds that are promising candidates for medicinal chemistry. PMID:22644268

  20. Evaluation of variability and quality control procedures for a receptor-binding assay for paralytic shellfish poisoning toxins.

    PubMed

    Ruberu, S R; Langlois, G W; Masuda, M; Perera, S Kusum

    2012-01-01

    The receptor-binding assay (RBA) method for determining saxatoxin (STX) and its numerous analogues, which cause paralytic shellfish poisoning (PSP) in humans, was evaluated in a single laboratory study. Each step of the assay preparation procedure including the performance of the multi-detector TopCount® instrument was evaluated for its contribution to method variability. The overall inherent RBA variability was determined to be 17%. Variability within the 12 detectors was observed; however, there was no reproducible pattern in detector performance. This observed variability among detectors could be attributed to other factors, such as pipetting errors. In an attempt to reduce the number of plates rejected due to excessive variability in the method's quality control parameters, a statistical approach was evaluated using either Grubbs' test or the Student's t-test for rejecting outliers in the measurement of triplicate wells. This approach improved the ratio of accepted versus rejected plates, saving cost and time for rerunning the assay. However, the potential reduction in accuracy and the lack of improvement in precision suggests caution when using this approach. The current study has recommended an alternate quality control procedure for accepting or rejecting plates in place of the criteria currently used in the published assay, or the alternative of outlier testing. The recommended procedure involves the development of control charts to monitor the critical parameters identified in the published method (QC sample, EC₅₀, slope of calibration curve), with the addition of a fourth critical parameter which is the top value (100% binding) of the calibration curve.

  1. Elucidation of the binding mechanism of renin using a wide array of computational techniques and biological assays.

    PubMed

    Tzoupis, Haralambos; Leonis, Georgios; Avramopoulos, Aggelos; Reis, Heribert; Czyżnikowska, Żaneta; Zerva, Sofia; Vergadou, Niki; Peristeras, Loukas D; Papavasileiou, Konstantinos D; Alexis, Michael N; Mavromoustakos, Thomas; Papadopoulos, Manthos G

    2015-11-01

    We investigate the binding mechanism in renin complexes, involving three drugs (remikiren, zankiren and enalkiren) and one lead compound, which was selected after screening the ZINC database. For this purpose, we used ab initio methods (the effective fragment potential, the variational perturbation theory, the energy decomposition analysis, the atoms-in-molecules), docking, molecular dynamics, and the MM-PBSA method. A biological assay for the lead compound has been performed to validate the theoretical findings. Importantly, binding free energy calculations for the three drug complexes are within 3 kcal/mol of the experimental values, thus further justifying our computational protocol, which has been validated through previous studies on 11 drug-protein systems. The main elements of the discovered mechanism are: (i) minor changes are induced to renin upon drug binding, (ii) the three drugs form an extensive network of hydrogen bonds with renin, whilst the lead compound presented diminished interactions, (iii) ligand binding in all complexes is driven by favorable van der Waals interactions and the nonpolar contribution to solvation, while the lead compound is associated with diminished van der Waals interactions compared to the drug-bound forms of renin, and (iv) the environment (H2O/Na(+)) has a small effect on the renin-remikiren interaction.

  2. A fluorescent microplate assay quantifies bacterial efflux and demonstrates two distinct compound binding sites in AcrB.

    PubMed

    Iyer, Ramkumar; Ferrari, Annette; Rijnbrand, R; Erwin, Alice L

    2015-04-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.

  3. A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

    PubMed Central

    Ferrari, Annette; Rijnbrand, R.; Erwin, Alice L.

    2015-01-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux. PMID:25645845

  4. Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species

    EPA Science Inventory

    ABSTRACT In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human (hERα), quai...

  5. Quenching methods for background reduction in luminescence-based probe-target binding assays

    DOEpatents

    Cai, Hong; Goodwin, Peter M; Keller, Richard A.; Nolan, Rhiannon L.

    2007-04-10

    Background luminescence is reduced from a solution containing unbound luminescent probes, each having a first molecule that attaches to a target molecule and having an attached luminescent moiety, and luminescent probe/target adducts. Quenching capture reagent molecules are formed that are capable of forming an adduct with the unbound luminescent probes and having an attached quencher material effective to quench luminescence of the luminescent moiety. The quencher material of the capture reagent molecules is added to a solution of the luminescent probe/target adducts and binds in a proximity to the luminescent moiety of the unbound luminescent probes to quench luminescence from the luminescent moiety when the luminescent moiety is exposed to exciting illumination. The quencher capture reagent does not bind to probe molecules that are bound to target molecules and the probe/target adduct emission is not quenched.

  6. 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.

  7. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    PubMed

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.

  8. Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody.

    PubMed

    Fricke, W A; Brinkhous, K M; Garris, J B; Roberts, H R

    1985-09-01

    An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil-T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF-binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF-binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those

  9. Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay.

    PubMed

    Aminian, Mahdi; Nabatchian, Fariba; Vaisi-Raygani, Asad; Torabi, Mojgan

    2013-03-15

    The Bradford protein assay is a popular method because of its rapidity, sensitivity, and relative specificity. This method is subject to some interference by nonprotein compounds. In this study, we describe the interference of cetyltrimethylammonium bromide (CTAB) with the Bradford assay. This interference is based on the interaction of Coomassie Brilliant Blue G-250 (CBB) with this cationic detergent. This study suggests that both electrostatic and hydrophobic interactions are involved in the interaction of CTAB and CBB. The anionic and neutral forms of CBB bind to CTAB by electrostatic attraction, which accelerates hydrophobic interactions of these CBB forms and the hydrophobic tail of CTAB. Consequently, the hydrophobic regions of the dominant free cationic form of CBB dye compete for the tail of CTAB with two other forms of the dye and gradually displace the primary hydrophobic interactions and rearrange the primary CBB-CTAB complex. This interaction of CTAB and CBB dye produces a primary 650-nm-absorbing complex that then gradually rearranges to a complex that shows an absorbance shoulder at 800-950 nm. This study conclusively shows a strong response of CBB to CTAB that causes a time-dependent and nearly additive interference with the Bradford assay. This study also may promote an application of CBB for CTAB quantification.

  10. Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay

    PubMed Central

    Ngo, Liza; Wu, Jiang; Yang, Chao

    2015-01-01

    Abstract Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [3H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor–substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators. PMID:26065557

  11. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties

    PubMed Central

    Baxter, Sarah K.; Lambert, Abigail R.; Scharenberg, Andrew M.; Jarjour, Jordan

    2014-01-01

    A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. PMID:23423888

  12. Evaluation of Primary Binding Assays for Presumptive Serodiagnosis of Swine Brucellosis in Argentina

    PubMed Central

    Paulo, P. Silva; Vigliocco, A. M.; Ramondino, R. F.; Marticorena, D.; Bissi, E.; Briones, G.; Gorchs, C.; Gall, D.; Nielsen, K.

    2000-01-01

    An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%. PMID:10973463

  13. Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreen-based assay for antagonists of methyl-lysine binding proteins.

    PubMed

    Wigle, Tim J; Herold, J Martin; Senisterra, Guillermo A; Vedadi, Masoud; Kireev, Dmitri B; Arrowsmith, Cheryl H; Frye, Stephen V; Janzen, William P

    2010-01-01

    The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.

  14. Decoupling competing surface binding kinetics and reconfiguration of receptor footprint for ultrasensitive stress assays

    NASA Astrophysics Data System (ADS)

    Patil, Samadhan B.; Vögtli, Manuel; Webb, Benjamin; Mazza, Giuseppe; Pinzani, Massimo; Soh, Yeong-Ah; McKendry, Rachel A.; Ndieyira, Joseph W.

    2015-10-01

    Cantilever arrays have been used to monitor biochemical interactions and their associated stress. However, it is often necessary to passivate the underside of the cantilever to prevent unwanted ligand adsorption, and this process requires tedious optimization. Here, we show a way to immobilize membrane receptors on nanomechanical cantilevers so that they can function without passivating the underlying surface. Using equilibrium theory, we quantitatively describe the mechanical responses of vancomycin, human immunodeficiency virus type 1 antigens and coagulation factor VIII captured on the cantilever in the presence of competing stresses from the top and bottom cantilever surfaces. We show that the area per receptor molecule on the cantilever surface influences ligand-receptor binding and plays an important role on stress. Our results offer a new way to sense biomolecules and will aid in the creation of ultrasensitive biosensors.

  15. In vitro detection of NEMO-ubiquitin binding using DELFIA and microscale thermophoresis assays.

    PubMed

    Vincendeau, Michelle; Krappmann, Daniel; Hadian, Kamyar

    2015-01-01

    Canonical NF-κB signaling in response to various stimuli converges at the level of the IκB kinase (IKK) complex to ultimately activate NF-κB. To achieve this, the IKK complex uses one of its regulatory subunit (IKKγ/NEMO) to sense ubiquitin chains formed by upstream complexes. Various studies have shown that different Ubiquitin chains are involved in the binding of NEMO and thereby the activation of NF-κB. We have utilized two distinct biochemical methods, i.e., Dissociation-Enhanced Lanthanide Fluorescence Immunoassay (DELFIA) and Microscale Thermophoresis (MST), to detect the interaction of NEMO to linear and K63-linked Ubiquitin chains, respectively. Here, we describe the brief basis of the methods and a detailed underlying protocol.

  16. Assay Development and High-Throughput Screening for Inhibitors of Kaposi's Sarcoma-Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes.

    PubMed

    Beauchemin, Chantal; Moerke, Nathan J; Faloon, Patrick; Kaye, Kenneth M

    2014-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

  17. Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation.

    PubMed

    Santa-Coloma, T A; Bley, M A; Charreau, E H

    1987-08-01

    The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.

  18. Current industrial practices and regulatory requirements to assess analyte and reagent stability using ligand-binding assays.

    PubMed

    Wang, Jin; Nowatzke, William; Ma, Mark

    2015-01-01

    Specific guidelines on bioanalytical method validation for drug development support are recommended by regulatory agencies. Regarding stability assessment, US FDA states that 'Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at the intended storage temperature) and short-term (bench-top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process'. Additional regulatory considerations are discussed including topics such as analyte and reagent stability. This article reviews the regulatory requirements as issued by the USA (FDA), Europe (EMA) and Japan (MHLW), for stability studies where bioanalytical methods are used to support drug development programs and summarizes the current industry standard for conducting stability studies when utilizing ligand-binding assays.

  19. Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

    PubMed

    Lee, Hae Kyung; Kim, Tae Sung; Kim, Chang Yeong; Kang, Il Hyun; Kim, Mi Gyeong; Jung, Ki Kyung; Kim, Hyung Sik; Han, Soon Young; Yoon, Hae Jung; Rhee, Gyu Seek

    2012-01-01

    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17β-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17β-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

  20. Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

    PubMed

    Kecskés, Miklós; Kumar, T Santhosh; Yoo, Lena; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-08-15

    Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

  1. A Cell-Based Pharmacokinetics Assay for Evaluating Tubulin-Binding Drugs

    PubMed Central

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

    2014-01-01

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

  2. Calculations for Adjusting Endogenous Biomarker Levels During Analytical Recovery Assessments for Ligand-Binding Assay Bioanalytical Method Validation.

    PubMed

    Marcelletti, John F; Evans, Cindy L; Saxena, Manju; Lopez, Adriana E

    2015-07-01

    It is often necessary to adjust for detectable endogenous biomarker levels in spiked validation samples (VS) and in selectivity determinations during bioanalytical method validation for ligand-binding assays (LBA) with a matrix like normal human serum (NHS). Described herein are case studies of biomarker analyses using multiplex LBA which highlight the challenges associated with such adjustments when calculating percent analytical recovery (%AR). The LBA test methods were the Meso Scale Discovery V-PLEX® proinflammatory and cytokine panels with NHS as test matrix. The NHS matrix blank exhibited varied endogenous content of the 20 individual cytokines before spiking, ranging from undetectable to readily quantifiable. Addition and subtraction methods for adjusting endogenous cytokine levels in %AR calculations are both used in the bioanalytical field. The two methods were compared in %AR calculations following spiking and analysis of VS for cytokines having detectable endogenous levels in NHS. Calculations for %AR obtained by subtracting quantifiable endogenous biomarker concentrations from the respective total analytical VS values yielded reproducible and credible conclusions. The addition method, in contrast, yielded %AR conclusions that were frequently unreliable and discordant with values obtained with the subtraction adjustment method. It is shown that subtraction of assay signal attributable to matrix is a feasible alternative when endogenous biomarkers levels are below the limit of quantitation, but above the limit of detection. These analyses confirm that the subtraction method is preferable over that using addition to adjust for detectable endogenous biomarker levels when calculating %AR for biomarker LBA.

  3. Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents.

    PubMed

    O'Hara, Denise M; Theobald, Valerie; Egan, Adrienne Clements; Usansky, Joel; Krishna, Murli; TerWee, Julie; Maia, Mauricio; Spriggs, Frank P; Kenney, John; Safavi, Afshin; Keefe, Jeannine

    2012-06-01

    Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

  4. In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

    PubMed Central

    Lee, Sangkyou; Lee, Ilkyun; Jung, Yoonsuh; McConkey, David; Czerniak, Bogdan

    2012-01-01

    The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems. PMID:23272234

  5. In-frame cDNA library combined with protein complementation assay identifies ARL11-binding partners.

    PubMed

    Lee, Sangkyou; Lee, Ilkyun; Jung, Yoonsuh; McConkey, David; Czerniak, Bogdan

    2012-01-01

    The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5'-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5'-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an "in-frame cDNA library." We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5'-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

  6. A 155-plex high-throughput in vitro coregulator binding assay for (anti-)estrogenicity testing evaluated with 23 reference compounds.

    PubMed

    Wang, Si; Houtman, René; Melchers, Diana; Aarts, Jac; Peijnenburg, Ad; van Beuningen, Rinie; Rietjens, Ivonne; Bovee, Toine F

    2013-01-01

    To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor α was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERα binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects.

  7. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    PubMed

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses.

  8. An update on the von Willebrand factor collagen binding assay: 21 years of age and beyond adolescence but not yet a mature adult.

    PubMed

    Favaloro, Emmanuel J

    2007-11-01

    von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. It is diagnosed after a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant (FVIII:C), von Willebrand factor (VWF) protein (antigen; VWF:Ag), and VWF function, which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focused on another functional VWF assay, the collagen binding (VWF:CB) assay, as a possible replacement for the VWF:RCo assay or as a supplementary test of VWF adhesive "activity." Additional laboratory testing can comprise a battery of confirmatory and VWD subtype assisting assays, including assessment of VWF:multimers. This review updates our knowledge of VWD diagnostics with a particular emphasis on the VWF:CB assay. There is good evidence now in place that an optimized VWF:CB assay can significantly reduce the diagnostic error rate otherwise arising from the use of a test panel restricted to including the VWF:RCo assay as the sole functional VWF assay. Nevertheless, the VWF:CB assay should not be used to wholly replace the VWF:RCo assay in phenotypic testing but rather as a supplementary assay. However, with some thought and justification, the VWF:CB assay can be used to partly replace the VWF:RCo assay in some "screening" applications and can also be used to abrogate the need to perform routine VWF:multimers in most test cases.

  9. 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

  10. A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library.

    PubMed

    Watkins, Derrick; Jiang, Liuwei; Nahar, Smita; Maiti, Souvik; Arya, Dev P

    2015-01-01

    MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.

  11. High-performance mass spectrometry as a drug discovery tool: a high-throughput screening assay to identify RNA-binding ligands

    NASA Astrophysics Data System (ADS)

    Sannes-Lowery, Kristin A.; Drader, Jared J.; Griffey, Richard H.; Hofstadler, Steven A.

    2001-04-01

    Fourier transform mass spectrometry (FTMS) is increasingly being used as a drug discovery tool. We describe the development of a parallel high-throughput screening (HTS) strategy to identify small molecules that bind RNA targets using FTMS as an alternative to classical high-throughput biological screening methods for combinatorial libraries. The Multitarget Affinity/Specificity Screening (MASS) assay takes advantage of the "intrinsic mass" label of each compound and target RNA by employing high resolution, high precision mass measurements. The ability to analyze complex mixtures allows large compound libraries to be screened in the presence of multiple RNA targets simultaneously. The identity of the small molecule(s) which bind, the RNA target to which it binds, the compound-specific binding affinity and the location of the binding site on the RNA can be determined in one set of rapid experiments. The MASS technology detects complexes with dissociation constants of < 5 mM, with high sensitivity.

  12. Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.

    EPA Science Inventory

    Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell bin...

  13. An Undergraduate Laboratory Experiment that Utilizes a Glass Fiber Filter Assay to Determine the Steroid Specificity and Equilibrium Binding Properties of Glucocorticoid Receptors.

    ERIC Educational Resources Information Center

    John, Nancy J.; Firestone, Gary L.

    1987-01-01

    Describes two complementary laboratory exercises that use the glass fiber assay to assess receptor specificity and hormone binding affinity in rat liver cytoplasmic extracts. Details the methods, materials and protocol of the experiments. Discusses the basic concepts illustrated and the feasibility of using the experiments at the undergraduate…

  14. Using electrophoretic mobility shift assays to measure equilibrium dissociation constants: GAL4-p53 binding DNA as a model system.

    PubMed

    Heffler, Michael A; Walters, Ryan D; Kugel, Jennifer F

    2012-01-01

    An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K(D) ) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel; the DNA migrates more slowly when bound to a protein. To determine a K(D) the amount of unbound and protein-bound DNA in the gel is measured as the protein concentration increases. By performing this experiment, students will be introduced to making affinity measurements and gain experience in performing quantitative EMSAs. The experiment describes measuring the K(D) for the interaction between the chimeric protein GAL4-p53 and its DNA recognition site; however, the techniques are adaptable to other DNA binding proteins. In addition, the basic experiment described can be easily expanded to include additional inquiry-driven experimentation. © 2012 by The International Union of Biochemistry and Molecular Biology.

  15. Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells.

    PubMed

    Dirscherl, Cindy; Palankar, Raghavendra; Delcea, Mihaela; Kolesnikova, Tatiana A; Springer, Sebastian

    2017-02-02

    Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2K(b) , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2K(b) and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.

  16. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  17. Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.

    PubMed

    Song, Ge; Wu, Qing-Ping; Xu, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Zhang, Shi-Fu; Guo, Zhan-Yun

    2015-12-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies.

  18. Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay*

    PubMed Central

    Risse, Emmanuel; Nicoll, Andrew J.; Taylor, William A.; Wright, Daniel; Badoni, Mayank; Yang, Xiaofan; Farrow, Mark A.; Collinge, John

    2015-01-01

    The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrPC) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrPC may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). There has been considerable interest in identification of compounds that bind to PrPC, stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrPC could also inhibit the binding of toxic Aβ species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of Aβ oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both Aβ binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting Aβ binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit Aβ binding and reduce prion levels was established in cell-based assays. PMID:25995455

  19. Enzyme-linked PNA lectin binding assay compared with CA19-9 and CEA radioimmunoassay as a diagnostic blood test for pancreatic cancer.

    PubMed Central

    Ching, C. K.; Rhodes, J. M.

    1989-01-01

    Previous studies have shown that sera from patients with pancreatic cancer often contain a mucus glycoprotein that expresses the oncofetal antigen galactose 1-3, N-acetyl galactosamine, which is the T blood group antigen and the binding site for the lectin peanut agglutinin (PNA). An enzyme-linked lectin assay has been developed to quantify PNA-binding glycoproteins in serum and has been evaluated as a serological test for pancreatic cancer. Sera were studied from 53 patients with pancreatic cancer and 154 controls, including benign obstructive jaundice, acute and chronic pancreatitis, chronic liver disease and inflammatory bowel disease. The enzyme-linked peanut lectin assay proved highly reproducible and has 77% sensitivity and 83% specificity for pancreatic cancer, results that are very similar to those achieved in the same sera by CA19-9 radioimmunoassay (75% sensitivity, 82% specificity with the upper limit of normal set at 37 u ml-1). CEA assay proved less useful (60% sensitivity, 47% specificity). In this study better results were obtained if an upper limit of normal of 50 u ml-1 was used for CA19-9 (75% sensitivity, 92% specificity). Combination of CA19-9 assay with the upper limit set at 50 u ml-1 and the peanut lectin assay improved the sensitivity to 85% with only a slight fall in specificity (85%). These results compare well with published results for ultrasound and CT scanning. PMID:2736232

  20. Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses.

    PubMed

    Barrett, Susan; Mohr, Peter G; Schmidt, Peter M; McKimm-Breschkin, Jennifer L

    2011-01-01

    The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.

  1. Interpretation of Ocular Melanin Drug Binding Assays. Alternatives to the Model of Multiple Classes of Independent Sites.

    PubMed

    Manzanares, José A; Rimpelä, Anna-Kaisa; Urtti, Arto

    2016-04-04

    Melanin has a high binding affinity for a wide range of drugs. The determination of the melanin binding capacity and its binding affinity are important, e.g., in the determination of the ocular drug distribution, the prediction of drug effects in the eye, and the trans-scleral drug delivery. The binding parameters estimated from a given data set vary significantly when using different isotherms or different nonlinear fitting methods. In this work, the commonly used bi-Langmuir isotherm, which assumes two classes of independent sites, is confronted with the Sips isotherm. Direct, log-log, and Scatchard plots are used, and the interpretation of the binding curves in the latter is critically analyzed. In addition to the goodness of fit, the emphasis is placed on the physical meaning of the binding parameters. The bi-Langmuir model imposes a bimodal distribution of binding energies for the sites on the melanin granules, but the actual distribution is most likely continuous and unimodal, as assumed by the Sips isotherm. Hence, the latter describes more accurately the distribution of binding energies and also the experimental results of melanin binding to drugs and metal ions. Simulations are used to show that the existence of two classes of sites cannot be confirmed on the sole basis of the shape of the binding curve in the Scatchard plot, and that serious doubts may appear on the meaning of the binding parameters of the bi-Langmuir model. Experimental results of melanin binding to chloroquine and metoprolol are used to illustrate the importance of the choice of the binding isotherm and of the method used to evaluate the binding parameters.

  2. Comparison of GD2 Binding Capture ELISA Assays for Anti-GD2-Antibodies Using GD2-Coated Plates and a GD2-Expressing Cell-Based ELISA

    PubMed Central

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Mitra, Gautam

    2011-01-01

    Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2’-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment. PMID:21893062

  3. A naturally occurring mutation within the probe-binding region compromises a molecular-based West Nile virus surveillance assay for mosquito pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Dannen, Maureen; Anishchenko, Michael; Reisen, William K

    2012-07-01

    A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C-->T transition 6nt from the 5' end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.

  4. Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus.

    PubMed

    Wen, Guoyuan; Zhang, Tao; Yang, Jun; Luo, Qingping; Liao, Yonghong; Hu, Zhibin; Zhang, Rongrong; Wang, Hongling; Ai, Diyun; Luo, Ling; Song, Nianhua; Shao, Huabin

    2011-09-01

    A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.

  5. Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor.

    PubMed

    Swift, Jody L; Burger, Melanie C; Massotte, Dominique; Dahms, Tanya E S; Cramb, David T

    2007-09-01

    Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.

  6. Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: the case of melanocortin 4 receptors.

    PubMed

    Veiksina, Santa; Kopanchuk, Sergei; Rinken, Ago

    2014-01-01

    We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.

  7. An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.

    PubMed

    Duval, Romain; Fritsch, Lauriane; Bui, Linh-Chi; Berthelet, Jérémy; Guidez, Fabien; Mathieu, Cécile; Dupret, Jean-Marie; Chomienne, Christine; Ait-Si-Ali, Slimane; Rodrigues-Lima, Fernando

    2015-10-01

    CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

  8. A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.

    PubMed

    Yin, Liusong; Stern, Lawrence J

    2014-04-01

    HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

  9. An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy

    PubMed Central

    Selb, R.; Eckl-Dorna, J.; Vrtala, S.; Valenta, R.; Niederberger, V.

    2017-01-01

    Background It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy. Methods In 18 patients treated by Bet v 1-fragment-specific immunotherapy, the effects of IgG antibodies specific for the fragments on the binding of IgE antibodies to Bet v 1 were measured by ELISA. Blocking and possible enhancing effects on IgE binding were compared with skin sensitivity to Bet v 1 after treatment. Results We found that fragment-specific IgG enhanced IgE binding to Bet v 1 in two patients who also showed an increase of skin sensitivity to Bet v 1. Conclusion Our results indicate that it may be possible to develop serological tests which predict the induction of unfavourable IgG antibodies enhancing the binding of IgE to Bet v 1 during immunotherapy. PMID:23998344

  10. Identification of promethazine as an amyloid-binding molecule using a fluorescence high-throughput assay and MALDI imaging mass spectrometry☆

    PubMed Central

    McClure, Richard A.; Chumbley, Chad W.; Reyzer, Michelle L.; Wilson, Kevin; Caprioli, Richard M.; Gore, John C.; Pham, Wellington

    2013-01-01

    The identification of amyloid-binding compounds is a crucial step in the development of imaging probes and therapeutics for the detection and cure of Alzheimer's disease. Unfortunately, the process typically lags during the translation from in vitro to in vivo studies due to the impenetrable nature of the blood brain barrier (BBB). Here, we integrate fluorescence assay with MALDI imaging mass spectrometry to screen known compounds and repurpose their properties to enable the second function of binding to amyloid plaques. Through this approach, we identified an antihistamine compound, promethazine, that can bind to amyloid plaques. Finally, we demonstrate that promethazine is retained in the amyloid-burdened brain compared to a normal brain and that its distribution within the brain corroborates with that of amyloid plaques. PMID:24179813

  11. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    NASA Astrophysics Data System (ADS)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  12. Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays.

    PubMed

    Ahn, J H; Chiou, C J; Hayward, G S

    1998-03-27

    The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in-vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped

  13. WGA-coated yttrium oxide beads enable an imaging-based adenosine 2a receptor binding scintillation proximity assay suitable for high throughput screening.

    PubMed

    Bryant, Robert; McGuinness, Debra; Turek-Etienne, Tammy; Guyer, Deborah; Yu, Liming; Howells, Leighton; Caravano, Joseph; Zhai, Ying; Lachowicz, Jean

    2004-06-01

    Adenosine receptors belong to the superfamily of G protein-coupled receptors and are involved in a variety of physiologic functions. Traditionally, binding assays to detect adenosine 2a (A2a) antagonists and agonists have used filtration methods that are cumbersome to run and not amenable to HTS. We developed scintillation proximity assays (SPA trade mark ) utilizing HEK293 RBHA2AM cell membranes, either wheat germ agglutinin (WGA)-coated yttrium silicate (YSi) or red-shifted yttrium oxide (YO) beads and the A2a-selective radioligand [(3)H]SCH 58261. Both beads gave windows (total binding/nonspecific binding) of >5 and K(d) values of 2-3 nM for the radioligand, in agreement with results obtained by filtration. In contrast, WGA-polyvinyltoluene as well as other bead types had windows of <3 and significant radioligand binding to the uncoated beads. A 384-well WGA-YO bead SPA was optimized utilizing a LEADseeker imaging system and an automated trituration process for dispensing the dense yttrium-based beads. Signals were stable after 4 h, and Z' values were 0.7-0.8. The LEADseeker imaging assay tolerated 2% dimethyl sulfoxide and generated IC(50) values of 3-5 nM for the A2a antagonist CGS 15943, comparable to that obtained by the filtration method. A number of adenosine and xanthine analogues were identified as hits in the Library of Pharmacologically Active Compounds (LOPAC). This imaging-based A2a SPA enables HTS and is a major improvement over the filtration method.

  14. Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay.

    PubMed

    Karpenko, Iuliia A; Margathe, Jean-François; Rodriguez, Thiéric; Pflimlin, Elsa; Dupuis, Elodie; Hibert, Marcel; Durroux, Thierry; Bonnet, Dominique

    2015-03-12

    The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

  15. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

    PubMed

    Sonobe, M H; Trezena, A G; Guilhen, F B; Takano, V L; Fratelli, F; Sakauchi, D; Morais, J F; Prado, S M A; Higashi, H G

    2007-01-01

    A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  16. Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay.

    PubMed

    Ohashi, Atsushi; Kotera, Hirohisa; Hori, Hideo; Hibiya, Makoto; Watanabe, Koji; Murakami, Kazutaka; Hasegawa, Midori; Tomita, Makoto; Hiki, Yoshinobu; Sugiyama, Satoshi

    2005-01-01

    Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERalpha). Our results revealed that, while DEHP has some binding affinity for ERalpha, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERalpha. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERalpha was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERalpha, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERalpha.

  17. Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

    ERIC Educational Resources Information Center

    Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

    2012-01-01

    An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

  18. Mapping IgE-binding epitopes of Ric c 1 and Ric c 3, allergens from Ricinus communis, by mast cell degranulation assay.

    PubMed

    Felix, S P; Mayerhoffer, R O; Damatta, R A; Verícimo, M A; Nascimento, V V; Machado, O L T

    2008-04-01

    Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.

  19. Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

    PubMed

    Tang, W X; Bazaraa, H M; Magiera, H; Cooke, N E; Haddad, J G

    1996-06-01

    Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

  20. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

    PubMed Central

    Occhialini, A; Urdaci, M; Doucet-Populaire, F; Bébéar, C M; Lamouliatte, H; Mégraud, F

    1997-01-01

    Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes. PMID:9420046

  1. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

    PubMed

    Occhialini, A; Urdaci, M; Doucet-Populaire, F; Bébéar, C M; Lamouliatte, H; Mégraud, F

    1997-12-01

    Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.

  2. Acetylcholine Receptor Binding Characteristics of Snake and Cone Snail Venom Postsynaptic Neurotoxins: Further Studies with a Non-Radioactive Assay

    DTIC Science & Technology

    1993-01-01

    binding) results (BURDEN et at.. 1975: PETERSON, 1989: BARCHAN et al.. 1992). Obviously, the most reliable correlation between an in vivo and in vitro...SCHIMIDT "as particularly appreciated REFERENCES BARCHAN . D.. KACHALSKY. S.. NEUMANNv. D_. VOGFL.ý Z.. OVADIA. NI.. KOCHIXA. E. and [--.(itis. S. 1 192...Peptides isolated from the venom of’ Coitus xi’ographus block neuromuscular transmission. Neurosca, Lett. 24, 57-62. NEUMANN. D., BARCHAN . D.. FRIDKIN

  3. Combined QSAR-based virtual screening and fluorescence binding assay to identify natural product mediators of Interferon Regulatory Factor 7 (IRF-7) in pulmonary infection.

    PubMed

    Liu, Y; Huang, L; Ye, H; Lv, X

    2016-11-01

    Interferon regulatory factor-7 (IRF-7) is involved in pulmonary infection and pneumonia. Here, a synthetic strategy that combined quantitative structure-activity relationship (QSAR)-based virtual screening and in vitro binding assay was described to identify new and potent mediator ligands of IRF-7 from natural products. In the procedure, a QSAR scoring function was developed and validated using Gaussian process (GP) regression and a structure-based set of protein-ligand affinity data. By integrating hotspot pocket prediction, pharmacokinetics profile analysis and molecular docking calculations, the scoring function was successfully applied to virtual screening against a large library of structurally diverse, drug-like natural products. With the method we were able to identify a number of potential hits, from which several compounds were found to have moderate or high affinity to IRF-7 using fluorescence binding assays, with dissociation constants Kd at micromolar level. We have also examined the structural basis and noncovalent interactions of computationally modelled IRF-7 complex with its potent ligands. It is revealed that hydrophobic forces and van der Waals contacts play a central role in stabilization of the complex architecture, while few hydrogen bonds confer additional specificity for the protein-ligand recognition.

  4. Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

    PubMed Central

    Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

    2013-01-01

    Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

  5. A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

    PubMed

    Martin, Harry; Murray, Colleen; Christeller, John; McGhie, Tony

    2008-10-01

    A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

  6. Preparation and one-step purification of mono-125I-angiotensin II for radioligand binding assays

    SciTech Connect

    Speth, R.C.; Husain, A.

    1984-04-01

    A one-step purification of mono-/sup 125/I-angiotensin II prepared by the chloramine T procedure is described. The purification is effected on a cellulose cation exchange column with isocratic elution by 50 mM sodium acetate, pH 5.0. The purity of the mono-/sup 125/I-angiotensin II was determined by thin layer chromatography, high pressure liquid chromatography, enzymatic digestion, radioreceptor assay, and radioimmunoassay. Preparation and purification of mono-/sup 125/I-angiotensin II by this procedure offers significant advantages over existing methods for its preparation in terms of purity, simplicity, efficiency, and cost.

  7. A microplate assay for the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis.

    PubMed

    Kumar, Vidya P; Basavannacharya, Chandrakala; de Sousa, Sunita M

    2014-07-18

    A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.

  8. Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique.

    PubMed

    Erhardt, Juergen G; Estes, John E; Pfeiffer, Christine M; Biesalski, Hans K; Craft, Neal E

    2004-11-01

    The measurement of vitamin A (VA) and iron status is very important in the assessment of nutritional deficiencies. The objective of this research was to develop a sandwich ELISA technique for the simultaneous measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein (CRP) as indicators for VA and iron status. The inclusion of CRP as marker of infection allows for more accurate interpretation of VA and iron status. This is accomplished in a 30-microL serum or plasma sample using an ELISA with different capture and detection antibodies and different dilutions of the sample. Commercially available clinical serum controls were used for calibration purposes. The developed assays were compared to commercially available traditional tests. Regression coefficients comparing both assays were better than 0.84 (P < 0.001). Using a limited sample set, the sandwich ELISA assay produced very similar specificity and sensitivity compared to traditional methods when common cutoff values were applied. Intra- and interassay variability was between 5 and 14% for all tests. The cost of the materials for all 5 measurements decreases to less than $1/sample if a large number of samples is analyzed. Due to the low cost, high throughput, and comparability to traditional tests, this procedure has several advantages for assessing VA and iron status in population surveys.

  9. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    PubMed

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

  10. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

    USGS Publications Warehouse

    Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

    2014-01-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

  11. Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor.

    PubMed

    He, Sheng-Xiang; Song, Ge; Shi, Jia-Ping; Guo, Yu-Qi; Guo, Zhan-Yun

    2014-11-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.

  12. Analytical performance and clinical usefulness of two binding assays for growth hormone binding protein (GHBP) measurement: high performance liquid chromatography (HPLC)-gel filtration and dextran-coated charcoal adsorption.

    PubMed

    Llopis, M A; Granada, M L; Audí, L; Sanmartí, A; Bel, J; Sánchez-Planell, L; Formiguera, X; Marin, F; Corominas, A

    1997-11-28

    We compared two binding assays for growth hormone binding protein (GHBP) measurements, which differ in the method of bound and free GH separation: HPLC-gel filtration or dextran coated-charcoal adsorption (DCC). Two pools of sera (high and medium GHBP activity) were used for quality-control assessment. Moreover, 62 samples from 34 children and 28 adults with different nutritional status were studied. Total, between- and intra-iodination coefficients of variation (CVs) from the two methods were not different. Although percentage binding measured in the pool sera significantly differed, the concentrations assessed by Scatchard plot were comparable. Results obtained by the two methods in the 62 sera were significantly correlated (r = 0.77, P < 0.001). With both methods GHBP activity correlated with chronological age and body mass index (BMI) and differed among groups with different nutritional status. Although HPLC and DCC separation methods for GHBP measurement differ in their practicability, our study demonstrates that performance and the clinical usefulness of the two methods are comparable.

  13. Lack of binding to isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic assay, of the ultraviolet sunscreen filters Tinosorb M-active and Tinosorb S.

    PubMed

    Ashby, J; Tinwell, H; Plautz, J; Twomey, K; Lefevre, P A

    2001-12-01

    The presence of structurally diverse chemicals as contaminants in the environment has led to concerns regarding their possible endocrine disturbing effects. Recently, some ultraviolet absorbing components of sunscreen preparations have given positive responses in assays monitoring estrogen-like activity both in vitro and in vivo. Consequently, two recently developed sunscreen components, Tinosorb M-active and Tinosorb S, were evaluated using the in vitro estrogen and androgen receptor competitive binding assays. Neither compound gave a positive response in either of the assays, consistent with the large molecular dimensions of each chemical disfavoring binding to the hormone receptors. Both of the chemicals were inactive in immature rat uterotrophic assays conducted using the subcutaneous route of administration. It is concluded that neither of these agents possess intrinsic estrogenic/antiestrogenic or androgenic/antiandrogenic activity. The several positive control chemicals evaluated gave the expected positive responses in the assays used.

  14. A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

    PubMed

    Baranov, Konstantin; Volkova, Olga; Chikaev, Nikolai; Mechetina, Ludmila; Laktionov, Pavel; Najakshin, Alexander; Taranin, Alexander

    2008-03-20

    We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

  15. Application of cancer‐associated glycoforms and glycan‐binding probes to an in vitro diagnostic multivariate index assay for precise diagnoses of cancer

    PubMed Central

    Kang, Jeong Gu

    2016-01-01

    Personalized medicine has emerged as a widely accepted trend in medicine for the efficacious and safe treatment of various diseases. It covers every medical treatment tailored according to various properties of individuals. Cancer‐associated glycosylation mirrors cancer states more precisely, and this “sweet side of cancer” is thus intended to spur the development of an advanced in vitro diagnostic system. The changes of glyco‐codes are often subtle and thus not easy to trace, thereby making it difficult to discriminate changes from various compounding factors. Special glycan‐binding probes, often lectins, can be paired with aglycosylated antibodies to enable quantitative and qualitative measurements of glycoforms. With the in vitro diagnosis multivariate index assay (IVDMIA) considered to be capable of yielding patient‐specific results, the combinatorial use of multiple glycoproteins may be a good modality to ensure disease‐specific, personalized diagnoses. PMID:27005968

  16. Qualification of a homogeneous cell-based neonatal Fc receptor (FcRn) binding assay and its application to studies on Fc functionality of IgG-based therapeutics.

    PubMed

    Mathur, Abhishek; Arora, Taruna; Liu, Ling; Crouse-Zeineddini, Jill; Mukku, Venkat

    2013-04-30

    The Fc region of IgG-based molecules plays an important role in determining their in vivo pharmacokinetic profile by its pH-dependent binding to the neonatal Fc receptor (FcRn) which is expressed on the endothelial cells lining blood vessels. By virtue of this pH-specific interaction with IgG-Fc, FcRn mediates IgG homeostasis in human adults by maintaining serum IgG levels, and also transfers maternal IgGs from mother to fetus via the placenta. The Fc-FcRn interaction is also critical for keeping IgG-based therapeutic molecules in circulation thereby enhancing their serum half life. A homogeneous cell-based flow cytometric FcRn binding assay was established to characterize the Fc-FcRn interaction of therapeutic IgG-based molecules. It is a competition-based assay, wherein the IgG-Fc containing test molecule competes with a fixed concentration of fluorescently-labeled IgG-Fc moiety in solution for binding to the cell-expressed FcRn. The cell-bound fluorescence is read on a flow cytometer. Response of the test sample is analyzed relative to the standard sample and the results are reported as % relative binding. The assay is robust and meets the qualification criteria for specificity, method linearity, accuracy and precision over the relative binding range of 60%-160%. This assay was shown to effectively characterize altered Fc-FcRn interactions for photo-stressed, heat-stressed, oxidized, and Fc mutant samples. It was observed that the relative binding of the IgG-Fc to the cell-surface-expressed FcRn in the assay varies across different molecules, even within the same IgG subclass. This indicates that the Fc-FcRn binding can be influenced by the antigen-binding region of the molecules in addition to the IgG subclass. Overall, this assay is reflective of the in vivo mechanism of immunoglobulin binding to membrane-bound FcRn, and can be used as an analytical tool for assessing lot-to-lot consistency and stability testing across different batches of the same molecule

  17. Low agreement between radio binding assays in analyzing glutamic acid decarboxylase (GAD65Ab) autoantibodies in patients classified with type 2 diabetes.

    PubMed

    Daka, Bledar; Svensson, Maria K; Lernmark, Ke; Mincheva-Nilsson, Lucia; Hallmans, Goran; Rolandsson, Olov

    2009-09-01

    Autoantibodies against glutamic acid decarboxylase (GAD65Ab) are used in the classification of diabetes in adults. We assessed the concordance in GAD65 autoantibody levels within subjects between three different GAD65Ab radio binding assays (RBA). Plasma samples from 112 diabetes patients (median age 50 years) initially classified with type 2 diabetes was randomly selected from a local diabetes registry. Coded samples were analyzed with two RBA employing (35)S-labeled GAD65. The first used the pEx9 plasmid (pEx9 RBA), the second employed the pThGAD65 plasmid (pThGAD65 RBA) to label GAD65 by in vitro transcription translation. We also used a commercial kit employing plasmid pGAD17 labelled with (125)I (pGAD17 RBA). Subsequent analyses followed standard procedures. Two different cut-offs for GAD65Ab positivity were used in all three assays. We calculated the correlation, concordance, and agreement between the assays. The proportion of GAD65Ab positivity differed between assays when low cut-offs were used (pEx9 RBA 25%, pThGAD65 RBA 17.9%, and pGAD17 RBA 12.5%, respectively). When high cut-offs were applied, the concordance between the pEx9 RBA and the pThGAD65 RBA was 97.3 while their concordance to the pGAD17 RBA was lower (88.4 and 87.4, respectively). There was a low agreement between both pEx9 RBA and pGAD17 RBA (0.45, 95% CI 0.20-0.70) and between pThGAD65 RBA and pGAD17 RBA (0.43, 95% CI 0.18-0.68). We found discrepancies in determining the GAD65Ab positivity, which constitutes a problem when GAD65Ab are used clinically. Further methodological GAD65Ab assays studies are warranted.

  18. Influence of Vitamin D Binding Protein on Accuracy of 25-Hydroxyvitamin D Measurement Using the ADVIA Centaur Vitamin D Total Assay

    PubMed Central

    Freeman, James; Wilson, Kimberly; Spears, Ryan; Shalhoub, Victoria; Sibley, Paul

    2014-01-01

    Vitamin D status in different populations relies on accurate measurement of total serum 25-hydroxyvitamin D [25(OH)D] concentrations [i.e., 25(OH)D3 and 25(OH)D2]. This study evaluated agreement between the ADVIA Centaur Vitamin D Total assay for 25(OH)D testing (traceable to the NIST-Ghent reference method procedure) and a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for various populations with different levels of vitamin D binding protein (DBP). Total serum 25(OH)D concentrations were measured for 36 pregnant women, 40 hemodialysis patients, and 30 samples (DBP-spiked or not) from healthy subjects. ELISA measured DBP levels. The mean serum DBP concentrations were higher for pregnancy (415 μg/mL) and lower for hemodialysis subjects (198 μg/mL) than for healthy subjects and were highest for spiked serum (545 μg/mL). The average bias between the ADVIA Centaur assay and the LC-MS/MS method was −1.4% (healthy), −6.1% (pregnancy), and 4.4% (hemodialysis). The slightly greater bias for samples from some pregnancy and hemodialysis subjects with serum DBP levels outside of the normal healthy range fell within a clinically acceptable range—reflected by analysis of their low-range (≤136 μg/mL), medium-range (137–559 μg/mL), and high-range (≥560 μg/mL) DBP groups. Thus, the ADVIA Centaur Vitamin D Total assay demonstrates acceptable performance compared with an LC-MS/MS method for populations containing different amounts of DBP. PMID:25045351

  19. The CD11a binding site of efalizumab in psoriatic skin tissue as analyzed by Multi-Epitope Ligand Cartography robot technology. Introduction of a novel biological drug-binding biochip assay.

    PubMed

    Bonnekoh, B; Böckelmann, R; Pommer, A J; Malykh, Y; Philipsen, L; Gollnick, H

    2007-01-01

    -1) as its ligand was most prevalent in the dermal vessels. T lymphocytes (for which the markers were CD3, CD8, CD4, and CD45R0) were the major cellular targets of efalizumab. In contrast, NK cells were only a minor target of efalizumab. Our study demonstrated that efalizumab represents a treatment for psoriasis that primarily targets memory CD4+ and CD8+ T cells and has a high specificity for psoriatic disease activity. Moreover, we hereby introduce the novel principle of a biological drug-binding biochip assay being especially useful for the future monitoring of psoriatic skin lesions under efalizumab treatment conditions.

  20. Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates

    PubMed Central

    Arnold, A. R.; Ford, B. A.; McAllister, S. K.; Lonsway, D.; Albrecht, V.; Jerris, R. C.; Rasheed, J. K.; Limbago, B.

    2015-01-01

    The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively. PMID:26677248

  1. Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

    PubMed

    Arnold, A R; Burnham, C-A D; Ford, B A; Lawhon, S D; McAllister, S K; Lonsway, D; Albrecht, V; Jerris, R C; Rasheed, J K; Limbago, B; Burd, E M; Westblade, L F

    2016-03-01

    The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

  2. Identification, Expression Profiling and Fluorescence-Based Binding Assays of a Chemosensory Protein Gene from the Western Flower Thrips, Frankliniella occidentalis

    PubMed Central

    Zhang, Zhi-Ke; Lei, Zhong-Ren

    2015-01-01

    Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four—cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating

  3. Differential enzyme-linked immunosorbent assay and ligand-binding mass spectrometry for analysis of biotransformation of protein therapeutics: application to various FGF21 modalities.

    PubMed

    Hager, Todd; Spahr, Chris; Xu, Jing; Salimi-Moosavi, Hossein; Hall, Michael

    2013-03-05

    Novel protein therapeutics have become increasingly important modalities for treating diseases. Such therapeutics include recombinant fusions of pharmacoactive polypeptides to half-life extenders such as monoclonal antibodies, fragments of antibodies, and albumin. Half-life extension can also be achieved via chemical attachment to polymers such as polyethylene glycol. Any of these therapeutics may be susceptible to biotransformation, most notably in vivo proteolytic truncation, and it is vital to understand this phenomenon during early drug development to ensure correct pharmacokinetic profiling and optimize the in vivo stability through re-engineering. In this paper, we describe an integrated approach that combines differential enzyme-linked immunosorbent assay (ELISA) with ligand-binding-mass spectrometry (LB-MS) to provide a thorough understanding of the biotransformation of novel protein therapeutics. Differential ELISA allows for a fast, high-throughput means to reveal gross in vivo proteolytic liabilities. Ensuing LB-MS analysis provides higher resolution details such as specific vulnerable loci to allow design refinement of the molecule. In this work, the power of the approach is elucidated by application to the optimization of a promising drug candidate, FGF21.

  4. A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe.

    PubMed

    Meyners, Christian; Wawrzinek, Robert; Krämer, Andreas; Hinz, Steffen; Wessig, Pablo; Meyer-Almes, Franz-Josef

    2014-08-01

    High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide.

  5. Novel histamine H3 receptor antagonists: affinities in an H3 receptor binding assay and potencies in two functional H3 receptor models.

    PubMed

    Schlicker, E; Kathmann, M; Reidemeister, S; Stark, H; Schunack, W

    1994-08-01

    1. We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring-containing alkyl residue [corrected]. 2. The specific binding of [3H]-N alpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the ten compounds at pKi values ranging from 7.56 to 8.68. 3. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]-noradrenaline was antagonized by the ten compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.07 to 9.20. 4. The electrically induced contraction in guinea-pig ileum strips (which was abolished by atropine) was inhibited by the H3 receptor agonists R-(-)-alpha-methylhistamine (pEC50 7.76), N alpha-methylhistamine (7.90) and imetit (8.18). The concentration-response curve of R-(-)-alpha-methylhistamine was shifted to the right by thioperamide (apparent pA2 8.79) and by the ten novel compounds (range of pA2 values 6.64-8.81). 5. The affinities and potencies were compared by linear regression analysis. This analysis was extended to thioperamide, the standard H3 receptor antagonist, which is also capable of differentiating between H3A and H3B sites. Comparison of the apparent pA2 values in the two functional H3 receptor models yielded a regression coefficient of 0.77 (P<0.02). When the pA2 of the drugs in the mouse brain cortex were compared to the pXj for H3 sites (ten novel compounds) and for H3A sites (thioperamide), a significant correlation (r = 0.87; P<0.001) was obtained. There was, however, no significant correlation when the pKi of

  6. Key Role of Effector Memory CD4+ T Lymphocytes in a Short-Incubation Heparin-Binding Hemagglutinin Gamma Interferon Release Assay for the Detection of Latent Tuberculosis

    PubMed Central

    Wyndham-Thomas, Chloé; Corbière, Véronique; Dirix, Violette; Smits, Kaatje; Domont, Fanny; Libin, Myriam; Loyens, Marc; Locht, Camille

    2014-01-01

    The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4+ T lymphocytes, a finding suggestive of continuous HBHA presentation during latency. PMID:24391135

  7. Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide.

    PubMed

    Sano, Daisuke; Matsuo, Takahiro; Omura, Tatsuo

    2004-06-01

    The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H(2)N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology

  8. Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System

    PubMed Central

    Tanemura, Yutaro; Mochizuki, Yuki; Kumachi, Shigefumi; Nemoto, Naoto

    2015-01-01

    Constrained peptides are an attractive class as affinity reagents or drug leads owing to their excellent binding properties. Many kinds of these peptides, such as cyclic peptides containing disulfide bridges, are found in nature or designed artificially by directed evolution. However, confirming the binding properties of the disulfide-rich peptides can be generally difficult, because of oxidative folding problems in the preparation steps. Therefore, a method for evaluating the binding properties of such peptides rapidly and easily is required. Here, we report an easy and rapid method for preparing biotin-attached peptides containing disulfide bridges or a chemical cross-linker using a cell-free translation system and a puromycin-linker, which is applicable to pull-down assays for protein (or peptide) molecular interaction analysis. PMID:25738808

  9. Binding Assays for the Quantitative Detection of P. Brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

    DTIC Science & Technology

    1990-03-15

    Recommendations 27 VII. Literature Cited 28 I!. [i• Figure 1. Structures of the Brevetoxins 5 Figure 2. Non-Competitive Brevetoxin-Antibody- Protein A Urease ...Barracuda. 20 Figure 9. Toxin- Urease Assay 23 Figure 10. Toxin-Peroxidase Assay 23 Figure 11. Non-Competitive Peroxidase-Linked Sandwich Immuunoassay 24...8217. Thus, the greatest flexibility is gained employing such techniques, and many different variations may be developed to oset defined :riteria. 9 i Urease 2

  10. Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation.

    PubMed

    Oh, Seok-Jeong; Kim, Hwa; Liu, Yingqiu; Han, Hyo-Kyung; Kwon, Kyenghee; Chang, Kyung-Hwa; Park, Kwangsik; Kim, Younghun; Shim, Kyuhwan; An, Seong Soo A; Lee, Moo-Yeol

    2014-03-21

    A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.

  11. Proposal for the standardization of the serum unsaturated iron binding capacity assay, and results in groups of subjects with normal iron stores and with prelatent, latent, and manifest iron deficiency.

    PubMed

    Gabbe, E E; Heinrich, H C; Icagić, F

    1982-02-26

    An effort has been made to standardize the indirect iron saturation excess method for the determination of the serum unsaturated iron binding capacity (UIBC) and thus to relinquish the direct adsorption methods for the assay of the serum total iron binding capacity (TIBC) which give falsely high results due to unspecific binding of the saturating iron to serum proteins. In order to eliminate the interfering effects of hydrolytic polymerization of iron(III) on the saturation of apotransferrin in serum and on the colorimetric determination of the unbound iron excess at pH 8.3, conditions have been studied for the preparation of the iron-nitrilotriacetate-complex (Fe(NTA)2) solution at pH 8.3 with respect to its reactivity with the reductant sodium ascorbate and with the chromogen bathophenanthroline-disulfonate in photometric standards and in samples containing iron-saturated serum. The validity of the results for the UIBC thus obtained has been investigated (1) by direct spectrophotometric titration with Fe(NTA)2 of the apotransferrin in serum by measuring the absorbance of transferrin at 470 nm in 50-mm cuvettes, and of the UIBC using the modified indirect iron saturation excess assay, both of which gave the same saturation points, and (2) by the correlation of the TIBC obtained from serum iron determinations and the UIBC, with the transferrin concentration measured by the radial immunodiffusion assay. Results of UIBC determinations are presented along with serum iron concentration, TIBC, and transferrin saturation in groups of subjects with normal iron stores and prelatent, latent, and manifest iron deficiency.

  12. A new nonpeptide substrate of human sirtuin in a capillary electrophoresis-based assay. Investigation of the binding mode by docking experiments.

    PubMed

    Abromeit, Hans; Kannan, Srinivasaraghaven; Sippl, Wolfgang; Scriba, Gerhard K E

    2012-06-01

    Sirtuins are nicotinamide dinucleotide-dependent class III histone deacetylases catalyzing various physiological processes involved in cell proliferation, differentiation, apoptosis, and ageing. This makes them attractive targets in drug research. In order to simplify sirtuin substrates for assay development, two N(ɛ)-acetyllysine derivatives, N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine amide, and N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine methyl ester were synthesized and evaluated as substrates for human SIRT1 in a capillary electrophoresis-based enzyme assay. Substrate, deacetylated product, and the coproduct nicotinamide were separated in a 200 mM phosphate/Tris buffer at pH 2.85. Field-amplified sample injection was employed to achieve sufficient assay sensitivity. While the ester derivative was not recognized by the enzyme, the amide substrate was effectively converted to the deacetylated product. The assay was subsequently validated with respect to range, linearity, limit of detection, and limit of quantification. Michaelis-Menten kinetic parameters, K(m) = 83 μM and V(max) = 6.8 μM/min were determined. The applicability of the assay for inhibitor screening was demonstrated using the known inhibitors sirtinol and the suramin derivate NF258. Resveratrol did not increase the deacetylation rate at concentrations of up to 200 μM. Docking experiments revealed the necessity of an amide function at the C-terminus of nonpeptide substrates while more structural freedom is tolerated at the N-terminus of N(ɛ) -acetyllysine.

  13. Sensitive and accurate identification of protein–DNA binding events in ChIP-chip assays using higher order derivative analysis

    PubMed Central

    Barrett, Christian L.; Cho, Byung-Kwan

    2011-01-01

    Immuno-precipitation of protein–DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein–DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein–DNA interactions and for extending the usefulness horizon of the ChIP-chip platform. PMID:21051353

  14. THE DEVELOPMENT OF AN IN VITRO ASSAY FOR EVALUATING THE BINDING OF PERFLUOROALKYL ACIDS (PFAAS) TO THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS)

    EPA Science Inventory

    The purpose of this work was to evaluate the binding of PFAAs to PPAR receptors and determine the potential for activation or antagonism of the pathway during embryonic development. Activation of mouse and human PPAR isoforms by perfluorooctanoic acid (PFOA) and perfluorooctanes...

  15. Comparative binding of antitumor indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] to serum transport proteins assayed by capillary zone electrophoresis.

    PubMed

    Timerbaev, Andrei R; Rudnev, Alexander V; Semenova, Olga; Hartinger, Christian G; Keppler, Bernhard K

    2005-06-15

    The indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] coordination compound shows notable antiproliferative activity in different tumor models and has recently ended phase I clinical trials as a lead anticancer metallodrug candidate. Its approval could be greatly facilitated if more precise information was available on the rate and degree of the drug's transformation occurring upon interaction with serum transport proteins and on the stability of the adducts formed. With this objective, a new method has been developed for the determination of the protein-binding rate and association constants under simulated physiological conditions by capillary zone electrophoresis (CZE). These binding parameters were assessed by monitoring the time- and concentration-dependent changes in peak area responses of reaction components, constructing the corresponding binding curves, and conducting a mathematical analysis. Comparison of the apparent rate constants determined by CZE revealed that indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] binds to transferrin much faster than to albumin: k=39.5 x 10(-4) and 3.3 x 10(-4)s(-1), respectively. The corresponding association constants are indicative of moderate metal-protein coordination, with a somewhat higher affinity of the Ru complex toward albumin (9910 and 6460 M(-1), respectively). The results of our study confirm in a quantitative manner that, in real bloodstream circumstances, plasma albumin may serve as a reservoir and a natural carrier of the administered ruthenium drug and hence mediate its accumulation in tumors.

  16. Total Vitamin D Assay Comparison of the Roche Diagnostics “Vitamin D Total” Electrochemiluminescence Protein Binding Assay with the Chromsystems HPLC Method in a Population with both D2 and D3 forms of Vitamin D

    PubMed Central

    Abdel-Wareth, Laila; Haq, Afrozul; Turner, Andrew; Khan, Shoukat; Salem, Arwa; Mustafa, Faten; Hussein, Nafiz; Pallinalakam, Fasila; Grundy, Louisa; Patras, Gemma; Rajah, Jaishen

    2013-01-01

    This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (75%) had a D2 concentration >10 nmol/L while the remaining twenty four subjects had vitamin D2 concentration of less than 10 nmol/L by HPLC. Overall, the Roche Diagnostics method showed a negative bias of −2.59 ± 4.11 nmol/L on the e602 as compared to the HPLC with a concordance rate of 84%. The concordance rate was 91% in samples with D2 of less than 10 nmol/L and 82% in those with D2 concentration >10 nmol/L. The overall correlation had an r value of 0.77. The r value was higher in samples with D2 levels of less than 10 nmol/L, r = 0.96, as compared to those with D2 values of greater than 10 nmol/L, r = 0.74. The observed bias had little impact on clinical decision and therefore is clinically acceptable. PMID:23525081

  17. Total vitamin D assay comparison of the Roche Diagnostics "Vitamin D total" electrochemiluminescence protein binding assay with the Chromsystems HPLC method in a population with both D2 and D3 forms of vitamin D.

    PubMed

    Abdel-Wareth, Laila; Haq, Afrozul; Turner, Andrew; Khan, Shoukat; Salem, Arwa; Mustafa, Faten; Hussein, Nafiz; Pallinalakam, Fasila; Grundy, Louisa; Patras, Gemma; Rajah, Jaishen

    2013-03-22

    This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (75%) had a D2 concentration >10 nmol/L while the remaining twenty four subjects had vitamin D2 concentration of less than 10 nmol/L by HPLC. Overall, the Roche Diagnostics method showed a negative bias of -2.59 ± 4.11 nmol/L on the e602 as compared to the HPLC with a concordance rate of 84%. The concordance rate was 91% in samples with D2 of less than 10 nmol/L and 82% in those with D2 concentration >10 nmol/L. The overall correlation had an r value of 0.77. The r value was higher in samples with D2 levels of less than 10 nmol/L, r = 0.96, as compared to those with D2 values of greater than 10 nmol/L, r = 0.74. The observed bias had little impact on clinical decision and therefore is clinically acceptable.

  18. Assays for calcitonin receptors

    SciTech Connect

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

    1985-01-01

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

  19. Synthesis, characterization, X-ray crystal structure, DFT calculation, DNA binding, and antimicrobial assays of two new mixed-ligand copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Ebrahimipour, S. Yousef; Sheikhshoaie, Iran; Mohamadi, Maryam; Suarez, Sebastian; Baggio, Ricardo; Khaleghi, Moj; Torkzadeh-Mahani, Masoud; Mostafavi, Ali

    2015-05-01

    Two new Cu(II) complexes, [Cu(L)(phen)] (1), [Cu(L)(bipy)] (2), where L2- = (3-methoxy-2oxidobenzylidene)benzohydrazidato, phen = 1,10 phenanthroline, and bipy = 2,2‧ bipyridine, were prepared and fully characterized using elemental analyses, FT-IR, molar conductivity, and electronic spectra. The structures of both complexes were also determined by X-ray diffraction. It was found that, both complexes possessed square pyramidal coordination environment in which, Cu(II) ions were coordinated by donor atoms of HL and two nitrogens of heterocyclic bases. Computational studies were performed using DFT calculations at B3LYP/6-311+G(d,p) level of theory. DNA binding activities of these complexes were also investigated using electronic absorption, competitive fluorescence titration and cyclic voltammetry studies. The obtained results indicated that binding of the complexes to DNA was of intercalative mode. Furthermore, antimicrobial activities of these compounds were screened against microorganisms.

  20. Binding Assays for the Quantitative Detection of P. brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

    DTIC Science & Technology

    1990-05-15

    antibodies, sodium channels 0 6 01 receptor binding, brevetoxins. 1 ABSTRACT (Continue on reverse if necessary and identify by block number) The...redissolved in 0.3 volumes of the original volume 6 in phosphate buffered saline containing 0.01% sodium azide. IgG is stored for longer periods of time... sodium borotritiide used for reduction. Using 1 Ci of tritiated sodium borotritiide, we have been successful in producing between 200 and 250 mCi of

  1. In vitro and ex vivo binding to uterine progestin receptors of the rat as a tool to assay progestational activity of glucocorticoids.

    PubMed

    Luzzani, F; Gallico, L; Glässer, A

    1982-01-01

    The competition of some widely employed glucocorticoids with the binding of [3H]-promegestone, a highly potent synthetic progestagen, to uterine cytosol progestin receptors of the immature rat has been studied both in in vitro and ex vivo experiments. The relative binding affinities (RBA's) to progesterone were determined in vitro: fluocinolone acetonide greater than triamcinolone acetonide greater than betamethasone 17-valerate greater than prednisolone, betamethasone, triamcinolone and cortisol. After pretreating rats in vivo with progesterone or chlormadinone acetate (subcutaneously), a dose-dependent decrease in in vitro binding of [3H]-promegestone to uterine cytosol was evident. Similar decreases were obtained after pretreatment with some of the other glucocorticoids tested. Potency ratios to progesterone, arbitrarily set at 1.0, were: fluocinolone acetonide 86.7, triamcinolone acetonide 5.6, betamethasone valerate 4.1, chlormadinone acetate 2.6. Prednisolone, betamethasone, triamcinolone and cortisol were inactive. Both the in vitro and the ex vivo results clearly indicate that glucocorticoids interact with the uterine cytosol progestin receptor system, depending on their chemical structures; this interaction may account for some of their unwanted side-effects in the endocrine system. Moreover, this experimental system may prove to be a useful tool for evaluation of the progestational activities of glucocorticoids and other steroids, using the rat as an animal model.

  2. Equilibrium binding assays reveal the elevated stoichiometry and salt dependence of the interaction between full-length human sex-determining region on the Y chromosome (SRY) and DNA.

    PubMed

    Baud, Stephanie; Margeat, Emmanuel; Lumbroso, Serge; Paris, Françoise; Sultan, Charles; Royer, Catherine; Poujol, Nicolas

    2002-05-24

    In an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3epsilon gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRY(WT)) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRY(WT) in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRY(Y129N), identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well.

  3. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  4. Development of a TaqMan(R)-Minor Groove Binding Protein Assay for the Detection and Quantification of Crimean-Congo Hemorrhagic Fever Virus

    DTIC Science & Technology

    2007-01-01

    little data available on the L segment. Thirteen full-length CCHF genomes were completely sequenced and reported in Gen- bank.13–16 This diversity...illustrates the need for a broad-range detection assay specific to the CCHFV species. Human diagnosis of CCHF to date has relied on several methods. Clinical...fatal cases of CCHF .5,15,17 There is also the problem of cross-reactivity with other members of the Nairovirus family using ELISA.18 Several reverse

  5. Nimesulide binding site in the B0AT1 (SLC6A19) amino acid transporter. Mechanism of inhibition revealed by proteoliposome transport assay and molecular modelling.

    PubMed

    Pochini, Lorena; Seidita, Angela; Sensi, Cristina; Scalise, Mariafrancesca; Eberini, Ivano; Indiveri, Cesare

    2014-06-01

    The effect of pharmaceutical compounds on the rat kidney B0AT1 transporter in proteoliposomes has been screened. To this aim, inhibition of the transport activity by the different compounds was measured on Na(+)-[(3)H]glutamine co-transport in the presence of membrane potential positive outside. Most of the tested drugs had no effect on the transport activity. Some compounds exhibited inhibitory effects from 5 to 88% at concentration of 300μM. Among the tested compounds, only the anti-inflammatory drug nimesulide exerted potent inhibition on B0AT1. From dose response analysis, an IC50 value of 23μM was found. Inhibition kinetic analysis was performed: noncompetitive inhibition of the glutamine transport was observed while competitive behaviour was found when the inhibition was analyzed with respect to the Na(+) concentration. Several molecules harbouring functional groups of nimesulide (analogues) were tested as inhibitors. None among the tested molecules has the capacity to inhibit the transport with the exception of the compound NS-398, whose chemical structure is very close to that of whole nimesulide. The IC50 for this compound was 131μM. Inhibition kinetics showed behaviour of NS-398 identical to that of nimesulide, i.e., noncompetitive inhibition respect to glutamine and competitive inhibition respect to Na(+). Molecular docking of nimesulide suggested that this drug is able to bind B0AT1 in an external dedicated binding site and that its binding produces a steric hindrance effect of the protein translocation path abolishing the transporter activity.

  6. A convenient method for europium-labeling of a recombinant chimeric relaxin family peptide R3/I5 for receptor-binding assays.

    PubMed

    Zhang, Wei-Jie; Jiang, Qian; Wang, Xin-Yi; Song, Ge; Shao, Xiao-Xia; Guo, Zhan-Yun

    2013-06-01

    Relaxin family peptides have important biological functions, and so far, four G-protein-coupled receptors have been identified as their receptors (RXFP1-4). A chimeric relaxin family peptide R3/I5, containing the B-chain of relaxin-3 and the A-chain of INSL5, is a selective agonist for both RXFP3 and RXFP4. In a previous study, europium-labeled R3/I5, as a nonradioactive and low-background receptor-binding tracer, was prepared through a chemical synthesis approach. In the present study, we established a convenient alternative approach for preparing the europium-labeled R3/I5 tracer based on a recombinant R3/I5 designed to carry a solubilizing tag at the A-chain N-terminus and a pyroglutamate residue at the B-chain N-terminus. Because of the presence of a single primary amine moiety, the recombinant R3/I5 peptide was site-specifically mono-labeled at the A-chain N-terminus by a diethylenetriaminepentaacetic acid/europium moiety through a convenient one-step procedure. The diethylenetriaminepentaacetic acid/Eu3+-labeled R3/I5 bound both receptors RXFP3 and RXFP4 with high binding affinities and low nonspecific binding. Thus, we have presented a valuable nonradioactive tracer for future interaction studies on RXFP3 and RXFP4 with various natural or designed ligands. The present approach could also be adapted for preparing and labeling of other chimeric relaxin family peptides.

  7. ERα dimerization: a key factor for the weak estrogenic activity of an ERα modulator unable to compete with estradiol in binding assays.

    PubMed

    Leclercq, Guy; Laïos, Ioanna; Elie-Caille, Céline; Leiber, Denis; Laurent, Guy; Lesniewska, Eric; Tanfin, Zahra; Jacquot, Yves

    2017-04-01

    Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [(3)H]17β-estradiol ([(3)H]17β-E2) for binding to the estrogen receptor α (ERα), questioning its ability to interact with the receptor. However, detection by atomic force spectroscopy (AFS) of an ESTZ-induced ERα dimerization has eliminated any remaining doubts. The effect of the compound on the proliferation of ERα-positive and negative breast cancer cells confirmed the requirement of the receptor. The efficiency of ESTZ in MCF-7 cells was weak without any potency to modify the proliferation profile of estradiol and coumestrol. Growth enhancement was associated with a proteasomal degradation of ERα without substantial recruitment of LxxLL coactivators. This may be related to an unusual delay between the acquisition by the receptor of an ERE-binding capacity and the subsequent estrogen-dependent transcription. A complementary ability to enhance TPA-induced AP-1 transcription was observed, even at concentrations insufficient to activate the ERα, suggesting a partly independent mechanism. ESTZ also rapidly and transiently activated ERK1/2 likely through membrane estrogenic pathways provoking a reorganization of the actin network. Finally, the systematic absence of biological responses with an ESTZ derivative unable to induce ERα dimerization stresses the importance of this step in the action of the compound, as reported for conventional estrogens. In view of the existence of many other ERα modulators (endocrine disruptors such as, for example, pesticides, environmental contaminants or phytoestrogens) with extremely weak or similar apparent lack of binding ability, our work may appear as pilot investigation for assessing their mechanism of action.

  8. Proposed mechanisms whereby the T3U, dialyzed fraction (%FT4) and antibody extracted (AE) T4 normalize binding protein effects in free T4 (FT4) assays do not explain the findings in monthyroidally ill (NTI) patients

    SciTech Connect

    Witherspoon, L.R.; Shuler, S.E.; Gilbert, S.

    1984-01-01

    Apparently falsely low FT4 results are observed in patients with NTI. FT4 estimates in NTI using some AE assays are lower than corresponding FTI. Both are lower than equilibrium dialysis (ED) results. These three approaches to FT4 estimation are similar - the T3U, %FT4 and %AE all are inversely related to TBG concentration. A FT4 estimate may be obtained by multiplying any of these x total T4 concentration. The mass of AE T4 may be quantitated by competitive binding, either after separation of AE T4 from serum (two step) or by employing a radiolabeled T4 analog recognized by the antibody but not by TBG. The authors made FT4 estimates in euthyroid patients with high, normal or low TBG concentrations; in hyper- and hypothyroid patients; and in NTI patients presumed euthyroid. Each approach produces similar results in euthyroid patients. The T3U and some AE assays display TBG dependence at TBG <10 and>50 ..mu..g/ml. In hyperthyroidism, the T3U is high, %FT4 normal or high, while the %AE is low. All FT4 results are high in hyperthyroidism. In hypothyroidism the T3U is low, %FT4 normal or low, while the %AE is high. All FT4 results are low in hypothyroidism. In NTI, the T3U is appropriate for the TBG concentration, %FT4 is often high and %AE is low. Furthermore, methods for quantifying the AE mass do not agree - index results are low, analog results lowers, such that %B/B is>100%, while the two step results are normal. The suggestion that the T3U, %FT4 and %AE all serve to normalize TBG effects in FT4 assays is inadequate. AE assays should be employed clinically with caution until the apparent anomalies, especially those seen in the analog systems, are explained.

  9. Novel agonists of benzodiazepine receptors: design, synthesis, binding assay and pharmacological evaluation of 1,2,4-triazolo[1,5-a]pyrimidinone and 3-amino-1,2,4-triazole derivatives.

    PubMed

    Faizi, Mehrdad; Dabirian, Sara; Tajali, Hamed; Ahmadi, Fatemeh; Zavareh, Elham Rezaee; Shahhosseini, Soraya; Tabatabai, Sayyed Abbas

    2015-02-01

    Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical practice. In spite of their benefits they have several side effects, so synthesis of new agonists of these receptors to get more specific effect and better profile of adverse drug reactions is still continued. Novel BZD agonists were designed based on the pharmacophore/receptor model of BZD binding site of GABAA receptor. Energy minima conformers of the designed compounds and estazolam, a known BZD receptor agonist, were well superimposed in conformational analysis. Docking studies revealed that the carbonyl group of the compound 4c, 3-(2-chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, was near the nitrogen moiety of triazole ring of estazolam providing the hydrogen bond acceptor in proper direction in the BDZ-binding site of GABAA receptor model (α1β2ϒ2). The designed compounds were synthesized and their in vitro affinity for the central BZD receptor was determined. Most of the novel compounds had better affinity for the BZD site of action on GABAA receptor complex than diazepam. Finally, the novel compound 4c with the best affinity in radioligand receptor binding assay (Ki=0.42 nM and IC50=0.68 nM) was selected as candidate for in vivo evaluation. This compound showed significant hypnotic activity and weak anticonvulsant effect with no impairment on learning and memory performance in mouse. The pharmacological effects of the compound 4c were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects of the novel ligand.

  10. Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice.

    PubMed

    Wiley, Jenny L; Lefever, Timothy W; Marusich, Julie A; Grabenauer, Megan; Moore, Katherine N; Huffman, John W; Thomas, Brian F

    2016-11-01

    Anecdotal reports suggest that abused synthetic cannabinoids produce cannabis-like "highs," but some of their effects may also differ from traditional cannabinoids such as Δ(9)-tetrahydrocannabinol (THC). This study examined the binding affinities of first-generation indole-derived synthetic cannabinoids at cannabinoid and noncannabinoid receptors and their effects in a functional observational battery (FOB) and drug discrimination in mice. All seven compounds, except JWH-391, had favorable affinity (≤159 nM) for both cannabinoid receptors. In contrast, binding at noncannabinoid receptors was absent or weak. In the FOB, THC and the six active compounds disrupted behaviors in CNS activation and muscle tone/equilibrium domains. Unlike THC, however, synthetic cannabinoids impaired behavior across a wider dose and domain range, producing autonomic effects and signs of CNS excitability and sensorimotor reactivity. In addition, mice acquired JWH-018 discrimination, and THC and JWH-073 produced full substitution whereas the 5-HT2B antagonist mianserin did not substitute in mice trained to discriminate JWH-018 or THC. Urinary metabolite analysis showed that the compounds were extensively metabolized, with metabolites that could contribute to their in vivo effects. Together, these results show that, while first-generation synthetic cannabinoids shared some effects that were similar to those of THC, they also possessed effects that differed from traditional cannabinoids. The high nanomolar (or absent) affinities of these compounds at receptors for most major neurotransmitters suggests that these divergent effects may be related to the greater potencies and/or efficacies at CB1 receptors; however, action(s) at noncannabinoid receptors yet to be assessed or via different signaling pathways cannot be ruled out.

  11. Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals.

    PubMed

    Escher, Beate I; Cowan-Ellsberry, Christina E; Dyer, Scott; Embry, Michelle R; Erhardt, Susan; Halder, Marlies; Kwon, Jung-Hwan; Johanning, Karla; Oosterwijk, Mattheus T T; Rutishauser, Sibylle; Segner, Helmut; Nichols, John

    2011-07-18

    Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values

  12. Topoisomerase Assays

    PubMed Central

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

    2012-01-01

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

  13. Measurement of IgG, IgA and IgE antibodies to Dermatophagoides pteronyssinus by antigen-binding assay, using a partially purified fraction of mite extract (F4P1).

    PubMed Central

    Chapman, M D; Platts-Mills, T A

    1978-01-01

    An extract of Dermatophagoides pteronyssinus culture has been fractionated by chromatography on Sephadex G-100 and Pevikon block electrophoresis to obtain a partially purified allergen (F4P1). This preparation has a molecular weight of between 15--25,000 Dalton, migrates slowly on electrophoresis, and is colourless in solution. The skin-test reactivity of F1P1 was comparable to that of crude D. pteronyssinus extract. F4P1 was radio-labelled with 125I and used in an antigen-binding radioimmunoassay to measure IgG, IgA and IgE antibody (ab) to D. pteronyssinus. IgG, ab was detected in serum from 32/34 (94%) mite-allergic persons, and from 10/31 (30%) nonallergic persons. IgA ab and IgE ab were found in sera from 22/34 (65%) and 37/34 (79%) allergic persons respectively. Neither IgA nor IgE ab could be detected in sera from non-allergic persons. An excellent correlation was found between radioallergo-sorbent technique (RAST), using crude D. pteronyssinus extract and IgE-binding activity (BA) for F4P1, (r=0.94, P less than 0.001). The antigen-binding assay for IgE BA was as sensitive as RAST, but less sensitive than PK testing. There was a very good quantitative correlation between IgG BA and IgE BA (r = 0.84, P less than 0.001). IgG BA was shown to rise in the serum of three patients treated with injections of D. pteronyssinus extract. PMID:750116

  14. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  15. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  16. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  17. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

    PubMed Central

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

  18. Fluoresent Binding Assays for Antigen Detection.

    DTIC Science & Technology

    1983-09-09

    Raman scattering. Light is elastically scattered in solution; in the case of aqueous solutions, Raman scattering occurs approx- imately 300 wave numbers...on either side of the exciting light. The Stokes shift of many fluorescent markers is approximately 300 wave numbers, and thus, Raman scattering is...registered by the photomultiplier using the excita- tion and emission filters for the fluorescent marker. Raman scattering can be minimized by using

  19. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  20. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  1. Ureaplasma urealyticum binds mannose-binding lectin.

    PubMed

    Benstein, Barbara D; Ourth, Donald D; Crouse, Dennis T; Shanklin, D Radford

    2004-10-01

    Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.

  2. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

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

  3. (/sup 3/H)Batrachotoxinin A 20 alpha-benzoate binding to voltage-sensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs

    SciTech Connect

    McNeal, E.T.; Lewandowski, G.A.; Daly, J.W.; Creveling, C.R.

    1985-03-01

    (/sup 3/H)Batrachotoxinin A benzoate ((/sup 3/H)BTX-B) binds with high affinity to sites on voltage-dependent sodium channels in a vesicular preparation from guinea pig cerebral cortex. In this preparation, local anesthetics competitively antagonize the binding of (/sup 3/H)BTX-B. The potencies of some 40 classical local anesthetics and a variety of catecholamine, histamine, serotonin, adenosine, GABA, glycine, acetylcholine, and calcium antagonists, tranquilizers, antidepressants, barbiturates, anticonvulsants, steroids, vasodilators, antiinflammatories, anticoagulants, analgesics, and other agents have been determined. An excellent correlation with the known local anesthetic activity of many of these agents indicate that antagonism of binding of (/sup 3/H)BTX-B binding provides a rapid, quantitative, and facile method for the screening and investigation of local anesthetic activity.

  4. Combined application of a laser ablation-ICP-MS assay for screening and ESI-FTICR-MS for identification of a Cd-binding protein in Spinacia oleracea L. after exposure to Cd.

    PubMed

    Polatajko, Aleksandra; Feldmann, Ingo; Hayen, Heiko; Jakubowski, Norbert

    2011-10-01

    We have studied the binding of the toxic element Cd to plant proteins and have used for this purpose spinach (Spinacia oleracea L.) plants treated with 50 μM Cd(II) as a model system. Laser ablation ICP-MS has been applied for the screening of Cd-binding proteins after separation by native anodal polyacrylamide gel electrophoresis (AN-PAGE) and electroblotting onto membranes. The main Cd-carrying protein band was isolated and investigated by nano-electrospray ionization-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry after tryptic digestion. By this procedure, the main Cd-binding protein was identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The latter enzyme has been discussed in the literature to be affected in its activity by oxidative stress induced by Cd. However, in this paper it is demonstrated for the first time that RuBisCO directly binds Cd and thus may be directly altered by this toxic element. A commercially available protein standard was used to verify direct binding of Cd(II) to the protein, even without metabolisation. The resulting metal-protein complex was shown to be stable enough to survive AN-PAGE separation and electroblotting. By the use of size exclusion chromatography coupled with ICP-MS it was demonstrated that the RuBisCO protein standard shows similar metal binding properties to Cd. Furthermore, essential elements such as Mn(II), Fe(II) and Cu(II), which are known to possibly replace the RuBisCO activator Mg(II), were investigated in addition to Zn(II). Again, similar binding properties in comparison to the plant protein were observed.

  5. FCA does not bind abscisic acid.

    PubMed

    Risk, Joanna M; Macknight, Richard C; Day, Catherine L

    2008-12-11

    The RNA-binding protein FCA promotes flowering in Arabidopsis. Razem et al. reported that FCA is also a receptor for the phytohormone abscisic acid (ABA). However, we find that FCA does not bind ABA, suggesting that the quality of the proteins assayed and the sensitivity of the ABA-binding assay have led Razem et al. to erroneous conclusions. Because similar assays have been used to characterize other ABA receptors, our results indicate that the ABA-binding properties of these proteins should be carefully re-evaluated and that alternative ABA receptors are likely to be discovered.

  6. ATP sulfurylase-dependent assays for inorganic pyrophosphate: applications to determining the equilibrium constant and reverse direction kinetics of the pyrophosphatase reaction, magnesium binding to orthophosphate, and unknown concentrations of pyrophosphate.

    PubMed

    Daley, L A; Renosto, F; Segel, I H

    1986-09-01

    A continuous, coupled, spectrophotometric assay is described in which the enzyme ATP sulfurylase is employed to measure the concentration of inorganic pyrophosphate (PPi) at equilibrium with known concentrations of inorganic orthophosphate (Pi) in the presence of excess inorganic pyrophosphatase (PPitase). In agreement with previous reports, the apparent equilibrium constant (Keq,app) of the PPi hydrolysis reaction was shown to decrease as the concentration of Mg2+ is increased. At pH 7.3, 30 degrees C, in the presence of 150 mM NaCl and 1 mM free Mg2+, Keq,app (calculated as [Pi]t2/[PPi]t) was 1950. Measurements of Keq,app at different total concentrations of Mg2+ and Pi permitted the determination of K0, the dissociation constant of the Mg-Pi complex. In 0.05 M Tris-Cl, pH 8.0, at 30 degrees C, K0 was 3.6 mM. In the presence of excess ATP sulfurylase, yeast PPitase catalyzed PPi formation from Pi with a specific activity (Vmax) of 9 units X mg protein-1 at pH 8.0, 30 degrees C, and 1 mM free Mg2+. Half-maximum reverse reaction velocity was observed at a total Pi concentration of 18 mM. (Under the same conditions, Vmax of the PPi hydrolysis reaction was 530 units X mg protein-1.) A radiochemical end point ("reaction-to-completion") assay for measuring unknown concentrations of PPi was devised. In the presence of excess 35S-adenosine-5'-phosphosulfate ([35S]APS) as the cosubstrate, 35SO2-4 formation was stoichiometric with added PPi. (The 35SO2-4 and [35S]APS are separated by adsorption of the latter onto charcoal.) The sensitivity of the assay can be adjusted by varying the specific radioactivity of the [35S]APS. In the absence of interfering substances, as little as 2 pmol of PPi per 1.0 ml assay volume can be measured. The sensitivity of the assay is reduced in the presence of ATP plus perchlorate (which synergistically inhibit the enzyme). However, if the bulk of the ATP is removed from perchloric acid extracts of tissues with glucose and hexokinase, initial

  7. Comparison of fluorigenic peptide substrates PL50, SNAPTide, and BoTest A/E for BoNT/A detection and quantification: exosite binding confers high-assay sensitivity.

    PubMed

    Ouimet, Tanja; Duquesnoy, Sophie; Poras, Hervé; Fournié-Zaluski, Marie-Claude; Roques, Bernard P

    2013-07-01

    Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

  8. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  9. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  10. C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

    PubMed

    Hiser, C; Mills, D A; Schall, M; Ferguson-Miller, S

    2001-02-13

    To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

  11. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin–DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody

    PubMed Central

    Dou, Shuping; Virostko, John; Greiner, Dale L.; Powers, Alvin C.; Liu, Guozheng

    2016-01-01

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ~95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  12. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

    PubMed

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-08-03

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  13. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  14. Binding Procurement

    NASA Technical Reports Server (NTRS)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  15. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  16. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  17. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

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

  18. Rotor assembly and assay method

    DOEpatents

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

    1993-09-07

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

  19. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

  20. Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.

    PubMed

    Chennathukuzhi, V M; Kurihara, Y; Bray, J D; Yang, J; Hecht, N B

    2001-11-01

    The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

  1. Parallel Force Assay for Protein-Protein Interactions

    PubMed Central

    Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

  2. Absolute nuclear material assay

    DOEpatents

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

    2012-05-15

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

  3. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

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

  4. Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity† †Electronic supplementary information (ESI) available: NMR chemical shift characterisation of modified heparins, protein sequence alignment methodology and data, protein binding and activity assay dose-response curves. See DOI: 10.1039/c6mb00432f Click here for additional data file.

    PubMed Central

    Ahmed, Yassir A.; Yates, Edwin A.; Moss, Diana J.; Loeven, Markus A.; Hussain, Sadaf-Ahmahni; Hohenester, Erhard; Turnbull, Jeremy E.

    2016-01-01

    Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit–Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo. PMID:27502551

  5. Assays of thyroid-stimulating antibody

    SciTech Connect

    McKenzie, J.M.; Zakarija, M.

    1985-01-01

    A comparison is presented of the two major assay methods of thyroid-stimulating antibody (TSAb) of Graves' disease. The basic procedures involve: (1) some index of thyroid stimulation, usually in vitro, using TSAb to indicate its activity; and (2) indirect recognition by assessment of the inhibition of binding of radioiodinated thyrotropin (TSH) to a preparation of its receptor, i.e., TSH-binding inhibition or TBI. There is potential for misinterpretation of data acquired by testing patients' sera by one or the other basic procedure.

  6. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. Results Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite

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

    PubMed

    Schultis, T; Metzger, J W

    2004-12-01

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

  8. Methods and devices for protein assays

    DOEpatents

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  9. Polymeric assay film for direct colorimetric detection

    DOEpatents

    Charych, Deborah; Nagy, Jon; Spevak, Wayne

    2002-01-01

    A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

  10. Polymeric assay film for direct colorimetric detection

    DOEpatents

    Charych, Deborah; Nagy, Jon; Spevak, Wayne

    1999-01-01

    A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

  11. Production and assay of forskolin antibodies

    SciTech Connect

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

    1986-05-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  12. Protein-binding assays in biological liquids using microscale thermophoresis.

    PubMed

    Wienken, Christoph J; Baaske, Philipp; Rothbauer, Ulrich; Braun, Dieter; Duhr, Stefan

    2010-10-19

    Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.

  13. An assay for intermolecular exchange of alpha crystallin

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    An affinity column of alpha crystallin linked to cyanogen bromide-activated Sepharose was developed to study the exchange of alpha subunits. Alpha crystallin bound to the Sepharose-alpha complex was dissociated with 8 mol/l urea, followed by quantitation using high-performance reverse-phase liquid chromatography. The time course of binding at 37 degrees C showed a hyperbolic binding pattern reaching equilibrium between 6-18 hr. Under these conditions, binding of beta and gamma crystallins to the same matrix was less than 10% of the alpha values, as was binding of alpha to glycine-coupled Sepharose. This assay was used to demonstrate changes in the subunit exchange of alpha crystallins present in high molecular weight versus lower molecular weight aggregates of the human lens. These results show that this binding procedure was a specific reproducible assay that might be used to study intermolecular interactions of the alpha crystallins.

  14. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  15. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  16. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

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

  17. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  18. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  19. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  20. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

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

    NASA Astrophysics Data System (ADS)

    Kidwell, David A.

    1993-05-01

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

  2. Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

    PubMed

    Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

    2015-02-01

    One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

  3. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  4. Galectin-3-Binding and Metastasis

    PubMed Central

    Nangia-Makker, Pratima; Balan, Vitaly; Raz, Avraham

    2013-01-01

    i. Summary Galectin-3 is a member of a family of carbohydrate-binding proteins. It is present in the nucleus, the cytoplasm and also extracellular matrix of many normal and neoplastic cell types. Arrays of reports show an upregulation of this protein in transformed and metastatic cell lines (1, 2). Moreover, in many human carcinomas, an increased expression of galectin-3 correlates with progressive tumor stages (3–6). Several lines of analysis have demonstrated that the galectins participate in cell-cell and cell-matrix interactions by recognizing and binding complimentary glycoconjugates and thereby play a crucial role in normal and pathological processes. Elevated expression of the protein is associated with an increased capacity for anchorage-independent growth, homotypic aggregation, and tumor cell lung colonization (7–9). In this chapter we describe the methods of purification of galectin-3 from transformed E. coli and some of the commonly used functional assays for analyzing galectin-3 binding. PMID:22674139

  5. Rover waste assay system

    SciTech Connect

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

    1997-11-01

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

  6. Clonogenic Assay: Adherent Cells

    PubMed Central

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

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  7. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  8. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  9. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  10. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R; Benett, William J; Coleman, Matthew A; Pearson, Francesca S; Nasarabadi, Shanavaz L

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  11. Is there a link between selectivity and binding thermodynamics profiles?

    PubMed

    Tarcsay, Ákos; Keserű, György M

    2015-01-01

    Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding.

  12. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  13. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  14. Analytical applications of MIPs in diagnostic assays: future perspectives.

    PubMed

    Bedwell, Thomas S; Whitcombe, Michael J

    2016-03-01

    Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed-with a focus on work conducted from 2005 to date.

  15. A modified fluorescent intercalator displacement assay for RNA ligand discovery

    PubMed Central

    Asare-Okai, Papa Nii; Chow, Christine S.

    2010-01-01

    Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic-acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not have to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR, h31, and H69) were identified. PMID:20863807

  16. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

    PubMed Central

    Abo, Hirohito; Soga, Keisuke; Tanaka, Atsuhiro; Tateno, Hiroaki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units. PMID:26714191

  17. The prion protein binds thiamine.

    PubMed

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.

  18. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  19. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  20. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  1. Radioreceptor assay analysis of tamsulosin and terazosin pharmacokinetics

    PubMed Central

    Taguchi, Katsunari; Schäfers, Rafael F; Michel, Martin C

    1998-01-01

    Aims A radioreceptor assay has been developed for α1-adrenoceptor subtypes and applied to a pharmacokinetic analysis of tamsulosin and terazosin. Methods Young, male, healthy volunteers received 0.4 mg tamsulosin (as Omnic® modified release capsules) or 5 mg terazosin (as Flotrin® tablets) in a randomized, cross-over design. Before and after 1, 3, 5, 7, 10 and 23.5 h plasma was analyzed by radioreceptor assay using cloned human α1A-, α1B- and α1D-adrenoceptors and specific h.p.l.c. analysis. Results Following ingestion of tamsulosin median peak plasma levels of 16 ng ml−1 were reached after 5 h and declined to 2 ng ml−1 at 23.5 h. The time course in the radioreceptor assay was similar, and at most time points binding to α1A-adrenoceptors was significantly greater than to α1B- and α1D-adrenoceptors. Following ingestion of terazosin median peak plasma levels of 91 ng ml−1 were reached after 1 h and declined to 11 ng ml−1 at 23.5 h. In the radioreceptor assay binding also peaked at 1 h and declined thereafter, but even after 23.5 h considerable binding activity remained detectable at all three subtypes. At most time points binding to the α1A- and α1D-adrenoceptor was significantly greater than to the α1B-adrenoceptor. Conclusions We conclude that α1-adrenoceptor antagonist pharmacokinetics can be monitored by radioreceptor assays in a subtype-selective manner. Tamsulosin and terazosin exhibit subtype selective receptor binding ex vivo. The discordance between terazosin blood levels as determined by h.p.l.c. and radioreceptor assay at late time points indicates the possible involvement of metabolites in in vivo terazosin effects. PMID:9489594

  2. Implementation of a radioreceptor assay for dexmedetomidine.

    PubMed

    Salonen, M; Maze, M

    1993-11-01

    We have implemented a radioreceptor assay for dexmedetomidine, a novel alpha 2-adrenoceptor agonist. Receptor-bearing membranes were prepared from rat cerebral cortex and 3H-clonidine, 4 nM, was used as the labeled ligand. Dexmedetomidine displaced 3H-clonidine in a linear fashion over a concentration of 2 x 10(-10) to 2 x 10(-8)M. The detection limit of dexmedetomidine (i.e. 10% of radiolabeled ligand displaced) in this assay was 50 pg.ml-1 which is comparable to that seen with the reference method which utilizes gas chromotography with mass spectrometer (GC/MS) in series (Vuorilehto et al. 1989). Endogenous catecholamines, which can displace the radiolabeled ligand from its binding site, could easily be eliminated with a one-step extraction procedure. A comparison was made with the reference method (GC/MS) in 47 human plasma samples; the correlation coefficient (r2) was 0.61 (P < 0.001). The radioreceptor assay was also successfully applied for determining dexmedetomidine concentration in rabbit samples. These data indicate that the radioreceptor assay can be utilized for characterizing the pharmacokinetics of novel alpha 2 agonists which are now being introduced into the clinical practice of anaesthesia.

  3. Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes

    SciTech Connect

    Hansen, S.I.; Holm, J.; Nexo, E.

    1987-08-01

    Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L.

  4. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

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

  5. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  6. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

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

  7. Aminopyralid binds more tightly to soil than clopyralid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laboratory studies were conducted to compare the soil adsorption of aminopyralid and clopyralid using batch slurry and centrifugation assays. The calculated soil binding constants for both herbicides varied between the two techniques, but the centrifugation assay had a lower coefficient of variati...

  8. Capture-stabilize approach for membrane protein SPR assays.

    PubMed

    Chu, Ruiyin; Reczek, David; Brondyk, William

    2014-12-08

    Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.

  9. A novel and sensitive radioreceptor assay for serum melatonin levels

    SciTech Connect

    Tenn, C.; Niles, L. )

    1991-01-01

    A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-({sup 125}I)iodomelatonin (({sup 125}I)MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally related compounds. The sensitivity of the assay is {approximately}1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.

  10. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  11. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

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

  12. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  13. Assays for mechanistic investigations of protein/histone acetyltransferases.

    PubMed

    Berndsen, Christopher E; Denu, John M

    2005-08-01

    Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

  14. Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents.

    PubMed

    Staack, Roland F; Stracke, Jan O; Stubenrauch, Kay; Vogel, Rudolf; Schleypen, Julia; Papadimitriou, Apollon

    2011-03-01

    Ligand-binding assays are the standard technology used for bioanalysis of therapeutic proteins, for example, for drug quantification (pharmacokinetics assays) and immunogenicity testing (antidrug antibody assays). Besides the selection of the most suitable technology platform (e.g., ELISA, electrochemiluminescence assays and surface plasmon resonance assays) and assay procedure, a pivotal prerequisite for good assay performance on any technology platform is the design, production and characterization of high quality reagents. To enable bioanalytical project support over the complete product life cycle, an appropriate long-term reagent supply is needed. This perspective describes our opinion on the requirements for generation and QC of critical reagents used in ligand-binding assays for drug quantification and antidrug antibody detection to enable high-quality assays and long-term supply, including reagent batch switches. The critical parameters during reagent design, production and long-term supply, along with the appropriate analytical methods for QC testing and appropriate certification, are discussed.

  15. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  16. Radioreceptor assay of narcotic analgesics in serum.

    PubMed

    Grevel, J; Thomas, J; Richards, M L; Sadée, W

    1984-09-01

    A sensitive radioreceptor assay (RRA) to determine the serum concentrations of fentanyl, pentazocine and morphine was developed on the basis of the drug's competition with a labeled tracer ((3)H-naloxone) for the membrane bound opioid receptor in rat brain homogenates. The binding data were computer-fitted to a standard curve by means of nonlinear least square regression. Sensitivity of the assay applied directly to serum samples without extraction was limited to approximately 3, 5 and 25 ng/ml for fentanyl, morphine and pentazocine, respectively, because of endogenous plasma constituents that interfere with the opioid receptor binding. With the use of petrol-ether extraction the sensitivity was improved to 0.3 ng/ml fentanyl and 3 ng/ml pentazocine (0.3 ml serum samples). No RRA-active metabolites were detectable after HPLC separation of serum from a patient treated with fentanyl. The plasma concentration time course of fentanyl in a patient, measured by RRA, was similar to that obtained by a radioimmunoassay (RIA). The RRA represents a general procedure for the detection of clinically used opioid analgesics and their active metabolites.

  17. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  18. Survival assays using Caenorhabditis elegans

    PubMed Central

    Park, Hae-Eun H.; Jung, Yoonji; Lee, Seung-Jae V.

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

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

    SciTech Connect

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

    2014-09-01

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

  20. Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

    EPA Science Inventory

    We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

  1. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  2. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  3. Rho family and Rap GTPase activation assays.

    PubMed

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

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

  4. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  5. Kinetic viability assays using DRAQ7 probe.

    PubMed

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

    2013-07-01

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

  6. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  7. Magnesium ion is an effective inhibitor of the binding of Escherichia coli to heparin

    PubMed Central

    Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G.

    2007-01-01

    We investigated the effects of ions and temperature on the binding of E. coli to heparin using a chemiluminescence electrophoretic mobility shift assay. We found that magnesium ion is an effective inhibitor of the binding. The method can be readily applied to discover agents that can block the binding. PMID:17967492

  8. Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    SciTech Connect

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

    1982-06-01

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

  9. Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    SciTech Connect

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

    1982-06-01

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

  10. Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma

    SciTech Connect

    Cumin, F.; de Gasparo, M.; Wood, J.M.; Schnell, C.; Frueh, F.; Graf, P. )

    1989-10-01

    A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r = 0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 microM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with CGP 38 560 A (50 micrograms/kg). Similar values for the concentrations of renin inhibitor in plasma were obtained with the radioinhibitor binding assay and the enzyme inhibitor assay, and there was a significant correlation between values obtained with the two different methodologies (r = 0.94). The plasma levels of renin inhibitor reached a maximum at the end of infusion and then decreased rapidly, indicating a short plasma half-life. The changes in biochemical parameters, plasma renin activity, and plasma concentration of active renin could be related to the concentrations of CGP 38 560 measured in the plasma.

  11. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  12. The binding interactions of imidacloprid with earthworm fibrinolytic enzyme

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Qing; Zhang, Hong-Mei; Chen, Tao

    2014-08-01

    In this paper, several studies were conducted to elucidate the binding mechanism of earthworm fibrinolytic enzyme (EFE) with imidocloprid (IMI) by using theoretical calculation, fluorescence, UV-vis, circular dichroism spectroscopy and an enzymatic inhibition assay. The spectral data showed that the binding interactions existed between IMI and EFE. The binding constants, binding site, thermodynamic parameters and binding forces were analyzed in detail. The results indicate a single class of binding sites for IMI in EFE and that this binding interaction is a spontaneous process with the estimated enthalpy and entropy changes being 2.195 kJ mol-1 and 94.480 J mol-1 K-1, respectively. A single class of binding site existed for IMI in EFE. The tertiary or secondary structure of EFE was partly destroyed by IMI. The visualized binding details were also exhibited by the theoretical calculation and the results indicated that the interaction between IMI and Phe (Tyr, or Trp) or EFE occurred. Combining the experimental data with the theoretical calculation data, we showed that the binding forces between IMI and EFE were mainly hydrophobic force accompanied by hydrogen binding, and π-π stacking. In addition, IMI did not obviously influence the activity of EFE. In a word, the above analysis offered insights into the binding mechanism of IMI with EFE and could provide some important information for the molecular toxicity of IMI for earthworms.

  13. A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses

    PubMed Central

    Langereis, Eveline J.; Wagemans, Tom; Kulik, Wim; Lefeber, Dirk J.; van Lenthe, Henk; Oussoren, Esmee; van der Ploeg, Ans T.; Ruijter, George J.; Wevers, Ron A.; Wijburg, Frits A.; van Vlies, Naomi

    2015-01-01

    Introduction Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III). Methods Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs. Results The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them. Conclusion The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay. PMID:26406883

  14. Scatter factor binds to thrombospondin and other extracellular matrix components.

    PubMed Central

    Lamszus, K.; Joseph, A.; Jin, L.; Yao, Y.; Chowdhury, S.; Fuchs, A.; Polverini, P. J.; Goldberg, I. D.; Rosen, E. M.

    1996-01-01

    Scatter factor (SF) is an angiogenic growth factor that stimulates motility and invasion of carcinoma cells. SF is present in the extracellular matrix (ECM) of breast cancers, where it might act to promote tumor cell invasion and angiogenesis. To investigate how SF is incorporated into the ECM, we studied the binding of SF to various ECM components using a solid-phase binding assay based on the SF enzyme-linked immunosorbent assay. We found that SF binds to a variety of ECM molecules, with different binding capacities. The highest SF binding capacities were observed for thrombospondin-1 (TSP-1), fibronectin (Fn), and heparan sulfate proteoglycan, although SF did not bind to albumin. Mature two-chain SF and precursor single-chain SF bound approximately equally well to TSP-1 and Fn. Moreover, two SF alpha-chain peptides (NK1 and NK2) both bound to TSP-1 and Fn, suggesting that the whole SF molecule is not required for binding. Based on binding competition assays, TSP-1 exhibited higher affinity for SF than did nine other ECM molecules, including Fn and heparan sulfate proteoglycan. Although heparin in solution potently inhibited the binding of SF to TSP-1-coated surfaces, even very high concentrations of heparin could not elute SF already bound to TSP-1. SF binding was modulated by binding interactions among ECM molecules (TSP-1-Fn, TSP-1-collagen I, and Fn-collagen I), suggesting that the matrix capacity to bind SF depends upon its exact composition. SF bound in a dose-dependent fashion to ECMs secreted by three human breast carcinoma cell lines. Binding of SF to matrices from all three cell lines was significantly inhibited by preincubation of the matrices with antibodies against TSP-1, whereas antibodies against several other ECM components were less effective or ineffective in inhibiting SF binding. In addition, TSP-1 markedly inhibited chemotaxis of microvascular endothelial cells toward SF and SF-induced angiogenesis in the rat cornea neovascularization assay

  15. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  16. Assaying environmental nickel toxicity using model nematodes

    USGS Publications Warehouse

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

    2013-01-01

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

  17. Assaying environmental nickel toxicity using model nematodes.

    PubMed

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

    2013-01-01

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

  18. Assaying Environmental Nickel Toxicity Using Model Nematodes

    PubMed Central

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

    2013-01-01

    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species. PMID:24116204

  19. In vitro peptidoglycan synthesis assay with lipid II substrate.

    PubMed

    Biboy, Jacob; Bui, Nhat Khai; Vollmer, Waldemar

    2013-01-01

    Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these reactions. Here, we describe an in vitro peptidoglycan synthesis assay utilizing radiolabeled lipid II substrate to monitor simultaneously peptidoglycan glycosyltransferase and transpeptidase activities. The products of the reaction are separated by high-pressure liquid chromatography and quantified by flow-through scintillation counting.

  20. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  1. First 25-hydroxyvitamin D assay for general chemistry analyzers.

    PubMed

    Saida, Fakhri B; Chen, Xiaoru; Tran, Kiet; Dou, Chao; Yuan, Chong

    2015-03-01

    25-Hydroxyvitamin D [25(OH)D], the predominant circulating form of vitamin D, is an accurate indicator of the general vitamin D status of an individual. Because vitamin D deficiencies have been linked to several pathologies (including osteoporosis and rickets), accurate monitoring of 25(OH)D levels is becoming increasingly important in clinical settings. Current 25(OH)D assays are either chromatographic or immunoassay-based assays. These assays include HPLC, liquid chromatography-tandem mass spectrometry (LC-MS/MS), enzyme-immunosorbent, immunochemiluminescence, immunofluorescence and radioimmunoassay. All these assays use heterogeneous formats that require phase separation and special instrumentations. In this article, we present an overview of these assays and introduce the first homogeneous assay of 25(OH)D for use on general chemistry analyzers. A special emphasis is put on the unique challenges posed by the 25(OH)D analyte. These challenges include a low detection limit, the dissociation of the analyte from its serum transporter and the inactivation of various binding proteins without phase separation steps.

  2. Two different approaches for developing immunometric assays of haptens.

    PubMed

    Grassi, J; Créminon, C; Frobert, Y; Etienne, E; Ezan, E; Volland, H; Pradelles, P

    1996-09-01

    To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

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

    PubMed

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

    2011-08-01

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

  4. A homologous radioreceptor assay for rat follicle-stimulating hormone

    SciTech Connect

    Findley, W.E.; Steinberger, A.

    1983-09-01

    A homologous radioreceptor assay (RRA) for rat FSH (rFSH), which is both sensitive and easy to perform, is described. The receptor preparation is isolated from a 12,000 X G pellet of rat testes homogenate prepared with a high speed tissue grinder using Tris buffer. The sensitivity of the assay extends below 10 ng rFSH RP-1 (approximately 0.13 ng purified I-3 rat FSH), and the range of the assay spans 2 orders of magnitude. The specific binding of the (/sup 125/I)rFSH tracer is 22-28% of the total tracer added. Such binding, which exceeds previously published values by 2- to 4-fold, allows the addition of relatively small amounts of total tracer radioactivity, which, in turn, contributes to a low nonspecific binding and highly reproducible values for replicates (coefficient of variation, approximately 3.0%). This represents the first homologous RRA for rFSH using testicular receptors. Likewise, the sensitivity and reproducibility exceed those of previous RRAs for rFSH.

  5. A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

    PubMed

    Robertson, Neil O; Shah, Manan; Matthews, Jacqueline M

    2016-10-10

    We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10(-9) -10(-5)  m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.

  6. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  7. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

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

    PubMed

    Morton, R E; Greene, D J

    1994-11-01

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

  9. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    PubMed

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander

    2013-10-18

    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.

  10. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  11. Partial characterization of GTP-binding proteins in Neurospora

    SciTech Connect

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-08-14

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. (/sup 35/S)GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of (/sup 35/S)GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.

  12. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  13. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats.

    PubMed

    Peel, Alison J; McKinley, Trevelyan J; Baker, Kate S; Barr, Jennifer A; Crameri, Gary; Hayman, David T S; Feng, Yan-Ru; Broder, Christopher C; Wang, Lin-Fa; Cunningham, Andrew A; Wood, James L N

    2013-11-01

    Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or 'cutoff', for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of 'negative' control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

  14. The systematic investigation and development of the histamine radioenzymatic assay

    SciTech Connect

    Verburg, K.M.

    1984-01-01

    The radioenzymatic assay for histamine is a widely used analytical procedure based on the enzymatic conversion of histamine to ({sup 3}H)tele-methylhistamine utilizing histamine N-methyltransferase (HNMT) and S-adenosyl-L-({sup 3}H-methyl) methionine (({sup 3}H)SAME). Despite numerous modifications of this method, the assay lacks the sensitivity and specificity required to quantify histamine from many important biologic samples such as human plasma. The objective of this study was to investigate systematically the radioenzymatic assay for histamine and develop a highly sensitive and specific assay for use in basic or clinical studies. HNMT was purified 260-fold from rat kidney and the use of purified HNMT in the histamine radioenzymatic assay improved specificity of this method and also improved sensitivity by eliminating the enzyme-dependent blank and permitting the inclusion of high specific activity ({sup 3}H)SAME. The adsorption of histamine to glass surfaces was characterized and strategies were developed to prevent binding. Finally, optimization of the reaction allowed the development of a simplified product isolation procedure. The histamine radioenzymatic assay developed in this study has a sensitivity of 2.0 pg and is specific for histamine as judged by direct product identification and cross-contamination studies. The assay was utilized to establish reference values for the concentration of histamine in human plasma and the 24-hour urinary excretion of histamine for normal human subjects. In summary, a sensitive and specific radioenzymatic assay for histamine was developed as a result of the systematic investigation of this methodology.

  15. Ocelot and oncilla spermatozoa can bind hen egg perivitelline membranes.

    PubMed

    de Araujo, Gediendson Ribeiro; de Paula, Tarcizio Antônio Rego; Deco-Souza, Thyara de; Garay, Rafael de Morais; Letícia Bergo, C F; Csermak-Júnior, Antônio Carlos; da Silva, Leanes Cruz; Alves, Saullo Vinícius Pereira

    2015-12-01

    We evaluated the capacity of ocelot and oncilla spermatozoa to bind to the perivitelline membranes (PVMs) of hen eggs in a sperm binding assay (S-PVM). In addition, a device that improves the standardization of the assay was developed. The number of sperm bound to the PVM in fresh (T1) and frozen-thawed (T2) semen from both species was compared to the sperm quality observed in routine tests. The PVM was stretched on a circular silicone device to create a standardized area for analysis. In both treatments and for both species, the spermatozoa were able to bind to the PVM, indicating that PVM may be used for a sperm binding assay in ocelot and oncilla. The S-PVM assay did not differ in fresh and frozen-thawed ocelot sperm (p>0.05). However, fewer oncilla sperm (p<0.05) were bound to the PVM in T2, indicating that the proposed test may be able to detect injuries that compromise sperm binding abilities. The device maintained the PVM stretched during the processing and defined the evaluation area.

  16. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  17. Characterization of the Binding Properties of Molecularly Imprinted Polymers.

    PubMed

    Ansell, Richard J

    2015-01-01

    The defining characteristic of the binding sites of any particular molecularly imprinted material is heterogeneity: that is, they are not all identical. Nonetheless, it is useful to study their fundamental binding properties, and to obtain average properties. In particular, it has been instructive to compare the binding properties of imprinted and non-imprinted materials. This chapter begins by considering the origins of this site heterogeneity. Next, the properties of interest of imprinted binding sites are described in brief: affinity, selectivity, and kinetics. The binding/adsorption isotherm, the graph of concentration of analyte bound to a MIP versus concentration of free analyte at equilibrium, over a range of total concentrations, is described in some detail. Following this, the techniques for studying the imprinted sites are described (batch-binding assays, radioligand binding assays, zonal chromatography, frontal chromatography, calorimetry, and others). Thereafter, the parameters that influence affinity, selectivity and kinetics are discussed (solvent, modifiers of organic solvents, pH of aqueous solvents, temperature). Finally, mathematical attempts to fit the adsorption isotherms for imprinted materials, so as to obtain information about the range of binding affinities characterizing the imprinted sites, are summarized.

  18. Novel DNA-binding properties of the RNA-binding protein TIAR.

    PubMed

    Suswam, Esther A; Li, Yan Yan; Mahtani, Harry; King, Peter H

    2005-01-01

    TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR-RNP complex formation with IL-8 and VEGF 3'-untranslated regions (3'-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3'-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA (K(d)app = 1.6x10(-9) M versus 9.4 x 10(-9) M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands.

  19. The in vitro DNA binding properties of NDP kinase are related to its oligomeric state.

    PubMed

    Mesnildrey, S; Agou, F; Véron, M

    1997-11-24

    Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK-B binds specifically single-strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild-type does not. These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.

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

    PubMed

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

    2017-01-05

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

  1. Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains.

    PubMed

    Sabbadini, Priscila Soares; Genovez, Marcia Rocha Novais; Silva, Cecília Ferreira da; Adelino, Thelma Lúcia Novaes; Santos, Cintia Silva dos; Pereira, Gabriela Andrade; Nagao, Prescilla Emy; Dias, Alexandre Alves de Souza de Oliveira; Mattos-Guaraldi, Ana Luiza; Hirata Júnior, Raphael

    2010-08-01

    The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

  2. Lectin-binding properties of Aeromonas caviae strains

    PubMed Central

    Rocha-de-Souza, Cláudio M.; Hirata-Jr, Raphael; Mattos-Guaraldi, Ana L.; Freitas-Almeida, Angela C.; Andrade, Arnaldo F. B.

    2008-01-01

    The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37°C. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22°C. Binding studies with 125I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains. PMID:24031204

  3. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  4. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

    PubMed Central

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-01-01

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval. PMID:27669303

  5. Barcoded microchips for biomolecular assays.

    PubMed

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

    2015-01-20

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

  6. RNA binding and replication by the poliovirus RNA polymerase

    SciTech Connect

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.

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

    SciTech Connect

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

    1980-01-01

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

  8. Melanin-binding radiopharmaceuticals

    SciTech Connect

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  9. Homogeneous assay for whole blood folate using photon upconversion.

    PubMed

    Arppe, Riikka; Mattsson, Leena; Korpi, Krista; Blom, Sami; Wang, Qi; Riuttamäki, Terhi; Soukka, Tero

    2015-02-03

    Red blood cell folate is measured for folate deficiency diagnosis, because it reflects the long-term folate level in tissues, whereas serum folate only represents the dietary intake. Direct homogeneous assay from whole blood would be ideal but conventional fluorescence techniques in blood suffer from high background and strong absorption of light at ultraviolet and visible wavelengths. In this study, a new photon upconversion-based homogeneous assay for whole blood folate is introduced based on resonance energy transfer from upconverting nanophosphor donor coated with folate binding protein to a near-infrared fluorescent acceptor dye conjugated to folate analogue. The sensitized acceptor emission is measured at 740 nm upon 980 nm excitation. Thus, optically transparent wavelengths are utilized for both donor excitation and sensitized acceptor emission to minimize the sample absorption, and anti-Stokes detection completely eliminates the Stokes-shifted autofluorescence. The IC50 value of the assay was 6.0 nM and the limit of detection (LOD) was 1 nM. The measurable concentration range was 2 orders of magnitude between 1.0-100 nM, corresponding to 40-4000 nM folate in the whole blood sample. Recoveries of added folic acid were 112%-114%. A good correlation was found when compared to a competitive heterogeneous assay based on the DELFIA-technology. The introduced assay provides a simple and fast method for whole blood folate measurement.

  10. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  11. Decreased plasma arachidonic acid binding capacity in neonates.

    PubMed

    Sadowitz, P D; Walenga, R W; Clark, D; Stuart, M J

    1987-01-01

    Arachidonic acid (AA) metabolites have been implicated in neonatal pathologic states such as respiratory distress syndrome (RDS). Since free (nonprotein bound) AA is the substrate for synthesis of these compounds, a decreased capacity to bind AA in neonatal plasma could contribute to these disorders. AA binding was assayed by equilibrium dialysis in plasma samples from healthy adults and various infant groups. Plasma from these infant groups bound significantly less AA than adult plasma. Premature infants with RDS and premature infants receiving intralipid had the lowest capacity to bind AA. The increased availability of free AA may be important in neonatal pathophysiologic states involving arachidonate metabolites.

  12. CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

    PubMed

    Jay, Anthony G; Chen, Alexander N; Paz, Miguel A; Hung, Justin P; Hamilton, James A

    2015-02-20

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

  13. Improved internal control for molecular diagnosis assays.

    PubMed

    Vinayagamoorthy, T; Maryanski, Danielle; Vinayagamoorthy, Dilanthi; Hay, Katie S L; Yo, Jacob; Carter, Mark; Wiegel, Joseph

    2015-01-01

    The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. •The analyte and internal control have the same PCR and sequencing annealing sequences.•This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.•This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.

  14. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    PubMed

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-12-21

    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  15. Putting the pieces together: contribution of fluorescence polarization assays to small-molecule lead optimization

    NASA Astrophysics Data System (ADS)

    Keating, Susan M.; Marsters, Jim; Beresini, Maureen; Ladner, Carmen; Zioncheck, Kim; Clark, Kevin; Arellano, Fred; Bodary, Sarah

    2000-04-01

    Fluorescence polarization assays with both purified receptor and intact cells have been developed to assess potency and selectivity of antagonists of the interaction of the lymphocyte receptor, LFA-1, and its endothelial ligand, ICAM-1. Fluorescein isothiocyanate conjugated small molecule probes were optimized for use in binding assay with LFA-1 and a closely related receptor, MAC-1. In the assays, the antagonists compete with the fluorescent probe for binding to the receptor. This enables the determination of IC50 and consequently Ki values of the antagonists for each of the receptors. Routine use of polarization assay with tranfected cells, in addition to purified receptors, has become feasible with the availability of sensitive plate readers that are able to detect 1 nM fluorescent probe in 15 (mu) l sample volumes with good signal to noise. These measurements aid in the iterative synthesis of more potent and selective compounds.

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

    PubMed

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

    2008-07-01

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

  17. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  18. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  19. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  20. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  1. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  2. RNA binding domain of Jamestown Canyon virus S segment RNAs.

    PubMed

    Ogg, Monica M; Patterson, Jean L

    2007-12-01

    Jamestown Canyon virus (JCV) is a member of the Bunyaviridae family, Orthobunyavirus genus, California serogroup. Replication and, ultimately, assembly and packaging rely on the process of encapsidation. Therefore, the ability of viral RNAs (vRNAs) (genomic and antigenomic) to interact with the nucleocapsid protein (N protein) and the location of this binding domain on the RNAs are of interest. The questions to be addressed are the following. Where is the binding domain located on both the vRNA and cRNA strands, is this RNA bound when double or single stranded, and does this identified region have the ability to transform the binding potential of nonviral RNA? Full-length viral and complementary S segment RNA, as well as 3' deletion mutants of both vRNA and cRNA, nonviral RNA, and hybrid viral/nonviral RNA, were analyzed for their ability to interact with bacterially expressed JCV N protein. RNA-nucleocapsid interactions were examined by UV cross-linking, filter binding assays, and the generation of hybrid RNA to help define the area responsible for RNA-protein binding. The assays identified the region responsible for binding to the nucleocapsid as being contained within the 5' half of both the genomic and antigenomic RNAs. This region, if placed within nonviral RNA, is capable of altering the binding potential of nonviral RNA to levels seen with wild-type vRNAs.

  3. Assaying cooperativity of protein-DNA interactions using agarose gel electrophoresis.

    PubMed

    Williams, Tanya L; Levy, Daniel L

    2013-01-01

    DNA-binding proteins play essential roles in many cellular processes. Understanding on a molecular level how these proteins interact with their cognate sequences can provide important functional insights. Here, we describe a band shift assay in agarose gel to assess the mode of protein binding to a DNA molecule containing multiple protein-binding sites. The basis for the assay is that protein-DNA complexes display retarded gel electrophoresis mobility, due to their increased molecular weight relative to free DNA. The degree of retardation is higher with increasing numbers of bound protein molecules, thereby allowing resolution of complexes with differing protein-DNA stoichiometries. The DNA is radiolabeled to allow for visualization of both unbound DNA and all the different DNA-protein complexes. We present a quantitative analysis to determine whether protein binding to multiple sites within the same DNA molecule is independent or cooperative.

  4. Binding of endotoxin to macrophages: distinct effects of serum constituents.

    PubMed

    Tahri-Jouti, M A; Chaby, R

    1991-07-01

    The respective roles of serum lipoproteins, and of the complement component C3, in the binding of endotoxin (LPS) to macrophages were analyzed by an in vitro assay using [3H]LPS. The addition of an anti-C3 serum in the medium induced an apparent abolishment of the specific binding of LPS to mouse macrophages, but this effect appeared to be due to an actual increase of nonspecific binding. Isolated complexes of LPS with lipoproteins of high density (HDL3) and of very high density (VHDL) did not bind to macrophages. Furthermore, addition of HDL3 and VHDL in the incubation medium inhibited the specific binding of LPS to macrophages. These results suggest that C3 reduces nonspecific interactions between LPS and macrophages whereas associations between LPS and HDL3 or VHDL inhibit specific LPS-macrophage interactions.

  5. A mercury saturation assay for measuring metallothionein in fish

    SciTech Connect

    Dutton, M.D. . Dept. of Zoology); Stephenson, M. . Environmental Science Branch); Klaverkamp, J.F. )

    1993-07-01

    An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution of enzymatic and nonenzymatic thiols, including rabbit liver MT-2, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of [sup 203]Hg in 10% trichloroacetic acid. Excess Hg was bound to an removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with [sup 203]Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.

  6. Rat α-Fetoprotein binding affinities of a large set of structurally diverse chemicals elucidated the relationships between structures and binding affinities.

    PubMed

    Hong, Huixiao; Branham, William S; Dial, Stacey L; Moland, Carrie L; Fang, Hong; Shen, Jie; Perkins, Roger; Sheehan, Daniel; Tong, Weida

    2012-11-19

    Endocrine disrupting chemicals interfere with the endocrine system in animals, including humans, to exert adverse effects. One of the mechanisms of endocrine disruption is through the binding of receptors such as the estrogen receptor (ER) in target cells. The concentration of any chemical in serum is important for its entry into the target cells to bind the receptors. α-Fetoprotein (AFP) is a major transport protein in rodent serum that can bind with estrogens and thus change a chemical's availability for entrance into the target cell. Sequestration of an estrogen in the serum can alter the chemical's potential for disrupting estrogen receptor-mediated responses. To better understand endocrine disruption, we developed a competitive binding assay using rat amniotic fluid, which contains very high levels of AFP, and measured the binding to the rat AFP for 125 structurally diverse chemicals, most of which are known to bind ER. Fifty-three chemicals were able to bind the rat AFP in the assay, while 72 chemicals were determined to be nonbinders. Observations from closely examining the relationship between the binding data and structures of the tested chemicals are rationally explained in a manner consistent with proposed binding regions of rat AFP in the literature. The data reported here represent the largest data set of structurally diverse chemicals tested for rat AFP binding. The data assist in elucidating binding interactions and mechanisms between chemicals and rat AFP and, in turn, assist in the evaluation of the endocrine disrupting potential of chemicals.

  7. Broad base biological assay using liquid based detection assays

    SciTech Connect

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

    2000-10-31

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

  8. Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents Active against Na Channel Neurotoxins

    DTIC Science & Technology

    1991-04-12

    to produce approximately half-maximal effects mediated through these different sodium channel sites in the assay. Thus, the binding of [3HJBTX-B should...experiments with I3H]STX, yielding the unexpected result that effects of HM-197 are not mediated through the TTX/STX sodium channel binding site. Additional...Scorpion toxin; Screening; nA Pyrethroids; Radioligand binding; Synaptoneurosomes; RA 1 ; nA I ~ I ITherapeutic agents; Sodium channel 19. ABSTRACT

  9. Development of a multiplex non-radioactive receptor assay: the benzodiazepine receptor, the serotonin transporter and the beta-adrenergic receptor.

    PubMed

    de Jong, Lutea A A; Jeronimus-Stratingh, C Margot; Cremers, Thomas I F H

    2007-01-01

    Binding assays still form a fundamental part of modern drug development. Receptor binding assays are mostly based on radioactivity because of their speed, ease of use and reproducibility. Disadvantages, such as health hazards and production of radioactive waste, have prompted the development of non-radioactive receptor binding assays. This application therefore focuses on measuring receptor-ligand interactions using mass spectrometry. Moreover, the novelty of this approach originates in determining multiple analytes in a single assay (multiplexing). The proof of principle of a non-radioactive multiplex receptor assay is demonstrated using a pool of receptors from rat cortical tissue with flunitrazepam, MADAM and pindolol in one vial with or without their respective displacers. Flunitrazepam, MADAM and pindolol bound specifically at 73%, 30% and 40% to their respective receptors. This corresponds to specific binding sites of 0.61 pmol/mg protein, 0.07 pmol/mg protein and 0.06 pmol/mg protein, respectively. We propose to measure the bound fraction instead of the free fraction in order to reach a significant difference in measured signals (total binding versus non-specific binding). The bound fraction can be obtained after dissociating the ligand from the receptor-ligand complex using 50% methanol in water. The current setup of the assay calls for further improvement with respect to the measurement of binding constants for a multitude of receptors in one assay with sufficient accuracy and precision.

  10. Assay strategies and methods for phospholipases

    SciTech Connect

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

    1991-01-01

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

  11. Validation and application of an assay for deoxyribonucleic acid to estimate concentrations of bull sperm.

    PubMed

    Fenton, S E; Ax, R L; Cowan, C M; Coyle, T; Gilbert, G R; Lenz, R W

    1990-11-01

    Spectrophotometers are used for estimating sperm concentrations from raw ejaculates in semen processing laboratories. Unfortunately, these instruments have a limited detection spectrum and do not permit accurate quantification of sperm numbers in highly diluted or concentrated samples. The objectives of this study were to validate a DNA assay for quantification of sperm numbers in extended or undiluted semen samples and to determine precision of the assay. The principle of the assay is based upon a fluorescent dye that binds to adenine-thymine base pairs in double-stranded DNA. Semen samples and calf thymus DNA standards were sonicated in 2 M NaCl buffer with 1 mM EDTA. The DNA content of samples was compared to standards of calf thymus DNA using fluorometry. Sensitivity of the assay was determined to be 1.4 x 10(5) sperm cells. Concentrations of sperm estimated from DNA assay values did not differ from flow cytometric cell counts. Assays were performed in three different laboratories, using different equipment, to assess the assay's repeatability. Estimates of sperm concentrations determined by the DNA assay were similar, regardless of location and source of equipment used to perform the assays. This assay fulfills statistical criteria for being sensitive, accurate, and repeatable, and it can be employed in laboratories processing semen for artificial insemination as a tool for spectrophotometer calibration, a check for straw filling accuracy, or to quantify sperm numbers in extended, packaged semen.

  12. Protein binding elements in the human beta-polymerase promoter.

    PubMed Central

    Englander, E W; Wilson, S H

    1990-01-01

    The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein. Images PMID:2315044

  13. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  14. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  15. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  16. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  17. Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains▿

    PubMed Central

    Guillén, D.; Santiago, M.; Linares, L.; Pérez, R.; Morlon, J.; Ruiz, B.; Sánchez, S.; Rodríguez-Sanoja, R.

    2007-01-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch. PMID:17468268

  18. Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains.

    PubMed

    Guillén, D; Santiago, M; Linares, L; Pérez, R; Morlon, J; Ruiz, B; Sánchez, S; Rodríguez-Sanoja, R

    2007-06-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.

  19. E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

    PubMed Central

    Rabinovich, Alina; Jin, Victor X.; Rabinovich, Roman; Xu, Xiaoqin; Farnham, Peggy J.

    2008-01-01

    We have previously shown that most sites bound by E2F family members in vivo do not contain E2F consensus motifs. However, differences between in vivo target sites that contain or lack a consensus E2F motif have not been explored. To understand how E2F binding specificity is achieved in vivo, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) assays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are (1) the site must be in a core promoter and (2) the region must be utilized as a promoter in that cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including (1) indirect recruitment, (2) looping to the core promoter mediated by an E2F bound to a distal motif, and (3) assisted binding of E2F to a site that weakly resembles an E2F motif. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated fragments. Our findings suggest that in vivo (1) a consensus motif is not sufficient to recruit E2Fs, (2) E2Fs can bind to isolated regions that lack a consensus motif, and (3) binding can require regions other than the best match to the E2F motif. PMID:18836037

  20. Dynamics of TBP binding to the TATA box

    NASA Astrophysics Data System (ADS)

    Schluesche, Peter; Heiss, Gregor; Meisterernst, Michael; Lamb, Don C.

    2008-02-01

    Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

  1. Binding of environmental carcinogens to asbestos and mineral fibres.

    PubMed Central

    Harvey, G; Pagé, M; Dumas, L

    1984-01-01

    A rapid method has been developed for measuring the binding capacity of asbestos and other mineral fibres for environmental carcinogens. Benzo(alpha)pyrene (B(alpha)P), nitrosonornicotine (NNN), and N-acetyl-2-aminofluorene (NAAF) were assayed in the presence of Canadian grade 4T30 chrysotile, chrysotile A, amosite, crocidolite, glass microfibres, glasswool, attapulgite, and titanium dioxide. Chrysotile binds significantly more carcinogens than the other mineral fibres. This binding assay is reproducible with coefficients of variation of less than 8% and 6% respectively for inter and intra assay. The influence of pH was also studied, and there is good correlation between the carcinogen binding and the charge of the tested mineral fibres. The in vitro cytotoxicity on macrophage like cell line P388D1 and the haemolytic activity of various mineral fibres were also measured; a good correlation was found between the binding capacity and the cytotoxicity of tested mineral fibres on P388D1 cells. These results give some explanations for the reported synergism between exposure to asbestos and the smoking habits of workers. PMID:6331497

  2. Metallochaperones: bind and deliver

    SciTech Connect

    Rosenzweig, A.C.

    2010-03-08

    Metallochaperones deliver metal ions directly to target proteins via specific protein-protein interactions. Recent research has led to a molecular picture of how some metallochaperones bind metal ions, recognize their partner proteins, and accomplish metal ion transfer.

  3. SHBG (Sex Hormone Binding Globulin)

    MedlinePlus

    ... as: Testosterone-estrogen Binding Globulin; TeBG Formal name: Sex Hormone Binding Globulin Related tests: Testosterone , Free Testosterone, ... I should know? How is it used? The sex hormone binding globulin (SHBG) test may be used ...

  4. Lactation-induced cadmium-binding proteins

    SciTech Connect

    Bhattacharyya, M.H.; Solaiman, D.; Garvey, J.S.; Miyazaki, W.Y.

    1987-01-01

    Previously we have demonstrated an increase during midlactation in /sup 109/Cd adsorption and increased retention by the duodenum, kidney, and mammary tissue of mouse dams receiving environmental levels of cadmium//sup 109/Cd via drinking water, with little change in /sup 109/Cd retention in liver and jejunum compared to nonpregnant controls. Results are reported here of a study of cadmium deposition during midlactation as associated with induction of metallothionein (MT). A cadmium/hemoglobin (Cd/Hb) assay and radioimmunoassay for MT which measures heat-stable cadmium binding capacity in tissues was used to determine MT concentrations in fractions of kidney, liver, duodenum, and jejunum from female mice. Both assays demonstrated clear lactation-induced increases in MT concentrations in liver, kidney, and duodenum, with MT concentrations falling rapidly to control levels after weaning. 4 refs., 1 tab.

  5. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  6. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  7. Important Norwegian crude assays updated

    SciTech Connect

    Corbett, R.A

    1990-03-12

    New assays on two important Norwegian North Sea crude oils, Statfjord and Gullfaks, are presented. Both are high-quality, low-sulfur crudes that will yield a full range of good-quality products. All assay data came from industry-standard test procedures. The Statfjord field is the largest in the North Sea. Production started in 1979. Statfjord is a typical North Sea crude, produced from three separate platforms and three separate loading buoys with interconnecting lines. Current production is about 700,000 b/d. Gullfaks is produced from a large field in Block 34/10 of the Norwegian sector of the North Sea production area. Gullfaks crude oil is more biodegraded than other crudes from the region. Biodegradation has removed most of the waxy normal paraffins, resulting in a heavier, more naphthenic and aromatic crude.

  8. Comet Assay in Cancer Chemoprevention.

    PubMed

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  9. [Visible spectrophotometric assay of ranitidine].

    PubMed

    Apostu, M; Dorneanu, V; Bibire, Nela

    2003-01-01

    Ranitidine, belonging to H2-antagonist group, is a compound containing a furanic moiety and is used in peptic ulcer therapy. This paper debates the possibility of developing a new visible spectrophotometric assessment by using the reaction between ranitidine and eosine. We carried out our determinations at 505 nm, where the absorbency of ranitidine-eosine complex is maximal, and we have established the optimal reaction conditions. This method was successfully applied for ranitidine assay from pharmaceutical dosage forms.

  10. Two offshore Australian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-09

    Two light, sweet crudes from offshore Australia have been assayed. Gippsland crude, also called Bass Strait, is produced off the coast of Victoria, in southeastern Australia. The 47 API, 0.09% sulfur crude was analyzed in mid-1993. Skua, a 42 API, 0.06 wt % sulfur crude, is produced in the Timor Sea. Data are given on the whole crude and fractions for both deposits. Both chemical and physical properties are listed.

  11. Bioluminescence assay for cell viability.

    PubMed

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

    2015-06-01

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

  12. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  13. Lipid binding capacity of spider hemocyanin.

    PubMed

    Cunningham, M; Gómez, C; Pollero, R

    1999-09-01

    The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.

  14. The Rabbit Corneal Pocket Assay.

    PubMed

    Morbidelli, Lucia; Ziche, Marina

    2016-01-01

    The rabbit corneal micropocket angiogenesis assay uses the avascular cornea as a substrate canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 1-2 weeks and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor and a synthetic polymer to allow slow release. A micropocket is surgically created in the rabbit cornea under anesthesia and a pellet implanted. On the days later, the angiogenic response is measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay are used to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter, the experimental details of the rabbit cornea assay and technical implementations to the original protocol are described.

  15. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

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

  16. Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.

    2010-01-01

    We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

  17. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

    EPA Science Inventory

    In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

  18. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

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

    PubMed Central

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

    2016-01-01

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

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

    DOEpatents

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

    2016-04-05

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

  1. Vitamin D assays: past and present debates, difficulties, and developments.

    PubMed

    Fraser, William D; Milan, Anna M

    2013-02-01

    Clinical interest in Vitamin D and its purported roles not only in calcium and bone metabolism but in several other medical conditions (diabetes, cardiovascular disease, multiple sclerosis, cancer, psychiatric disorders, neuro-muscular disease) has led to a surge in laboratory requests for 25 hydroxy vitamin D and 1,25 dihydroxy vitamin D measurement. Circulating 25 hydroxy vitamin D concentration is routinely used as the best indicator of vitamin D status, but measurement of other metabolites, especially the physiologically active 1,25 dihyroxy vitamin D, are of clinical value. Over the last 40 years the development of assays for vitamin D and its metabolites from early competitive binding assays through to immunoassay and liquid chromatography aligned to mass spectrometry have demonstrated various analytical challenges, the advantages and disadvantages of each method are constantly changing with new technological developments. Immunoassay remains the predominant mode of measurement for 25-hydroxy vitamin D although problems with equimolar recovery of the D2 and D3 metabolites remain an issue. Standardisation of all assays has been improved but not resolved with the currently available reference materials as evidenced by the international vitamin D external quality assurance scheme, DEQAS. The choice of method for each laboratory remains a balance mainly between turn around time, convenience, cost and the specificity and accuracy of the information obtained. With increasing discussion and clinical interest surrounding other vitamin D metabolites the vitamin D assay debate is set to continue.

  2. Aptamer-phage reporters for ultrasensitive lateral flow assays

    PubMed Central

    Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagström, Anna E. V.; Kourentzi, Katerina; Conrad, Jacinta C.; Willson, Richard C.

    2015-01-01

    We introduce the modification of bacteriophage particles with aptamers for the use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ~100 times lower than those of previously reported IgE assays. PMID:26456715

  3. Targets and assays for discovering novel antibacterial agents.

    PubMed

    Donadio, Stefano; Carrano, Lucia; Brandi, Letizia; Serina, Stefania; Soffientini, Adolfo; Raimondi, Elena; Montanini, Nicoletta; Sosio, Margherita; Gualerzi, Claudio O

    2002-11-13

    The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

  4. Evaluation of Colorimetric Assays for Analyzing Reductively Methylated Proteins: Biases and Mechanistic Insights

    PubMed Central

    Brady, Pamlea N.; Macnaughtan, Megan A.

    2015-01-01

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins’ molar extinction coefficients at 280 nm. For the Bradford assay, the response (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color-formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines, compared to the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

  5. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    PubMed

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.

  6. Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species.

    PubMed

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-12-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with (125)I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with (125)I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K(d) (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R(t) (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

  7. Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species▿

    PubMed Central

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-01-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of Kd (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding Rt (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins. PMID:18931285

  8. Non-steady-state measurement of in vivo radioligand binding with positron emission tomography: specificity analysis and comparison with in vitro binding

    SciTech Connect

    Perlmutter, J.S.; Moerlein, S.M.; Hwang, D.R.; Todd, R.D. )

    1991-05-01

    We previously have developed a non-steady-state method for in vivo measurement of radioligand-receptor binding in brain using positron emission tomography (PET) and {sup 18}F-spiperone ({sup 18}F-SP). This method has proven to be highly sensitive to the detection of decreases in the apparent number of available specific binding sites. The purposes of this investigation are to demonstrate the specificity of this PET assay and compare findings to in vitro binding assays. Three to six studies were performed in each of five male baboons. Each animal was pretreated with either ketanserin (serotonergic (S2)), eticlopride (dopaminergic (D2)), or unlabeled SP to compete with {sup 18}F-SP for specific binding sites. Sequential PET scans and arterial-blood samples were collected for 3 hr after intravenous injection of {sup 18}F-SP. Data were analyzed with a three-compartment model that considered the accumulation of radiolabeled metabolites in arterial blood. Five baboons were killed, and radioligand-receptor binding in vitro was measured by homogenate techniques. There was no detectable in vitro or in vivo specific binding of SP in cerebellum. The specific binding of SP in striatal tissue in vitro was approximately 74% to D2 sites and 26% to S2 sites, whereas ketanserin displaced all specific binding in frontal cortex. In close agreement, specific binding measured in vivo with PET revealed that 68% of apparent striatal binding could be blocked by pretreatment with eticlopride, and 34% by ketanserin.

  9. Multiplexed Serologic Assay for Nine Anogenital Human Papillomavirus Types▿

    PubMed Central

    Opalka, David; Matys, Katie; Bojczuk, Paul; Green, Tina; Gesser, Richard; Saah, Alfred; Haupt, Richard; Dutko, Frank; Esser, Mark T.

    2010-01-01

    A multiplexed human papillomavirus (HPV) immunoassay has been developed for the detection of human IgG antibodies to HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 virus-like particle (VLP) types in serum following natural infection or immunization with VLP-based vaccines. The VLP antigens were covalently conjugated to carboxyl Luminex microspheres (MS) using a carbodiimide chemistry. Antibody (Ab) titers were determined in a direct binding format, in which an IgG1- to -4-specific, phycoerythrin (PE)-labeled monoclonal antibody (MAb) (HP6043) binds to human serum IgG antibodies. Pooled serum samples from rhesus macaques immunized with a 9-valent VLP-based vaccine served as the reference standard. The overall specificity of the assay was >99%, and the linearity (parallelism) of the assay was <7% per 10-fold dilution. Total assay precision was <19% across 3 different VLP-microsphere lots, 2 secondary antibody lots, and 2 different operators over a period of 3 weeks. Three different methods were used to evaluate serostatus cutoffs (SCO): (i) a clinical sensitivity/specificity analysis based on “likely negative” and “likely positive” samples from nonvaccinees, (ii) stringent upper tolerance limits on samples from “likely negatives,” and (iii) stringent upper tolerance limits from the same “likely negative” sample set after VLP adsorption. Depending on the method to set the serostatus cutoff, the percentage of seropositive samples at the month 48 time point following vaccination with the HPV 6/11/16/18 quadrivalent vaccine ranged from 70% to 100%. This assay has proven useful for measuring the levels of serum antibody to the nine HPV VLPs following natural infection or administration of VLP-based vaccines. PMID:20237197

  10. Multiplexed serologic assay for nine anogenital human papillomavirus types.

    PubMed

    Opalka, David; Matys, Katie; Bojczuk, Paul; Green, Tina; Gesser, Richard; Saah, Alfred; Haupt, Richard; Dutko, Frank; Esser, Mark T

    2010-05-01

    A multiplexed human papillomavirus (HPV) immunoassay has been developed for the detection of human IgG antibodies to HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 virus-like particle (VLP) types in serum following natural infection or immunization with VLP-based vaccines. The VLP antigens were covalently conjugated to carboxyl Luminex microspheres (MS) using a carbodiimide chemistry. Antibody (Ab) titers were determined in a direct binding format, in which an IgG1- to -4-specific, phycoerythrin (PE)-labeled monoclonal antibody (MAb) (HP6043) binds to human serum IgG antibodies. Pooled serum samples from rhesus macaques immunized with a 9-valent VLP-based vaccine served as the reference standard. The overall specificity of the assay was >99%, and the linearity (parallelism) of the assay was <7% per 10-fold dilution. Total assay precision was <19% across 3 different VLP-microsphere lots, 2 secondary antibody lots, and 2 different operators over a period of 3 weeks. Three different methods were used to evaluate serostatus cutoffs (SCO): (i) a clinical sensitivity/specificity analysis based on "likely negative" and "likely positive" samples from nonvaccinees, (ii) stringent upper tolerance limits on samples from "likely negatives," and (iii) stringent upper tolerance limits from the same "likely negative" sample set after VLP adsorption. Depending on the method to set the serostatus cutoff, the percentage of seropositive samples at the month 48 time point following vaccination with the HPV 6/11/16/18 quadrivalent vaccine ranged from 70% to 100%. This assay has proven useful for measuring the levels of serum antibody to the nine HPV VLPs following natural infection or administration of VLP-based vaccines.

  11. Use of luminescent Leptospira interrogans for enumeration in biological assays.

    PubMed

    Murray, Gerald L; King, Amy M; Srikram, Amporn; Sermswan, Rasana W; Adler, Ben

    2010-06-01

    Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10(4) leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R(2) = 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R(2) = 0.916, complement killing assay R(2) = 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.

  12. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites.

    PubMed

    Sena, Janete A D; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-04-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  13. Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites▿

    PubMed Central

    Sena, Janete A. D.; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-01-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af. PMID:19181834

  14. A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

    PubMed

    Figueroa-López, Alejandro Miguel; Cordero-Ramírez, Jesús Damián; Quiroz-Figueroa, Francisco Roberto; Maldonado-Mendoza, Ignacio Eduardo

    2014-07-01

    A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).

  15. Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

    PubMed

    Lieb, S; Littmann, T; Plank, N; Felixberger, J; Tanaka, M; Schäfer, T; Krief, S; Elz, S; Friedland, K; Bernhardt, G; Wegener, J; Ozawa, T; Buschauer, A

    2016-12-01

    A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

  16. Insulin binding sites in various segments of the rabbit nephron

    SciTech Connect

    Nakamura, R.; Emmanouel, D.S.; Katz, A.I.

    1983-07-01

    Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of /sup 125/I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between /sup 125/I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the ''diluting segment'' (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.

  17. Characterization and properties of steroid binding protein in Bufo arenarum serum.

    PubMed

    Fernández, S N; Mansilla-Whitacre, Z C; Miceli, D C

    1994-08-01

    Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

  18. Clostridium botulinum type A progenitor toxin binds to Intestine-407 cells via N-acetyllactosamine moiety.

    PubMed

    Kojima, Shoudou; Eguchi, Hironobu; Ookawara, Tomomi; Fujiwara, Noriko; Yasuda, Jun; Nakagawa, Kazuhiko; Yamamura, Takehira; Suzuki, Keiichiro

    2005-06-03

    Botulism is a highly fatal disease caused by the botulinum progenitor toxin. In this study, the role of oligosaccharides for the binding of botulinum type A progenitor toxin (type A PTX) to human intestinal cells was investigated. The binding of type A PTX to Intestine-407 cells was inhibited by the addition of N-acetyllactosamine, lactose, and galactose. Treatment of Intestine-407 cells with neuraminidase led to a significant increase in the binding of type A PTX, while further digestion of cell surface oligosaccharides by beta-galactosidase and beta-N-acetylhexosaminidase decreased the binding. These results indicate that the N-acetyllactosamine moiety is responsible for the binding of type A PTX. These findings were further confirmed by a binding assay using synthesized oligosaccharides. Interestingly, sialylation or fucosylation of oligosaccharides inhibited the binding of type A PTX. These data suggest that the type A PTX binds to intestinal cells via cell surface N-acetyllactosamine moiety.

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

    PubMed Central

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

    2014-01-01

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

  20. Specific triplex binding capacity of mixed base sequence duplex nucleic acids used for single-nucleotide polymorphism detection.

    PubMed

    Daksis, Jasmine I; Erikson, Glen H

    2005-01-01

    Specific base recognition and binding between native double-stranded DNA (dsDNA) and complementary single-stranded DNA (ssDNA) of mixed base sequence is presented. Third-strand binding, facilitated and stabilized by a DNA intercalator, YOYO-1, occurs within 5 min at room temperature. This triplex binding capability has been used to develop a homogeneous assay that accurately detects 1-, 2-, or 3-bp mutations or deletions in the dsDNA target. Every type of 1-bp mismatch can be identified. The assay can reliably distinguish homozygous from heterozygous polymerase chain reaction (PCR)-amplified genomic dsDNA, thus providing a highly sensitive clinical diagnostic assay.

  1. Four assay designs and on-chip calibration: gadgets for a sepsis protein array.

    PubMed

    Buchegger, Patricia; Preininger, Claudia

    2014-03-18

    A protein microarray for the early stage diagnosis of sepsis that allows the simultaneous detection of C-reactive protein (CRP) (2-200 μg/mL), procalcitonin (PCT) (0.2-50 ng/mL), and interleukin 6 (IL-6) (2-2000 pg/mL) has been developed. To enable the parallel detection of the differently abundant analytes, the low binding affinity between CRP and phosphocholine is exploited in a "low-sensitive" sandwich assay for CRP. The calibration is integrated directly on the chip resulting in a "one patient-one array" format, to provide a user-friendly and rapid diagnostic tool. Four different assay designs are introduced: (I) the classical assay that works with biotin-streptavidin chemistry, (II) the rapid assay that is performed in a single detection step, and two ultrasensitive assay designs accomplished either by (III) an enzymatic or (IV) an antibody mediated amplification resulting in high density labeling. The assay designs were evaluated by the repetitive measurement of low, medium, and high concentration levels of commercially available certified control sera. The precision was similar across all assay designs (coefficient of variation (CV), CVintra: 8-14%; CVinter: 18-34%), while the sensitivity (limits of detection (LODs)) increased by 1 order of magnitude for the ultrasensitive assays (III, IV) and the accuracy was analyte dependent but best for the classical (I) and the antibody amplified (IV) assays.

  2. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    SciTech Connect

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  3. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  4. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  5. Heme Binding to the Mammalian Circadian Clock Protein Period 2 is Non-Specific†

    PubMed Central

    Airola, Michael V.; Du, Jing; Dawson, John H.; Crane, Brian R.

    2010-01-01

    The mammalian circadian clock synchronizes physical and metabolic activity with the diurnal cycle through a transcriptional-posttranslational feedback loop. An additional feedback mechanism regulating clock timing has been proposed to involve oscillation in heme availability. Period 2 (PER2), an integral component in the negative feedback loop that establishes circadian rhythms in mammals, has been identified as a heme binding protein. However, the majority of evidence for heme binding is based upon in vitro heme binding assays. We sought to ascertain if these largely spectral assays could distinguish between specific and non-specific heme interactions. Heme binding properties by a number of other well-characterized proteins, all with no known biological role involving heme interaction, corresponded to those displayed by PER2. Site-directed mutants of putative heme-binding residues identified by MCD were unable to locate a specific heme-binding site on PER2. Protein film electrochemistry also indicates that heme binds PER2 non-specifically on the protein surface. Our results establish the inability of typical in vitro assays to easily distinguish between specific and non-specific heme binding. We conclude that heme binding to PER2 is likely to be non-specific and does not involve the hydrophobic pocket within the PER2 PAS domains that in other PAS proteins commonly recognizes cofactors. These findings also question the significance of in vivo studies that implicate heme interactions with the clock proteins PER2 and nPAS2 in biological function. PMID:20411915

  6. Development of filtration-based time-resolved fluorescence assay for the high-throughput screening of urotensin II receptor antagonist.

    PubMed

    Oh, Kwang-Seok; Lee, Sunghou; Lee, Byung Ho

    2011-10-01

    The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.

  7. Chromatin landscape dictates HSF binding to target DNA elements.

    PubMed

    Guertin, Michael J; Lis, John T

    2010-09-09

    Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. We used the ChIP-seq assay to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in "active" chromatin is a primary determinant of whether HSF can bind following heat shock.

  8. Aluminum binding by humus

    SciTech Connect

    Benedetti, M.F.; Hiemstra, T.; Riemsdijk, W. van; Kinniburgh, D.

    1996-10-01

    The need for qualitative and quantitative description of the chemical speciation of Al, in particular and other metal ions in general, is stressed by the increased mobilization of metal ions in water and soils due to acid rain deposition. In this paper we present new data of Al binding to two humic acids. These new data sets and the some previously published data will be analyzed with the NICA-Donnan model using one set of parameters to describe the Al binding to the different humic substances. Once the experimental data is described with the NICA-Donnan approach, we will show the effect of Ca on Al binding and surface speciation as well as the effect of Al on the charge of the humic particles. The parameters derived from the laboratory experiments will be used to describe the variation of the field based Al partition coefficient.

  9. Radioenzymatic assay for quinolinic acid

    SciTech Connect

    Foster, A.C.; Okuno, E.; Brougher, D.S.; Schwarcz, R.

    1986-10-01

    A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing (/sup 3/H)ATP, further to (/sup 3/H) deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

  10. Key learnings from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 in vitro assays.

    PubMed

    LeBaron, Matthew J; Coady, Katie K; O'Connor, John C; Nabb, Diane L; Markell, Lauren K; Snajdr, Suzanne; Sue Marty, M

    2014-02-01

    Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.

  11. Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay

    ERIC Educational Resources Information Center

    Umali, Alona P.; Anslyn, Eric V.; Wright, Aaron T.; Blieden, Clifford R.; Smith, Carolyne K.; Tian, Tian; Truong, Jennifer A.; Crumm, Caitlin E.; Garcia, Jorge E.; Lee, Soal; Mosier, Meredith; Nguyen, Chester P.

    2010-01-01

    The use of an indicator displacement assay permits the visualization of binding events between host and guest molecules. An undergraduate laboratory experiment is described to demonstrate the technique in the determination of citric acid content in commercially available beverages such as soda pop and fruit juices. Through the technique, students…

  12. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  13. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    2001-10-09

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  14. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    1999-01-01

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  15. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

    PubMed Central

    Ng, R; Carbon, J

    1987-01-01

    Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo. Images PMID:2830498

  16. Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.

    PubMed

    Wu, Bill X; Clarke, Christopher J; Matmati, Nabil; Montefusco, David; Bartke, Nana; Hannun, Yusuf A

    2011-06-24

    Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.

  17. Ligand-binding characteristics of feline insulin-binding immunoglobulin G

    PubMed Central

    SUZUKI, Takafumi; NISHII, Naohito; TAKASHIMA, Satoshi; MATSUBARA, Tatsuya; IWASAWA, Atsushi; TAKEUCHI, Hirofumi; TAHARA, Kohei; HACHISU, Tatsuyuki; KITAGAWA, Hitoshi

    2015-01-01

    Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e−4 M for fraction B (low affinity IgGs) and 2e−5 M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin. PMID:26062435

  18. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

  19. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

    PubMed Central

    García, Javier; Puentes, Alvaro; Rodríguez, Luis; Ocampo, Marisol; Curtidor, Hernando; Vera, Ricardo; Lopez, Ramses; Valbuena, John; Cortes, Jimena; Vanegas, Magnolia; Barrero, Carlos; Patarroyo, Manuel A.; Urquiza, Mauricio; Patarroyo, Manuel E.

    2005-01-01

    The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins. PMID:16131654

  20. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  1. Assay of potentially contaminated propellant

    SciTech Connect

    Koster, J.E.; Williams, H.E. III; Scott, W.S.

    1995-02-01

    One of the decontamination and decommissioning projects within DOD is demilitarization of an aging stockpile of munitions. A large portion of the stockpile contains depleted uranium (DU) as an armor piercing core and so these munitions must be assayed for the presence of uranium in other components. The assay method must be fast and preferably easy to implement. Presence of DU is indicated by its alpha decay. The alpha particles in turn produce ions in the ambient air. If a significant fraction of these ions can escape the quantity of propellant, the ions can be detected instead of the alpha particles. As a test of the feasibility of detecting alpha emissions from DU somewhere within a cartridge of propellant, the transmission of ions through layers of real propellant was measured. The propellant is in the form of graphite-coated cylindrical pellets. A 105nun cartridge was modified for use as a pellet chamber. A check source served as an ion source. The ion detector consisted of a grid held at 300V coupled to an ammeter. Results confirm that this is a promising technique for testing the propellant for the presence of DU quickly yet with sensitivity.

  2. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  3. Characterization of C1 inhibitor binding to neutrophils.

    PubMed Central

    Chang, N S; Boackle, R J; Leu, R W

    1991-01-01

    In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH. PMID:2045131

  4. Polycation binding to glomerular basement membrane. Effect of biochemical modification.

    PubMed

    Bertolatus, J A; Hunsicker, L G

    1987-02-01

    The polycation hexadimethrine (HDM) binds to anionic sites in the glomerular basement membrane (GBM) and causes heavy proteinuria when infused in vivo. An in vitro assay of 3H-HDM binding to isolated dog GBM was developed, to permit further analysis of the GBM components binding HDM. 3H-HDM binding to isolated GBM was saturable, reversible in dose-dependent fashion by competing polycations, and inhibited by increasing salt concentration and low pH. The pH dependence of binding suggested that most of the HDM binds to carboxyl groups rather than to the sulfate groups of proteoglycans. Removal of heparan sulfate by heparinase or purified heparatinase had no detectable effect on HDM binding. Treatment of GBM with neuraminidase, hyaluronidase, or chondroitinase reduced binding of HDM by a maximum of 20 to 38%. However, substitution of carboxyl anions with nonionizable glycine methyl ester residues resulted in complete elimination of HDM binding. Parallel results were obtained in studies of glomerular localization of cationized ferritin (CatF), pI 8.5. After carboxyl substitution, GBM did not bind CatF; heparinase-treated GBM bound CatF in a distribution not demonstrably different from normal. Cellulose acetate electrophoresis of glycosaminoglycan fractions prepared from treated GBM confirmed that carboxyl modification did not alter the content or charge of the heparan sulfate of GBM, but heparinase treatment removed at least 90% of heparan sulfate. The results indicate that carboxyl groups are quantitatively more important than heparan sulfate for binding of HDM in vitro. Since HDM causes proteinuria in vivo, carboxyl groups may be important for maintenance of normal permselectivity.

  5. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  6. Multiple approaches to assess pectin binding to galectin-3.

    PubMed

    Zhang, Tao; Zheng, Yi; Zhao, Dongyang; Yan, Jingmin; Sun, Chongliang; Zhou, Yifa; Tai, Guihua

    2016-10-01

    Although several approaches have been used to evaluate binding of carbohydrates to lectins, results are not always comparable, especially with larger polysaccharides. Here, we quantitatively assessed and compared binding of pectin-derived polysaccharides to galectin-3 (Gal-3) using five methods: surface plasmon resonance (SPR), bio-layer interferometry (BLI), fluorescence polarization (FP), competitive fluorescence-linked immunosorbance (cFLISA), and the well-known cell-based hemagglutination assay (G3H). Our studies revealed that whereas Gal-3-pectin binding parameters determined by SPR and BLI were comparable and correlated with inhibitory potencies from the G3H assay, results using FP and cFLISA assays were highly variable and depended greatly on the probe and mass of the polysaccharide. In the cFLISA assay, for example, pectins showed no inhibition when using the DTAF-labeled asialofetuin probe, but did when using a DTAF-labeled pectin probe. And the FP approach with the DTAF-lactose probe did not work on polysaccharides and large galactan chains, although it did work well with smaller galactans. Nevertheless, even though results derived from all of these methods are in general agreement, derived KD, IC50, and MIC values do differ. Our results reflect the variability using various techniques and therefore will be useful to investigators who are developing pectin-derived Gal-3 antagonists as anti-cancer agents.

  7. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors.

    PubMed

    Furtek, Steffanie L; Matheson, Christopher J; Backos, Donald S; Reigan, Philip

    2016-11-22

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.

  8. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors

    PubMed Central

    Furtek, Steffanie L.; Matheson, Christopher J.; Backos, Donald S.; Reigan, Philip

    2016-01-01

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors. PMID:27793003

  9. Binding of (/sup 3/H)Forskolin to rat brain membranes

    SciTech Connect

    Seamon, K.B.; Vaillancourt, R.; Edwards, M.; Daly, J.W.

    1984-08-01

    (12-/sup 3/H)Forskolin (27 Ci/mmol) has been used to study binding sites in rat brain tissue by using both centrifugation and filtration assays. The binding isotherm measured in the presence of 5 mM MgCl/sub 2/ by using the centrifugation assay is described best by a two-site model: K/sub d1/ = 15 nM, B/sub max/sub 1// (maximal binding) = 270 fmol/mg of protein; K/sub d2/ = 1.1 ..mu..M; B/sub max/sub 2// = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; K/sub d/ = 26 nM, B/sub max/ = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 4.6.1.1) do not compete effectively for (/sup 3/H)forskolin binding sites. Analogs of forskolin that are less potent than forskolin in activating adenylate cyclase are also less potent in competing for forskolin binding sites. The presence of 5 mM MgCl/sub 2/ or MnCl/sub 2/ was found to enhance binding. In the presence of 1 mM EDTA the amount of high-affinity binding is reduced to 110 fmol/mg of protein with no change in K/sub d/. There is no effect of CaCl/sub 2/ (20 mM) or NaCl (100 mM) on the binding. No high-affinity binding can be detected in membranes from ram sperm, which contains an adenylate cyclase that is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein. 23 references, 5 figures, 2 tables.

  10. Surface-binding autoantibodies to cerebellar neurons in opsoclonus syndrome.

    PubMed

    Blaes, Franz; Fühlhuber, Verena; Korfei, Martina; Tschernatsch, Marlene; Behnisch, Wolfgang; Rostasy, Kevin; Hero, Barbara; Kaps, Manfred; Preissner, Klaus T

    2005-08-01

    Childhood opsoclonus-myoclonus syndrome can occur with or without associated neuroblastoma. An autoimmune pathogenesis has been discussed for both forms. We show here that the majority of children with opsoclonus-myoclonus syndrome (10/14) have autoantibodies binding to the surface of isolated rat cerebellar granular neurons. In some patients, these antibodies are masked by IgG binding to ubiquitous surface antigens, which could be removed by preincubation with the nonneuronal control cell line HEK 293. A newly introduced competitive binding assay showed that the surface binding is directed against the same autoantigen in different patients. Therefore, we hypothesize that opsoclonus-myoclonus syndrome may be the result of an autoimmune process against a neuronal surface protein.

  11. Binding of a tritiated pepstatin analog to human renin

    SciTech Connect

    Cumin, F.; Nisato, D.; Gagnol, J.P.; Corvol, P.

    1987-01-01

    The interaction between human renin and a potent pepstatin analog, SR 42128, has been investigated using binding studies. Binding and enzymatic assays were performed at pH 5.7 and pH 7.4. We found one specific inhibitor binding site per molecule of renin at both pH's. The dissociation constant (KD) obtained at equilibrium was 14-fold lower at pH 5.7 than at pH 7.4, showing a pH effect on binding of (/sup 3/H)SR 42128. A similar decrease was measured in enzymatic studies. In nonequilibrium conditions, we demonstrated that only association kinetic constants have been affected by pH variations. Radioligands provided interesting tools to investigate enzyme-inhibitor relationships.

  12. Sizes of Mn-binding sites in spinach thylakoids.

    PubMed

    Takahashi, M; Asada, K

    1986-12-25

    The sizes of the Mn-binding sites in spinach thylakoids were estimated by target size analysis, assaying the membrane-bound Mn that was resistant to EDTA washing after radiation inactivation. The inactivation curve showed well the inactivation of two independent Mn-binding sites of different sizes: about two-thirds of the Mn coordinated to a binding site of 65 kDa, and the rest bound to a much smaller site of only about 3 kDa. In the large site, there was about 1 g atom of Mn/110 mol of chlorophyll in spinach thylakoids, which was constant in normally grown plants, although the Mn level in the small site depended on culture conditions. Thylakoids that had been incubated with hydroxylamine or in 0.8 M Tris lost Mn exclusively from the large binding site.

  13. Sizes of Mn-binding sites in spinach thylakoids

    SciTech Connect

    Takahashi, M.; Asada, K.

    1986-12-25

    The sizes of the Mn-binding sites in spinach thylakoids were estimated by target size analysis, assaying the membrane-bound Mn that was resistant to EDTA washing after radiation inactivation. The inactivation curve showed well the inactivation of two independent Mn-binding sites of different sizes: about two-thirds of the Mn coordinated to a binding site of 65 kDa, and the rest bound to a much smaller site of only about 3 kDa. In the large site, there was about 1 g atom of Mn/110 mol of chlorophyll in spinach thylakoids, which was constant in normally grown plants, although the Mn level in the small site depended on culture conditions. Thylakoids that had been incubated with hydroxylamine or in 0.8 M Tris lost Mn exclusively from the large binding site.

  14. A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

    PubMed

    Murphy, James M; Zhang, Qingwei; Young, Samuel N; Reese, Michael L; Bailey, Fiona P; Eyers, Patrick A; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L; Liu, Ling; Daly, Roger J; Manning, Gerard; Babon, Jeffrey J; Lucet, Isabelle S

    2014-01-15

    Protein kinase-like domains that lack conserved residues known