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Sample records for western blot kit

  1. Evaluation of an indigenous western blot kit for human immunodeficiency virus

    Directory of Open Access Journals (Sweden)

    Lakshmi V

    2002-01-01

    Full Text Available PURPOSE: The Western Blot test is considered a gold standard test for the confirmation of an ELISA and/or rapid assay screened reactive sample in the diagnosis of HIV infection, especially in the low risk population. In this study, an indigenously developed HIV W. Blot kit (J.Mitra & Co., New Delhi, India was compared for its performance characteristics with a widely used Western Blot kit, HIV Blot 2.2 (Genelabs, Singapore. Antigens of both HIV-1 and the indicator antigen gp36 of HIV-2 are included in the strips. METHODS: A panel of 150 clinical serum samples was used in the evaluation. All the sera were tested simultaneously by both the kits. RESULTS: The HIV W. Blot kit had high performance characteristics (100% sensitivity and 100% specificity, like the HIV Blot 2.2. The test procedure was easy to perform. There was clear delineation of the bands. CONCLUSIONS: The interpretation of the results on the HIV W. Blot was less prone to subjective errors. The test gave positive bands at even very high serum dilutions in the test kit. This fact indicates that HIV W. Blot probably has a potential application in early phases of infection, when the antibody concentrations are still very low.

  2. Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

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    Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

    2015-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method.

  3. Relative performance of Organon kit in comparison to Du Pont for confirmatory serological testing of HIV infection by western blot test in sera from blood donors.

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    Aggarwal, R K; Chatterjee, R; Chattopadhya, D; Kumari, S

    1992-06-01

    A total of 32 specimens with different categories of reactivity by Du Pont Western Blot kit comprising of specimens showing full spectrum of HIV-I antigen specific bands, 19 specimens showing total absence of bands and four specimens showing non-specific bands (without any interpretative importance) were subjected to Western Blot testing by Organon test. Of the nine specimens showing full spectrum of bands by Du Pont the correlation with Organon kit was 100 per cent based on WHO criteria. Four specimens with non-specific indeterminate band pattern by Du Pont failed to show any band in Organon kit, indicating that latter to be more specific.

  4. The western blot

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    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  5. Western blot analysis.

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    Hirano, Seishiro

    2012-01-01

    Electrophoresis and the following western blot analysis are indispensable to investigate biochemical changes in cells and tissues exposed to nanoparticles or nanomaterials. Proteins should be extracted from the cells and tissues using a proper method, especially when phosphorylated proteins are to be detected. It is important to select a good blocking agent and an appropriate pair of primary and peroxidase-tagged secondary antibodies to obtain good results in western blot analysis. One thing that may be specific to nanomaterials, and that you should keep in mind, is that some proteins may be adsorbed on the surface of particulate nanomaterials. In this chapter the whole process of western blot analysis, from sample preparation to quantitative measurement of target proteins, is described.

  6. Western Blot Techniques.

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    Kim, Brianna

    2017-01-01

    The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest. Here, we describe the transfer and detection of Outer surface protein A (OspA), a protein only found on the surface of Borrelia burgdorferi, the bacteria responsible for Lyme disease.

  7. Lectin-probed western blot analysis.

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    Sato, Takeshi

    2014-01-01

    Lectin-probed western blot analysis, the so-called lectin blot analysis, is a useful method to yield basic information on the glycan structures of glycoproteins, based on the carbohydrate-binding specificities of lectins. By lectin blot analysis, researchers can directly analyze the glycan structures without releasing the glycans from glycoproteins. Here, the author describes protocols for standard analysis, and applies analysis in combination with glycosidase digestion of blot.

  8. Comparison of the efficiency of two commercial kits - ELFA and Western blot in estimating the phase of Toxoplasma gondii infection in pregnant women.

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    Sroka, Jacek; Wójcik-Fatla, Angelina; Zając, Violetta; Sawczyn, Anna; Cisak, Ewa; Karamon, Jacek; Dutkiewicz, Jacek; Bojar, Iwona

    2016-12-23

    Sera of 89 pregnant women were selected according to the results of ELFA IgM, IgG and avidity IgG, and tested with commercial tests IgM, IgG and avidity IgG Western Blot (WB) to compare the efficacy of both techniques in determining the phase of T. gondii infection. In total, 81 of 89 tested sera (91.0%) were classified as positive, both in the ELFA and WB tests for the presence of anti-Toxoplasma antibodies of class IgG, indicating a past infection, while the prevalence of anti-Toxoplasma positive reactions associated with the antibodies of class IgM indicating a recent infection was much lower - 31.5% and 20.2%, respectively. Sera of 81 women were also tested in the ELFA and WB tests for avidity, e.g. ability of forming high-molecular IgG antibody complexes. Low or medium results in these tests (in this study all classified as low), indicating a recent infection, were detected by ELFA and WB in 22.2% and 45.7% of the total examined samples, respectively. The Spearman's rank test for correlation, performed for recognition of quantitative data of the ELFA and WB tests (index, units or points), revealed a highly significant correlation between the ELFA and WB tests for homologous classes of antibodies, both for IgM and IgG (p0.05), except for the WB test for IgM antibodies, which showed a significant correlation with the ELFA test for IgG antibodies (p<0.01). A highly significant negative correlation between the ELFA and WB test for IgM antibodies and ELFA and WB tests for IgG avidity was demonstrated (p<0.01), except for a relationship between the WB test for IgM and WB for avidity, which was not significant. Such negative correlations are theoretically expected, as strong complexes with the participation of IgG antibodies are absent in the early phase of toxoplasmosis when early antibodies of IgM class are present. Summarizing, this study indicates the high usefulness of the commercial ELFA and WB tests in serodiagnostics of toxoplasmosis in pregnant women

  9. Western blot: technique, theory, and trouble shooting.

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    Mahmood, Tahrin; Yang, Ping-Chang

    2012-09-01

    Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

  10. Single cell-resolution western blotting.

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    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

  11. SDS -PAGE and Western Blotting Techniques.

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    Blancher, C; Jones, A

    2001-01-01

    The goal of Western blotting, or more correctly, immunoblotting, is to identify with a specific antibody a particular antigen within a complex mixture of proteins that has been fractionated in a polyacrylamide gel and immobilized onto a membrane. Immunoblotting can be used to determine a number of important characteristics of protein antigens-the presence and quantity of an antigen, the relative molecular weight of the polypeptide chain, and the efficiency of extraction of the antigen.Immunoblotting occurs in six stages: (1) extraction and quantification of protein samples; (2) resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis (SDS-PAGE); (3) transfer of the separated polypeptides to a membrane support; (4) blocking nonspecific binding sites on the membrane; (5) addition of antibodies; and (6) detection.Sample preparation is important for obtaining accurate separation of the proteins on the basis of molecular weight. Depending on whether an antigen is primarily extracellular, cytoplasmic, or membrane-associated different procedures might be required to prepare the sample initially. Although there are exceptions, many soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers that contain the nonionic detergent Nonidet P-40 (NP-40) and either no salt at all or relatively high concentrations of salt (e.g., 0.5 M NaCl). However, the efficiency of extraction is often greatly affected by pH of the buffer and the presence or absence of chelating agents such EDTA.

  12. Selected Reaction Monitoring (SRM) frente a Western Blot

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    Heredia Ponce, Zahira Maria; Urbano Gámez, Jose Alberto

    2016-01-01

    We propose the SRM technology as a complementary method to the Western Blot for the detection and quantification of proteins in a sample. The technique Western Blot has its own limitations: i) only a protein-of-choice is detected, ignoring any non-relevant proteins, ii) the sensitivity of the technique depends on the specificity of the antibody and iii) Western Blot is expensive and time-consuming. The advantages of SRM with respect Western Blot are remarkable: i) you can detect up to h...

  13. Studying protein-protein interactions via blot overlay/far western blot.

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    Hall, Randy A

    2015-01-01

    Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

  14. A defined methodology for reliable quantification of Western blot data.

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    Taylor, Sean C; Berkelman, Thomas; Yadav, Geetha; Hammond, Matt

    2013-11-01

    Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

  15. The Design of a Quantitative Western Blot Experiment

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    Sean C. Taylor

    2014-01-01

    Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  16. The design of a quantitative western blot experiment.

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    Taylor, Sean C; Posch, Anton

    2014-01-01

    Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  17. Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

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    Mavin, S; McDonagh, S; Evans, R; Milner, R M; Chatterton, J M W; Ho-Yen, D O

    2011-01-01

    This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients.

  18. Clinical, immunohistochemical, Western blot, and genetic analysis in dystrophinopathy.

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    Na, Sang-Jun; Kim, Won-Joo; Kim, Seung Min; Lee, Kee Ook; Yoon, Bora; Choi, Young-Chul

    2013-08-01

    Dystrophin-deficient muscular dystrophies (dystrophinopathies) are the most common form of muscular dystrophy, with variable clinical phenotypes ranging from the severe Duchenne (DMD) to the milder Becker (BMD) forms. In this study, we investigated the relationship between clinical characteristics, findings at immunohistochemistry (IHC) and Western blot, and the pattern of exon deletions in 24 male patients with dystrophinopathies. We retrospectively reviewed findings from clinical and laboratory examinations, IHC for dystrophin of muscle biopsy tissue, Western blot analysis, and multiplex polymerase chain reaction (PCR) examination of genomic DNA. All tests were performed in every patient. PCR examination revealed exon deletions in 13 patients (54.2%). At Western blot analysis, 15 patients (62.5%) were negative at all three dystrophin domains. Most of these patients had a clinical presentation consistent with the DMD phenotype. Nine (37.5%) others were weakly positive at one or more domains. Most of these patients presented clinically as BMD phenotype. One patient whose clinical presentation was consistent with BMD phenotype had normal findings at IHC and was weakly positive at all three domains on Western blot analysis; however, with the exception of this patient, the findings at IHC and Western blot were consistent for individual patients. Based on these findings, we conclude that Western blot analysis appears useful for confirmation of dystrophinopathy in BMD patients with normal staining on IHC. Exon deletion analysis by multiplex PCR using peripheral blood is also a simple and useful test for the diagnosis of dystrophinopathy, although it has limited sensitivity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

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    Sackstein, Robert; Fuhlbrigge, Robert

    2015-01-01

    Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex

  20. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

  1. Application of Intermittent Microwave Irradiation to Western Blot Analysis.

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    Liu, Yu-Ting; Toyokuni, Shinya

    2015-01-01

    We established a shortened protocol for Western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

  2. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvanta

  3. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

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    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  4. Validation of western blot for Histoplasma capsulatum antibody detection assay.

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    Almeida, Marcos de Abreu; Pizzini, Cláudia Vera; Damasceno, Lisandra Serra; Muniz, Mauro de Medeiros; Almeida-Paes, Rodrigo; Peralta, Regina Helena Saramago; Peralta, José Mauro; Oliveira, Raquel de Vasconcelos Carvalhaes; Vizzoni, Alexandre Gomes; de Andrade, Carla Lourenço Tavares; Zancopé-Oliveira, Rosely Maria

    2016-02-24

    Histoplasmosis is worldwide systemic mycoses caused by the dimorphic fungus Histoplasma capsulatum. The isolation and identification of H. capsulatum in culture is the reference test for histoplasmosis diagnosis confirmation. However, in the absence of it, serology has been used as a presumptive diagnosis through antibody and antigen detection. The purpose of the present study was to validate an immunoassay method (western blot) for antibodies detection in the diagnosis of histoplasmosis. To validate the western blot (WB) a study was conducted using 118 serum samples from patients with histoplasmosis and 118 serum controls collected from January 2000 to December 2013 in residents of the Rio de Janeiro State, Brazil. Diagnostic validation parameters were calculated based on the categorization of results obtained in a 2 × 2 table and subjected to statistical analysis. In addition, the viability of deglycosylated histoplasmin antigen (ptHMIN) onto nitrocellulose membranes previously sensitized was evaluated during the same period. The WB test showed sensitivity of 94.9 %, specificity of 94.1 %, positive predictive value of 94.1 %, negative predictive value of 94.9 %, accuracy of 94.5 %, and almost perfect precision. Besides, the strips have proved to be viable for using at least 5 years after ptHMIN antigen sensitization. Western blot test using ptHMIN provides sensitive, specific, and faster results. Therefore, could be considered a useful tool in the diagnosis of histoplasmosis being used by public health system, even in situations where laboratory facilities are relatively limited.

  5. The diagnostic value of Western blot method in patients with cystic echinococcosis.

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    Aslan, Mustafa; Yüksel, Pelin; Polat, Erdal; Cakan, Huseyin; Ergin, Sevgi; Öner, Y Ali; Zengin, Kagan; Arıkan, Soykan; Saribas, Suat; Torun, Muzeyyen Mamal; Kocazeybek, Bekir

    2011-04-01

    Cystic echinococcosis (CE) is the larval cystic stage (called echinococcal cysts) of a small taeniid-type tapeworm (Echinococcus granulosus). Carnivores such as dogs are usually definitive hosts. Intermediate hosts are typically herbivores such as sheep and cattle. CE can be detected using various imaging techniques such as ultrasonography or radiology. Moreover the primary diagnosis has to be confirmed by serological tests since the clinical signs of the disease are non-specific. This study examined the antigenic band patterns useful for serologic diagnosis of hydatidosis. We also report on the post-operative evolution of patients treated for this disease and also determined the diagnostic performance of Western blot IgG kit. Twenty-five (16 females and 9 males) non-operated patients with hydatid cysts (NOP) and 33 (21 females and 12 males) operated patients with hydatid cysts (OP) were included as study group and 22 healthy individuals (14 females and 8 males) with no known chronic diseases were included as a control group. The ages of the patients and control group individuals were between 16-83 years. Patient and control groups were matched for age and sex. Cyst hydatid IgG antibodies were detected in the sera from all patient groups but no antibodies were found in the sera from the control group using ELISA IgG method. Twenty-three (92%) non-operated patients and 18 (54.5%) operated patients exhibited positive results when Western blot IgG kit was used. The P7 band pattern was detected in the sera from all operated and non-operated patients. Twenty-seven of these positive cases had p7 and (p7+p16/18), (p7+p24/26) or (p7+p16/18+p24/26). No antibodies against p7, p16/18 ve p24/26 band patterns were seen in sera from the control group A statistically significant difference was detected between operated and nonoperated patients for Western blot positivity.(pWestern blot kit for 25 cases with CE and 22 healthy controls were calculated as 92%, 100%, 100% and 91

  6. Evaluating strategies to normalise biological replicates of Western blot data.

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    Degasperi, Andrea; Birtwistle, Marc R; Volinsky, Natalia; Rauch, Jens; Kolch, Walter; Kholodenko, Boris N

    2014-01-01

    Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

  7. Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot.

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    Putra, Sulistyo Emantoko Dwi; Tsuprykov, Oleg; Von Websky, Karoline; Ritter, Teresa; Reichetzeder, Christoph; Hocher, Berthold

    2014-01-01

    Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04-5.71%) as assessed by coefficient of variation. ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.

  8. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

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    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  9. The fastest Western in town: a contemporary twist on the classic Western blot analysis.

    Science.gov (United States)

    Silva, Jillian M; McMahon, Martin

    2014-02-05

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

  10. Antibody performance in western blot applications is context-dependent.

    Science.gov (United States)

    Algenäs, Cajsa; Agaton, Charlotta; Fagerberg, Linn; Asplund, Anna; Björling, Lisa; Björling, Erik; Kampf, Caroline; Lundberg, Emma; Nilsson, Peter; Persson, Anja; Wester, Kenneth; Pontén, Fredrik; Wernérus, Henrik; Uhlén, Mathias; Ottosson Takanen, Jenny; Hober, Sophia

    2014-03-01

    An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    Science.gov (United States)

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

  12. Cy5 total protein normalization in Western blot analysis.

    Science.gov (United States)

    Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

    2015-10-01

    Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Characterization of Nora Virus Structural Proteins via Western Blot Analysis

    Directory of Open Access Journals (Sweden)

    Brad L. Ericson

    2016-01-01

    Full Text Available Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs. The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles and ~1.33 g/mL (complete virions. Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

  14. The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

    Science.gov (United States)

    Rojas-Espinosa, O; Silva-Miranda, M; Wek-Rodriguez, K; Arce-Paredes, P

    2006-01-01

    We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

  15. Blame it on Southern, but it's a western blot.

    Science.gov (United States)

    Klionsky, Daniel J

    2017-01-02

    Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

  16. Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

    Science.gov (United States)

    Kong, Weiwei; Li, Yan; Cheng, Shaohui; Yan, Chen; An, Shiping; Dong, Zheng; Yan, Lina; Yuan, Yuhua

    2016-01-01

    Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (pWestern blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (pWestern blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Richter, Erik A

    2011-01-01

    This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies.......This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies....

  18. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  19. Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis.

    Science.gov (United States)

    Takesue, Masataka; Osaka, Yuki; Muranaka, Masanori; Katayama, Yoshinari; Ikadai, Hiromi

    2016-01-01

    In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.

  20. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    Science.gov (United States)

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A duplex approach for immunochemical staining and typing of protein in western blots

    NARCIS (Netherlands)

    Kuczius, T.; Brandstädter, L.; Karch, H.; Langeveld, J.P.M.

    2011-01-01

    The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a

  2. Comparisons of ELISA and Western blot assays for detection of autophagy flux.

    Science.gov (United States)

    Oh, Sung-Hee; Choi, Yong-Bok; Kim, June-Hyun; Weihl, Conrad C; Ju, Jeong-Sun

    2017-08-01

    We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3).

  3. Immunochemische detectiemethoden na western blotting van cytochroom P-450 iso-enzymen

    NARCIS (Netherlands)

    Laan CA; Jansen EHJM

    1992-01-01

    In this report a number of staining techniques on Western blots have been compared with respect to sensitivity, background staining, practical applicability and cost aspects. After electrophoresis of a rat microsomal liver sample followed by blotting, an incubation was performed of a primary antibo

  4. Use of Nonradioactive Detection Method for North- and South-Western Blot.

    Science.gov (United States)

    Franke, Claudia; Gräfe, Daniel; Bartsch, Holger; Bachmann, Michael P

    2015-01-01

    Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

  5. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

    Science.gov (United States)

    Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

    2012-06-15

    Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    Science.gov (United States)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  7. Two-dimensional gel-based protein standardization verified by western blot analysis.

    Science.gov (United States)

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  8. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    Science.gov (United States)

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  9. A western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins...

  10. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    Science.gov (United States)

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

  11. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  12. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

    Science.gov (United States)

    Omotola, Oluwabukola B.; Heda, Rajiv P.; Avery, Jamie

    2016-01-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin’s transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting. PMID:27582639

  13. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    Science.gov (United States)

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  14. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    Science.gov (United States)

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  15. Western blotting as a method for studying cell-biomaterial interactions : The role of protein collection

    NARCIS (Netherlands)

    van Kooten, TG; Klein, CL; Kirkpatrick, CJ

    2001-01-01

    Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure

  16. Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot.

    Science.gov (United States)

    Xu, Dong; Mane, Sarthak; Sosic, Zoran

    2015-01-01

    This paper describes the application of an automated size-based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion-Fc protein characterization and evaluation of its purification process. The fusion-Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion-Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion-Fc product-related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    Science.gov (United States)

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.

  18. Native Electrophoresis and Western Blot Analysis (NEWeB): Methods and Applications.

    Science.gov (United States)

    Manoussopoulos, Ioannis N; Tsagris, Mina

    2015-01-01

    Native Electrophoresis and Western Blot Analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle-protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP-protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.

  19. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite

    OpenAIRE

    Son, Eui-Sun; Kim, Tong Soo; Nam, Ho-Woo

    2001-01-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce ...

  20. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

    Directory of Open Access Journals (Sweden)

    Palacios Ricardo

    1999-01-01

    Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

  1. Imported intraocular gnathostomiasis with subretinal tracks confirmed by western blot assay.

    Science.gov (United States)

    Yang, Ji Ho; Kim, Moosang; Kim, Eung Suk; Na, Byoung-Kuk; Yu, Seung-Young; Kwak, Hyung-Woo

    2012-03-01

    We report a case of intraocular gnathostomiasis diagnosed by western blot assay in a patient with subretinal tracks. A 15-year-old male patient complained of blurred vision in the right eye, lasting for 2 weeks. Eight months earlier, he had traveled to Vietnam for 1 week and ate raw wild boar meat and lobster. His best-corrected visual acuity was 20/20 in both eyes and anterior chamber examination revealed no abnormalities. Fundus examination showed subretinal tracks in the right eye. Fluorescein angiography and indocyanine green angiography showed linear hyperfluorescence of the subretinal lesion observed on fundus in the right eye. Ultrasound examination revealed no abnormalities. Blood tests indicated mild eosinophilia (7.5%), and there was no abnormality found by systemic examinations. Two years later, the patient visited our department again for ophthalmologic evaluation. Visual acuity remained 20/20 in both eyes and the subretinal tracks in the right eye had not changed since the previous examination. Serologic examination was performed to provide a more accurate diagnosis, and the patient's serum reacted strongly to the Gnathostoma nipponicum antigen by western blot assay, which led to a diagnosis of intraocular gnathostomiasis. This is the first reported case of intraocular gnathostomiasis with subretinal tracks confirmed serologically using western blot in Korea.

  2. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

    Directory of Open Access Journals (Sweden)

    Tappe D

    2009-09-01

    Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

  3. Use of a Western blot technique for the serodiagnosis of glanders

    Directory of Open Access Journals (Sweden)

    de Souza Marcilia MA

    2011-01-01

    Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  4. More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

    Science.gov (United States)

    Evans, Roger; Mavin, Sally; McDonagh, Susan; Chatterton, Jean M W; Milner, Rachel; Ho-Yen, Darrel O

    2010-08-01

    To identify further Western blot bands that may be specific in the diagnosis of Lyme borreliosis. The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined. 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test). Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

  5. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    Science.gov (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  6. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

    Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

  7. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    applications and that this influences the epitopes exposed on the target protein, which might have profound consequences for the ability of a given antibody to bind specifically to its target. As an example, proteins that are analyzed by immunohistochemistry (IHC) are normally first cross-linked with formalin....... In conclusion, western blot and protein array analyses can indeed be useful tools when selecting specific antibodies for other applications. The use of these methods is encouraged both for antibody providers and users, and antibodies with signs of cross-reactivity in these applications should be treated...

  8. Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

    Science.gov (United States)

    Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

    2015-01-01

    Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.

    Science.gov (United States)

    Kim, Jungho; Wang, Hye-Young; Kim, Seoyong; Park, Soon Deok; Yu, Kwangmin; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 10(3)CFU/mL for GP bacteria, 10(3)CFU/mL for GN bacteria, and 10(4)CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay. Copyright © 2016. Published by Elsevier B.V.

  10. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    Science.gov (United States)

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Western blot assay for quantitative and qualitative antigen detection in vaccine development.

    Science.gov (United States)

    Kumar, Sanjai; Zheng, Hong; Mahajan, Babita; Kozakai, Yukiko; Morin, Merribeth; Locke, Emily

    2014-05-01

    Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Copyright © 2014 John Wiley & Sons, Inc.

  12. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

  13. Analysis of sperm membrane antigens relevant to antisperm antibody using Western blot

    Institute of Scientific and Technical Information of China (English)

    WangHF

    2002-01-01

    Objective:To identify the sperm membrane antigens associated with antisperm antibody.Methods:The antisperm antibody in serum was tested by ELISA.Antisperm antibody positive sera from 18 infertile men and 15 infertile women were used.The molecular weight(MW) of sperm membrane antigens associated with the antisperm antibody was analyzed with antisperm antibody positive serum using Western blot.Results:Eight kinds of MW of sperm membrane antigens were identified.The ratio of identification on the 78 KD(60.7%),60KD(71.4%),51KD(14.9%) and 23KD(14.29%)sperm antigen was higher than other.Conclusion:sperm membrane antigens with MW of 78KD,60KD,51KD and 23KD were associated with antisperm antibody and immunological infertility.

  14. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  15. TSE strain differentiation in mice by immunohistochemical PrP(Sc) profiles and triplex Western blot.

    Science.gov (United States)

    van Keulen, Lucien J M; Langeveld, Jan P M; Dolstra, Corry H; Jacobs, Jorg; Bossers, Alex; van Zijderveld, Fred G

    2015-10-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies. © 2014 British Neuropathological Society.

  16. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.

    Science.gov (United States)

    Son, E S; Kim, T S; Nam, H W

    2001-06-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

  17. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  18. Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

    Science.gov (United States)

    Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

    2016-05-01

    Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis,

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    Jaqueline Dario Capobiango

    Full Text Available Abstract Objective: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii (IgG-WB in the serum of children with suspected congenital toxoplasmosis. Methods: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. Results: 15 children (15.1% met the criteria for congenital toxoplasmosis and 32 (32.3% had the diagnosis excluded. The symptoms were observed in 12 (80.0% children and the most frequent were cerebral calcification in 9 (60.0%, chorioretinitis in 8 (53.3%, and hydrocephalus in 4 (26.6%. IgM antibodies anti-T. gondii detected by chemiluminescence (CL were found in 6 (40.0% children and the polymerase chain reaction (PCR for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%. The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%. The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. Conclusions: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

  20. Identification of α1-Antitrypsin as a Potential Candidate for Internal Control for Human Synovial Fluid in Western Blot.

    Science.gov (United States)

    Wang, Shaowei; Zhou, Jingming; Wei, Xiaochun; Li, Pengcui; Li, Kai; Wang, Dongming; Wei, Fangyuan; Zhang, Jianzhong; Wei, Lei

    Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.

  1. Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

    Science.gov (United States)

    Fischer, Peter U; Curtis, Kurt C; Folk, Scott M; Wilkins, Patricia P; Marcos, Luis A; Weil, Gary J

    2013-06-01

    Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

  2. A new Western blot assay for the detection of porcine cytomegalovirus (PCMV).

    Science.gov (United States)

    Plotzki, Elena; Keller, Martina; Ivanusic, Daniel; Denner, Joachim

    2016-10-01

    Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection.

    Science.gov (United States)

    Feng, Xia; Wang, Jibao; Gao, Zhiyun; Tian, Yu; Zhang, Ling; Chen, Huichao; Zhang, Tong; Xiao, Lin; Yao, Jun; Xing, Wenge; Qiu, Maofeng; Jiang, Yan

    2017-03-01

    In China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions. The aim of this study was to evaluate the efficacy of a dry blood spots (DBS)-urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection. Plasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed. A diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa=0.954) between the two diagnostic strategies. The DBS-urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

    Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

  5. Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting.

    Science.gov (United States)

    Sudhakar, N R; Samanta, S; Sahu, Shivani; Raina, O K; Gupta, S C; Goswami, T K; Lokesh, K M; Kumar, Ashok

    2014-06-01

    Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384 kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105 kDa. Common antigens band were observed at 57 and 105 KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

  6. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.

  7. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

    Science.gov (United States)

    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  8. Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development.

    Science.gov (United States)

    Rustandi, Richard R; Loughney, John W; Hamm, Melissa; Hamm, Christopher; Lancaster, Catherine; Mach, Anna; Ha, Sha

    2012-09-01

    Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Novel chemiluminescent Western blot blocking and antibody incubation solution for enhanced antibody-antigen interaction and increased specificity.

    Science.gov (United States)

    Schwartz, Kimberly; Bochkariov, Dmitry

    2017-07-13

    Western blotting is a ubiquitous tool used in protein and molecular biology research, providing information about the presence, size, relative abundance, and state of a protein in a mixture. First, the proteins in a sample are separated by size using SDS-PAGE then transferred onto a membrane for detection with a set of primary and secondary antibodies. High-quality Western data requires high signal-to-noise ratios, which depend upon reduction of nonspecific antibody interactions. Blocking is a critical step in the Western blot method as it prevents the antibodies from binding nonspecifically to the membrane and irrelevant proteins. A solution of nonfat dry milk (NFDM) in physiological buffer is commonly used for this purpose, but does not perform well with every type of antibody and is not optimal for low-abundance proteins. We present a novel blocking solution for chemiluminescent Western blots, AdvanBlock™-chemi, which outperforms NFDM in experiments with 20 unique antibodies by increasing signal-to-noise ratios and minimizing nonspecific binding. This solution enhances protein detection by Western blot and provides consistent results for detection of low abundant and modified proteins. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Science.gov (United States)

    Ogden, Nicholas H; Arsenault, Julie; Hatchette, Todd F; Mechai, Samir; Lindsay, L Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  11. Western blot analysis of sera from dogs with suspected food allergy.

    Science.gov (United States)

    Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

    2017-04-01

    Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL(®) is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL(®) for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic(®) , Vet-Concept Kanguru(®) or Vet-Concept Dog Sana(®) ; 8% and 7% to Hill's d/d Duck and Rice(®) ; 8% and 21% to Hill's z/d Ultra Allergen Free(®) ; 53% and 64% to Eukanuba Dermatosis FP(®) ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL(®) for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL(®) was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL(®) test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

  12. Evaluation of a Western Blot Test in an Outbreak of Acute Pulmonary Histoplasmosis

    Science.gov (United States)

    Pizzini, Claudia V.; Zancopé-Oliveira, Rosely M.; Reiss, Errol; Hajjeh, Rana; Kaufman, Leo; Peralta, José Mauro

    1999-01-01

    A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test’s sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected. PMID:9874658

  13. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    Science.gov (United States)

    Kim, Dohyun; Karns, Kelly; Tia, Samuel Q; He, Mei; Herr, Amy E

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  14. Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

    Science.gov (United States)

    Hashemi, Maryam; Madani, Rasool; Razmi, Nematollah

    2014-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

  15. Enrichment of PrPSc in Formalin Fixed Paraffin Embedded Tissues Prior to Analysis by Western Blot

    Science.gov (United States)

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

  16. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  17. Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.

    Science.gov (United States)

    Rustandi, Richard R; Hamm, Melissa; Lancaster, Catherine; Loughney, John W

    2016-01-01

    Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.

  18. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    Science.gov (United States)

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  19. Investigation of telomerase activity and apoptosis on invasive ductal carcinoma of the breast using immunohistochemical and Western blot methods.

    Science.gov (United States)

    Simsek, B C; Turk, B A; Ozen, F; Tuzcu, M; Kanter, M

    2015-08-01

    Invasive ductal carcinoma (IDC) comprises the largest group of breast cancers. This study aimed to investigate telomerase activity and apoptosis using immunohistochemical and Western blot methods. In total, 75 cases that had been diagnosed as IDC and 20 cases that had undergone a freezing procedure were included. The histological sections were stained with Bax, Bcl-2, hTERT and BNIP3. The ages of the patients, as well as their hormonal status and tumour sizes and grades were evaluated, as well as the staining characteristics of the antibodies in question. A decrease in Bcl-2 positivity and an increase in Bax positivity were found immunohistochemically with increasing tumour grades. The data obtained by western blot method showed that Bcl-2 was highest in grade 1 tumours although these results were not statistically significant. The relationship between estrogen and progesterone receptor positivity and Bcl-2 was statistically significant, suggesting there is hormonal control through apoptosis. BNIP3 was found to be decreased with increasing tumour grades. Similarly, BNIP3 was found to be having the lowest value in grade 3 tumours by western blot method. Furthermore, hTERT was found to be increased with increasing tumour grades. In the western blot method, hTERT increased nearly four-fold compared to the control. In addition, hTERT, which was seen in very high levels in tumours, may be a helpful cancer marker. Both hTERT and BNIP3 are important markers that can provide information about prognosis. Big improvements can be achieved in tumour progression control with new treatment modalities that stop telomerase activity and hypoxic cell death.

  20. Western blot confirmation of the H+/K+-ATPase proton pump in the human larynx and submandibular gland.

    Science.gov (United States)

    Altman, Kenneth W; Kinoshita, Yayoi; Tan, Melin; Burstein, David; Radosevich, James A

    2011-11-01

    The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. Prospective controlled tissue analysis study. Academic medical institution. Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or β subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens. Consistent IHC staining was observed in the submandibular gland specimens for both α and β subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated β subunit protein, as well as the 52-kDa β subunit precursor for all specimens. The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

  1. Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

    Directory of Open Access Journals (Sweden)

    Mohammad Zibaei

    2013-01-01

    Full Text Available The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8% and 3 (3.5% persons exhibited Toxocara immunoglobulin G (IgG antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (, 3 (3.5% persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy.

  2. Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

    Science.gov (United States)

    Goasdoue, Kate; Awabdy, Doreen; Bjorkman, Stella Tracey; Miller, Stephanie

    2016-02-01

    A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

    Science.gov (United States)

    Melrose, J; Shen, B; Ghosh, P

    2001-12-01

    The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

  4. Transcription Factors Downstream of IL-4 and TGF-β Signals: Analysis by Quantitative PCR, Western Blot, and Flow Cytometry.

    Science.gov (United States)

    Sugimoto, Atsushi; Kawakami, Ryoji; Mikami, Norihisa

    2017-01-01

    IL-9-producing Th9 cell is a novel Th cell subset involved in type II allergic inflammations such as asthma. Th9 cells can be induced from naïve Th cells in the presence of IL-4 and TGF-β. It is also well established that downstream signals of IL-4 and TGF-β, including STAT6, IRF4, Smad, and PU.1, directly mediate IL-9 production in Th9 cells. In this chapter we describe the methods of flow cytometry, qPCR and western blot analysis to determine the expression or activation of these transcription factors downstream of IL-4 and TGF-β.

  5. How to Distinguish Between the Activity of HDAC1-3 and HDAC6 with Western Blot.

    Science.gov (United States)

    Beyer, Mandy; Kiweler, Nicole; Mahboobi, Siavosh; Krämer, Oliver H

    2017-01-01

    Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in their target proteins. This biochemical modification can have profound effects on the functions of these proteins and a dysregulation of HDAC activity contributes to severe diseases, including neoplastic transformation. In the following chapter, we present a strategy that allows to distinguish between the inhibition of the class I HDACs HDAC1, 2, and 3 and of the class IIb HDAC HDAC6. This method is based on Western blot and relies on the detection of hyperacetylated substrates of class I or class IIb HDACs in lysates from cells that were treated with histone deacetylase inhibitors (HDACi).

  6. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Science.gov (United States)

    Kale, Sonia; Kale, Anup; Gholap, Haribhau; Rana, Abhimanyu; Desai, Rama; Banpurkar, Arun; Ogale, Satishchandra; Shastry, Padma

    2012-03-01

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  7. [Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes].

    Science.gov (United States)

    Escalante, Hermes; Jara, César; Davelois, Kelly; Iglesias, Miguel; Benites, Adderly; Espinoza, Renzo

    2014-01-01

    Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

  8. Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

    Institute of Scientific and Technical Information of China (English)

    Hao-fei WANG; Zhu-qiong XIANG; Yi-xing WANG

    2003-01-01

    Objective To identify the sperm membrane proteins that are associated with antisperm antibodyMethods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody.Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5~6.2, 4.6,5.1,5.5~5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher.Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two-dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

  9. Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

    Science.gov (United States)

    Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

    1990-01-01

    The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors.

  10. Herpes simplex virus type 2 antibody detection performance in Kisumu, Kenya, using the Herpeselect ELISA, Kalon ELISA, Western blot and inhibition testing.

    Science.gov (United States)

    Smith, J S; Bailey, R C; Westreich, D J; Maclean, I; Agot, K; Ndinya-Achola, J O; Hogrefe, W; Morrow, R A; Moses, S

    2009-04-01

    In certain parts of Africa, type-specific herpes simplex virus type 2 (HSV-2) ELISAs may have limited specificity. To date, no study has been conducted to validate HerpeSelect and Kalon type-specific HSV-2 ELISAs using both the Western blot and recombinant gG ELISA inhibition testing as reference standards. A total of 120 men who were HIV seronegative (aged 18-24 years) provided blood samples. HSV-2 IgG serum antibodies were detected using four different methods: HerpeSelect HSV-2 ELISA (n = 120), Kalon HSV-2 ELISA (n = 120), University of Washington Western blot (n = 101) and a recombinant inhibition test (n = 93). HSV-2 seroprevalence differed significantly by HSV-2 detection method, ranging from 24.8% with the Western blot to 69.8% with the HerpeSelect ELISA. Using the Western blot as the reference standard, the HerpesSelect had the highest sensitivity for HSV-2 antibody detection (100%) yet lowest specificity (40%). Similar results were obtained using the inhibition test as the reference standard. The sensitivity and specificity of the Kalon test versus the Western blot were 92% and 79%, respectively, and 80% and 82% versus the inhibition test. Using the inhibition test as the reference standard, the sensitivity of the Western blot appeared low (49%). In men in western Kenya who were HIV seronegative, the HerpeSelect and Kalon type-specific ELISAs had high sensitivities yet limited specificities using the Western blot as reference standard. Overall, the Kalon ELISA performed better than the HerpeSelect ELISA in these young men from Kisumu. Further understanding is needed for the interpretation of HSV-2 inhibition or ELISA test positive/ Western blot seronegative results. Before HSV-2 seropositivity may be reliably reported in selected areas of Africa, performance studies of HSV-2 serological assays in individual geographical areas are recommended.

  11. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated capillary electrophoresis (CE) western blot.

    Science.gov (United States)

    Xu, Dong; Marchionni, Kentaro; Hu, Yunli; Zhang, Wei; Sosic, Zoran

    2017-10-25

    An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals. Appropriate analytical tools are needed to realize such goals by providing information on product quality at an early stage to help understanding and control of the manufacturing process. In this work, a fully automated, multi-capillary instrument is utilized for size-based separation and western blot analysis to provide an early readout on product quality in order to enable a more consistent manufacturing process. This approach aims at measuring two important qualities of a biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired data for isoform distribution can then be used to predict the corresponding values of the final drug substance, and potentially provide information for remedy through timely adjustment of the downstream purification process, should the expected values fall out of the accepted range. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation.

    Science.gov (United States)

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-12-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, "Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  13. Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.

    Science.gov (United States)

    Mielenz, M; Mielenz, B; Singh, S P; Kopp, C; Heinz, J; Häussler, S; Sauerwein, H

    2013-04-01

    Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro-differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.

    Science.gov (United States)

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui

    2014-10-01

    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  15. MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Bahrmand, Ahmadreza

    2015-12-01

    Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

  16. Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

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    Luiz Claudio Pereira Ribeiro

    2013-09-01

    Full Text Available Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1 antibodies (Abs represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP patients. Western blotting (WB for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I and gag (p24 proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

  17. Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection.

    Science.gov (United States)

    Cárdenas, Ana María; Baughan, Eleonore; Hodinka, Richard L

    2013-12-01

    In the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. To evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. A series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients>18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children≤18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. Multispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. Multispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Discrepancies between a new highly sensitive Toxoplasma gondii ELISA assay and other reagents: interest of Toxo IgG Western blot.

    Science.gov (United States)

    Leslé, F; Touafek, F; Fekkar, A; Mazier, D; Paris, L

    2011-10-01

    Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.

  19. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    Science.gov (United States)

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian

    2010-07-14

    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces. (c) 2009 Elsevier B.V. All rights reserved.

  20. Serodiagnosis of grass carp reovirus infection in grass carp Ctenopharyngodon idella by a novel Western blot technique.

    Science.gov (United States)

    He, Yongxing; Jiang, Yousheng; Lu, Liqun

    2013-12-01

    Frequent outbreaks of grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV) infection, pose as serious threats to the production of grass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnostic methods have been shown effective, lack of commercial monoclonal antibody against grass carp IgM has impeded the development of any reliable immunoassays in detection of GCRV infection. The present study describes the preparation and screening of monoclonal antibodies against the constant region of grass carp IgM protein, and the development of a Western blot (WB) protocol for the specific detection of antibodies against outer capsid VP7 protein of GCRV that serves as antibody-capture antigen in the immunoassay. In comparison to a conventional RT-PCR method, validity of the WB is further demonstrated by testing on clinical fish serum samples collected from a grass carp farm in Jiangxi Province during disease pandemic in 2011. In conclusion, the WB technique established in this study could be employed for specific serodiagnosis of GCRV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    Science.gov (United States)

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  3. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation

    Directory of Open Access Journals (Sweden)

    Haitham A. Badr

    2015-12-01

    Full Text Available This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  4. [Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

    Science.gov (United States)

    Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

    1995-01-01

    The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

  5. Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

    Directory of Open Access Journals (Sweden)

    NUNES Cáris Maroni

    1997-01-01

    Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

  6. Evaluation of western blotting for the diagnosis of enzootic bovine leukemia Avaliação da técnica de western blot no diagnóstico da leucose enzoótica bovina

    Directory of Open Access Journals (Sweden)

    E.T. Gonzalez

    1999-08-01

    Full Text Available A western blotting (WB procedure has been developed for detecting antibodies to bovine leukosis virus (BLV in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antigens (gp51 and p24, or against one of them. Other proteins (gp30, p15, p12 and p10 were not detected with any AGID positive sera, being observed occasionally three bands corresponding to the p24 protein. Using sera obtained by BLV experimental inoculation, the antibodies directed to p24 appeared early (between the 2nd and 4th week post inoculation and thereafter antibodies to gp51were detected in some animals. The analysis of field serum samples by AGID as compared to WB showed an agreement of 90.9%. Only 1.7% of sera were negative by AGID and positive by WB and 7.2% that were not conclusive by AGID and were defined by WB (4.2% as positive and 3.0% as negative.Um sistema de western blotting (WB foi desenvolvido para detecção de anticorpos contra o vírus da leucose em soros de bovinos. Foram utilizadas amostras de soros de 233 animais naturalmente infectados e soros de vacas experimentalmente infectadas. O teste de imunodifusão em ágar (AGID foi usado para comparação dos resultados. Graus diferentes de reatividade foram observados em soros positivos ao AGID, quando testados em WB frente a um ou aos dois antígenos mais importantes (gp51 e p24. Outras proteínas (gp30, p15, p12 e p10 não foram detectadas por nenhum soro positivo ao AGID, sendo que três bandas correspondentes à proteína p24 foram observadas ocasionalmente. Em soros obtidos por inoculação experimental, anticorpos contra a proteína p24 foram detectados entre a segunda e a quarta semanas

  7. Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

    Science.gov (United States)

    Cuttell, Leigh; Gómez-Morales, Maria Angeles; Cookson, Beth; Adams, Peter J; Reid, Simon A; Vanderlinde, Paul B; Jackson, Louise A; Gray, C; Traub, Rebecca J

    2014-01-31

    Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from

  8. Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

    Science.gov (United States)

    Desoubeaux, Guillaume; Pantin, Ana; Peschke, Roman; Joachim, Anja; Cray, Carolyn

    2017-02-01

    Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

  9. A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

    Science.gov (United States)

    Liu, Zhi Yun; Hao, Qin; Hou, Xue Xia; Jiang, Yi; Geng, Zhen; Wu, Yi Mou; Wan, Kang Lin

    2013-03-01

    To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  10. Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

    Science.gov (United States)

    Santana, Aline Elisa; Taborda, Carlos Pelleschi; Severo, Julia So; Rittner, Glauce Mary Gomes; Muñoz, Julian Esteban; Larsson, Carlos Eduardo; Larsson, Carlos Eduardo

    2017-03-11

    Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases.

    Science.gov (United States)

    Seyyedtabaei, Seyyed Javad; Rostami, Ali; Haghighi, Ali; Mohebali, Mehdi; Kazemi, Bahram; Fallahi, Shirzad; Spotin, Adel

    2017-01-01

    Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB) compared with indirect immunofluorescence test (IFAT) to serodiagnosis of leishmaniasis. This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups. WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  12. Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

    Science.gov (United States)

    Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

    2017-09-01

    Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

  13. Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting

    Directory of Open Access Journals (Sweden)

    Al-Bader Maie D

    2006-03-01

    Full Text Available Abstract Background High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified. Methods The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg. Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S, a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H and a polyclonal antibody raised against the amino terminus of ER beta. Results ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg. Conclusion This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage.

  14. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    Science.gov (United States)

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  15. Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis.

    Science.gov (United States)

    Giménez-Bachs, José M; Salinas-Sánchez, Antonio S; Serrano-Oviedo, Leticia; Nam-Cha, Syong H; Rubio-Del Campo, Antonio; Sánchez-Prieto, Ricardo

    2012-10-01

    This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression. An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC. The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade. Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

  16. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

    Directory of Open Access Journals (Sweden)

    Greenlee Justin J

    2011-09-01

    Full Text Available Abstract Background Three distinct forms of bovine spongiform encephalopathy (BSE, defined as classical (C-, low (L- or high (H- type, have been detected through ongoing active and passive surveillance systems for the disease. The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC and Western blot (WB BSE confirmatory protocols to detect C- and atypical (L- and H-type BSE forms. Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods. Results The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type. Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step. Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form. Conclusions The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE

  17. Evaluation of Western blot, ELISA and latex agglutination tests to detect Toxoplasma gondii serum antibodies in farmed red deer.

    Science.gov (United States)

    Patel, Kandarp Khodidas; Howe, Laryssa; Heuer, Cord; Asher, Geoffery William; Wilson, Peter Raymond

    2017-09-15

    Abortion due to Toxoplasma gondii has been suspected in New Zealand farmed red deer. However, knowledge around the epidemiology and prevalence of T. gondii in farmed red deer is limited. The aim of this study was to firstly, assess the sensitivity and specificity of two commercially available assays, ELISA and latex agglutination test (LAT), for use in deer and secondly, to estimate the sero-prevalence of T. gondii in red deer. A total of 252 sera from rising 2-year-old and adult hinds from 17 New Zealand red deer herds at early and late pregnancy scanning and from known aborted and/or non-aborted hinds were tested for the presence of T. gondii antibodies. Each assays' sensitivity and specificity was evaluated by both the Western Blot (WB) as a gold standard method and Bayesian latent class (BLC) analysis in the absence of a gold standard. The sensitivity and specificity for WB were 95.8% (95% credible interval: 89.5-99.2%) and 95.1% (95% credible interval: 90.6-98.1%), respectively. For the LAT at the manufacturer's recommended ≥1:32 cut-off titre, the sensitivity (88.7%, 95% credible interval: 80.8-94.7%) and specificity (74.3%, 95% credible interval: 67.5-80.5%) were lower and higher than the sensitivity (76.2%, 95% credible interval: 66.7-84.5%) and specificity (89.7%, 95% credible interval: 84.5-93.9%) at a ≥1:64 cut-off, using (BLC) analysis. Sensitivity and specificity of the LAT at cut-off titre of 1:32 were estimated to be 84.4% (95% CI: 74.9-90.9%) and 73.5% (95% CI: 65.8-79.9%) against WB. The LAT had better agreement with WB at cut-off titre of ≥1:64 than ≥1:32 (Kappa=0.63 vs 0.54). At optimised cut-off S/P of 15.5%, the sensitivity (98.8%, 95% credible interval 96.1-99.8%) and specificity (92.8%, 95% credible interval 88.9-95.7%) of the ELISA were higher and lower, respectively, than the sensitivity (85.1%, 95% credible interval 76.2-91.9%) and specificity (98.5%, 95% credible interval 96.9-99.4%) at manufacturer's cut-off S/P of 30%, from BLC

  18. Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

    Directory of Open Access Journals (Sweden)

    Nilton Thaumaturgo

    2002-10-01

    Full Text Available Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females, Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

  19. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    Science.gov (United States)

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

  20. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

    Science.gov (United States)

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

    2012-01-01

    Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

  1. Expansion of HIV screening to non-clinical venues is aided by the use of dried blood spots for Western blot confirmation.

    Science.gov (United States)

    Sullivan, Timothy J; Antonio-Gaddy, Mara San; Richardson-Moore, April; Styer, Linda M; Bigelow-Saulsbery, Deborah; Parker, Monica M

    2013-12-01

    HIV rapid testing programs in New York State (NYS) are required to collect a specimen for confirmation of a preliminary positive result; however, some venues have limited capacity to collect venous blood, and confirmation using oral fluid is restricted by cost and availability. To evaluate the feasibility of using dried blood spots (DBS) at non-clinical HIV rapid testing sites for Western blot testing. The New York State Department of Health facilitated registration of 48 non-clinical HIV test sites and provided training on DBS procedures. Following a reactive rapid test, DBS were collected by fingerstick onto filter paper cards, dried and mailed to the NYS public health laboratory for Western blot testing. From October 2010 to December 2012, 280 DBS specimens were submitted for confirmation. Four (1.4%) were unsatisfactory for testing and 276 (98.6%) DBS were tested. Of these, 235 (85.1%) were positive, 37 (13.4%) were negative and 4 (1.4%) were indeterminate. During this period, the laboratory also received 1033 venous blood specimens for rapid test confirmation, and 35 (3.4%) were unsatisfactory. Of the 998 tested by Western blot, 784 (78.6%) were positive, 197 (19.7%) were negative and 17 (1.7%) were indeterminate. Compared to venous blood, the percentage of rapid test referral specimens with a positive Western blot was significantly greater for DBS specimens and the frequency of unsatisfactory specimens did not differ significantly. These results indicate that DBS are a suitable alternative to venous blood for confirmation of HIV rapid tests conducted at non-clinical sites. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Analysis of the Crude Antigen of Hymenolepis nana from Mice by SDS-PAGE and the Determination of Specific Antigens in Protein Structure by Western Blotting

    OpenAIRE

    GÖNENÇ, Bahadır

    2002-01-01

    Protein bands of crude antigens of Hymenolepis nana were determined by SDS-PAGE and Western blotting. Thirty Swiss albino mice were allotted into two groups of 15 each as positive (infected with H. nana) and negative (non-infected with H. nana) groups. The natural infections of H. nana and other helminths were determined by centrifugal flotation of faeces. After bleeding, the mice were necropsied and their guts were examined for H. nana and other intestinal helminths. Sera from mice were test...

  3. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

    Science.gov (United States)

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-08-17

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

  4. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-09-11

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  5. Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.

    Science.gov (United States)

    Martinet, Wim; Abbeloos, Vanessa; Van Acker, Nathalie; De Meyer, Guido R Y; Herman, Arnold G; Kockx, Mark M

    2004-03-01

    In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells.

  6. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

    Science.gov (United States)

    Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

    2012-06-01

    With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

  7. Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression† ▿

    OpenAIRE

    Stanton, Richard James; McSharry, Brian Patrick; Rickards, Carole Ruth; Wang, Edward Chung Yern; Tomasec, Peter; Wilkinson, Gavin William Grahame

    2007-01-01

    Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotti...

  8. Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

    Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

  9. Study of a viral-dual infection in rainbow trout (Oncorhynchus mykiss) by seroneutralization, western blot and polymerase chain reaction assays.

    Science.gov (United States)

    Rodríguez, S; Vilas, M P; Alonso, M; Pérez, S I

    1995-12-01

    Viral-dual infections in fish are of interest to aquaculture practices but they are rarely described and studied. Several methods were applied in this work to demonstrate a case of coinfection in a reared rainbow trout (Oncorhynchus mykiss) population. Inoculation in cell cultures and cross-neutralization tests were the standard procedures that made it possible to isolate and identify a birnavirus, the infectious pancreatic necrosis virus (IPNV), and suspect of a second virus. Western blotting with both polyclonal and monoclonal antibodies, and reverse transcriptional-polymerase chain reaction (RT-PCR) demonstrate coexistence of both, IPNV and a rhabdovirus.

  10. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. APPLICATION OF WESTERN BLOTTING TECHNIQUE FOR EVALUATING THE EXPRESSION OF VASOPRESSIN RECEPTORS IN THE HEART CELLS; IMPORTANCE IN THE CARDIOVASCULAR SYSTEM

    Directory of Open Access Journals (Sweden)

    Manoj G Tyagi

    2012-08-01

    Full Text Available Vasopressin, a posterior pituitary hormone is responsible for water reabsorption by the kidneys and maintenance of cardio-vascular homeostasis. Vasopressin receptors are characterized as VR 1 (V1a, VR2 (V2, and VR3 (V1b. VR1, which is abundant in vascular smooth muscles, causes vasoconstriction by increasing intracellular calcium via the phosphatidylinositol bisphosphonate pathway and a positive inotropic effect in cardiac muscle. VR2 has also been shown to be expressed in the heart. There is emerging role for vasopressin receptors in health and disease. This study describes the application of Western blotting to elucidate the importance of vasopressin receptors in the heart cells.

  12. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    Science.gov (United States)

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ((25)ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ((27)FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

    Science.gov (United States)

    Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E; McIntosh, Mark A; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

  14. A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

    Science.gov (United States)

    Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

    2015-06-01

    To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

  15. A theoretical timeline for myocardial infarction: immunohistochemical evaluation and western blot quantification for Interleukin-15 and Monocyte chemotactic protein-1 as very early markers.

    Science.gov (United States)

    Turillazzi, Emanuela; Di Paolo, Marco; Neri, Margherita; Riezzo, Irene; Fineschi, Vittorio

    2014-07-02

    Experimental and human studies have demonstrated that innate immune mechanisms and consequent inflammatory reaction play a critical role in cardiac response to ischemic injury. Thus, the detection of immuno-inflammatory and cellular phenomena accompanying cardiac alterations during the early inflammatory phase of myocardial infarction (MI) may be an excellent diagnostic tool. Current knowledge of the chronology of the responses of myocardial tissue following the occurrence of ischemic insult, as well as the existence of numerous studies aiming to identify reliable markers in dating MI, induced us to investigate the myocardial specimens of MI fatal cases in order to better define the age of MI. We performed an immunohistochemical study and a Western blot analysis to evaluate detectable morphological changes in myocardial specimens of fatal MI cases and to quantify the effects of cardiac expression of inflammatory mediators (CD15, IL-1β, IL-6, TNF-α, IL-15, IL-8, MCP-1, ICAM-1, CD18, tryptase) and structural and functional cardiac proteins. We observed a biphasic course of MCP-1: it was strongly expressed in the very early phase (0-4 hrs), to diminish in the early period (after 6-8 hrs). Again, our choice of IL-15 is explained by the synergism with neutrophilic granulocytes (CD15) and our study shows the potential for striking cytokine synergy in promoting fast, local neutrophil response in damaged tissues. A progressively stronger immunoreaction for the CD15 antibody was visible in the areas where the margination of circulating inflammatory cells was detectable, up to very strong expression in the oldest ones (>12 hours). Further, the induction of CD15, IL-15, MCP-1 expression levels was quantified by Western blot analysis. The results were as follows: IL-15/β-actin 0.80, CD15/β-actin 0.30, and MCP-1/β-actin 0.60, matching perfectly with the results of immunohistochemistry. Control hearts from traumatic death cases did not show any immunoreactivity to the

  16. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    Science.gov (United States)

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. A comparison of antigenic peptides in muscle larvae of several Trichinella species by two-dimensional western-blot analysis with monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Dea-Ayuela M.A.

    2001-06-01

    Full Text Available The antigens recognised by mAb US5 specific to 53 kDa glycoprotein (gp 53 in T. spiralis L-1 muscle larvae (TSL1 antigens, mAb US9 specific to gp 53 in TSL1 from all encapsulated species and mAb US4 specific to a tyvelose containing tetrasaccharide present in TSL1, were investigated in crude extracts from muscle larvae of T. spiralis, T. nativa and T. britovi by 2D-electrophoresis and western-blot. At least four proteins of different pI were recognised by mAb US5 on T. spiralis antigens. Recognition profile of mAb US9 on T. spiralis antigens exhibited some variation with regard to that of the US5. Polymorphism was apparent in gp 53. High reactivity was shown by the mAb US4 with the three species.

  18. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Dybboe, Rie; Hansen, Christina Neigaard

    2015-01-01

    physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from...... SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level...... and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology....

  19. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    Science.gov (United States)

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  20. Use of rapid HIV assays as supplemental tests in specimens with repeatedly reactive screening immunoassay results not confirmed by HIV-1 Western blot.

    Science.gov (United States)

    Wesolowski, Laura G; Delaney, Kevin P; Meyer, William A; Blatt, Amy J; Bennett, Berry; Chavez, Pollyanna; Granade, Timothy C; Owen, Michele

    2013-09-01

    An alternate HIV testing algorithm has been proposed which includes a fourth-generation immunoassay followed by an HIV-1/HIV-2 antibody differentiation supplemental test for reactive specimens and a nucleic acid test (NAT) for specimens with discordant results. To evaluate the performance of five rapid tests (Alere Clearview, Bio-Rad Multispot, OraSure OraQuick, MedMira Reveal, and Trinity Biotech Unigold) as the supplemental antibody assay in the algorithm. A total of 3273 serum and plasma specimens that were third-generation immunoassay repeatedly reactive and Western blot (WB) negative or indeterminate were tested with rapid tests and NAT. Specimens were classified by NAT: (1) HIV-1 infected (NAT-reactive; n=184, 5.6%), (2) HIV-status unknown (NAT nonreactive; n=3078, 94.2%) or by Multispot, (3) HIV-2 positive (n=5), and (4) HIV-1 and HIV-2 positive (n=6). Excluding HIV-2 positive specimens, we calculated the proportion of reactive rapid tests among specimens with reactive and nonreactive NAT. The proportion of infected specimens with reactive rapid test results and negative or indeterminate WB ranged from 30.4% (56) to 47.8% (88) depending on the rapid test. From 1% to 2% of NAT-negative specimens had reactive rapid test results. In these diagnostically challenging specimens, all rapid tests identified infections that were missed by the Western blot, but only Multispot could differentiate HIV-1 from HIV-2. Regardless of which rapid test is used as a supplemental test in the alternative algorithm, false-positive algorithm results (i.e., reactive screening and rapid test in uninfected person) may occur, which will need to be resolved during the baseline medical evaluation. Published by Elsevier B.V.

  1. Pacientes neurológicos del noroeste del Perú con serología positiva por Western Blot a la larva de Taenia solium

    Directory of Open Access Journals (Sweden)

    Hermes Escalante A

    2004-04-01

    Full Text Available Objetivos: Determinar la frecuencia de pacientes con sintomatología neurológica de la zona noroeste del Perú que presentan serología positiva por Western Blot a la larva de Taenia solium. Material y Métodos: El estudio se realizó en 3515 pacientes de cero a noventa años de edad y de ambos sexos con sintomatología neurológica, procedentes de los departamentos de Piura, Lambayeque, Cajamarca, Ancash, La Libertad y algunas poblaciones de la zona selvática (5 a 10° LS; 77 a 82° LO. Dichos pacientes fueron atendidos en consultorios privados y en los servicios de Neurología de los Hospitales Belén y Regional de Trujillo desde enero de 1997 hasta diciembre de 2000. De cada uno de los pacientes se obtuvo una muestra sanguínea, cuyo suero fue procesado por la técnica de Western Blot, la cual tiene una sensibilidad de 91% y una especificidad de 100%. Resultados: Se detectó serología positiva en 562 pacientes, la cual representa una prevalencia de 16%, siendo el sexo masculino el que presentó mayor porcentaje (58,4%. Los grupos etáreos con mayor frecuencia de serología positiva fueron los de 41 a 50 años (18,7% y 31 a 40 años (17,4%. Los mayores porcentajes de serología positiva se obtuvo en pacientes procedentes de Piura y Lambayeque. Conclusiones: Los pacientes estudiados presentan una alta frecuencia de serología positiva a la larva de T. solium.

  2. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya.

    Science.gov (United States)

    Njiiri, Nyawira E; Bronsvoort, B Mark deC; Collins, Nicola E; Steyn, Helena C; Troskie, Milana; Vorster, Ilse; Thumbi, S M; Sibeko, Kgomotso P; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C; Woolhouse, Mark; Toye, Philip

    2015-05-30

    The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

  3. Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

    Institute of Scientific and Technical Information of China (English)

    Hao Chanjuan; Xu Chenggang; Zhang Zhiyun; Liang Aihua

    2008-01-01

    An insect excitatory toxin gene from Buthus martensii Karsch (BmKIT) was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Escherichia coli BL21 (DE3) host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.

  4. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

    Directory of Open Access Journals (Sweden)

    Kelly Davelois

    Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

  5. Effects of tetracyclines on neutrophil, gingival, and salivary collagenases. A functional and western-blot assessment with special reference to their cellular sources in periodontal diseases.

    Science.gov (United States)

    Sorsa, T; Ding, Y; Salo, T; Lauhio, A; Teronen, O; Ingman, T; Ohtani, H; Andoh, N; Takeha, S; Konttinen, Y T

    1994-09-06

    The characterization and regulation of matrix metalloproteinases (MMPs) have been studied to determine their role(s) in periodontal tissue destruction. Progress in elucidating the roles of MMPs in periodontal tissue destruction has led to a new concept involving the chemotherapeutic inhibition on MMPs, a therapeutic strategy which less than a decade ago was considered "a difficult and perhaps impossible task." Tetracyclines/doxycycline (DOXY) and their chemically modified nonantimicrobial derivatives (CMTs) are known to inhibit the matrix metalloproteinases, especially preferring human neutrophil collagenase (MMP-8), and prevent the oxidative activation of procollagenases. We characterized by Western blotting the molecular forms and cellular sources of gingival tissue, dental plaque, gingival crevicular fluid (GCF), and salivary MMPs associated with periodontitis. Also the molecular forms of tissue inhibitors of matrix metalloproteinases (TIMP-1 and TIMP-2) in periodontitis were studied by Western blot. Neutrophil (PMN)-derived MMPs were found to predominate in periodontitis, and phospholipase C present in increased amounts in periodontitis sites was found to be a potential inducer of PMN degranulation. We further studied the effects of DOXY on molecular forms of different latent and active MMPs purified from different cellular sources (PMNs, fibroblasts, keratinocytes) and present in vivo in oral exudates (gingival extracts, GCF, and saliva). DOXY inhibition of activated (oxidatively or proteolytically) MMPs were not associated with MMP fragmentation. Michaelis-Menten plots of initial rates of degradation of soluble type I collagen revealed an apparent Km value of 0.3-0.6 microM for MMP-8, and 75 microM DOXY inhibited MMP-8 in a manner which did not result in changes in apparent Km value but did prevent the initial degradation reaching Vmax providing evidence for noncompetitive inhibition. Treatment of patients with long-term DOXY medication results in decreased

  6. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    Science.gov (United States)

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  7. Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

    Science.gov (United States)

    Alenazi, Fahaad S H; Ibrahim, Baher A; Briski, Karen P

    2015-04-01

    Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Immunohistochemical and western blot analysis suggest that the soluble forms of FGF1-2 and FGFR1-2 sustain tail regeneration in the lizard.

    Science.gov (United States)

    Alibardi, Lorenzo

    2017-08-18

    Fibroblast Growth Factors 1-2 (FGF1-2) stimulate tail regeneration in lizards and therefore the distribution of their receptors, FGFR1-2, in the regenerating tail of the lizard. Podarcis muralis has been studied using immunofluorescence and western blotting. Immunoreactive protein bands at 15-16kDa for FGF1-2 in addition to those at 50-65kDa are detected in the regenerating epidermis, but weak bands at 35, 45 and 50kDa appear from the regenerating connective tissues. Strongly immunolabeled bands for FGFR1 at 32, 60, and 80kDa and less intense for FGFR2 only appear in the regenerating tail. In normal tail epidermis and dermis, higher MW forms are present at 80 and 115-140kDa, respectively, but they disappear in the regenerating epidermis and dermis where low MW forms of FGFR1-2 are found at 50-70kDa. Immunolocalization confirms that most FGFR1-2 are present in the wound epidermis, Apical Epidermal Peg, ependymal tube while immunolabeling lowers in regenerating muscles, blastema cells, cartilage and connectives tissues. The likely release of FGFs from the Apical Epidermal Peg and ependyma and the presence of their receptors in these tissues may determine the autocrine stimulation of proliferation and a paracrine stimulation of the blastema cells through their FGF Receptors. Copyright © 2017 Elsevier GmbH. All rights reserved.

  9. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    Science.gov (United States)

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  10. Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

    Science.gov (United States)

    Woźniak, Marta; Gamian, Elżbieta; Łaczmańska, Izabela; Sąsiadek, Maria M; Duś-Szachniewicz, Kamila; Ziółkowski, Piotr

    2014-05-01

    Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

  11. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

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    Terezinha de Jesus Teixeira Santos

    2003-06-01

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

  12. Evaluación de la técnica Western blot para la detección de antígenos de Hymenolepis nana

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    Flora Chávez-Salas

    2013-04-01

    Full Text Available Este trabajo tuvo como objetivo evaluar la técnica de inmunoelectrotransferencia (Western Blot para detectar los antígenos específicos de excreción/secreción de Hymenolepis nana en sueros de pacientes con himenolepiosis y con otras helmintiosis confirmadas. Se utilizó a Mesocricetus auratus “hamster” para obtener ejemplares adultos de H. nana. Los antígenos de excreción/secreción fueron obtenidos en el medio MEM (Minimum Essential Medium Eagle, y enfrentados con un grupo de sueros de pacientes con himenolepiosis confirmada para evaluar su calidad inmunológica y con sueros individuales de pacientes con himenolepiosis y con otras helmintiosis confirmadas para detectar mediante la técnica de “Western Blot”, los antígenos específicos de este cestode. El grupo de sueros de pacientes con himenolepiosis confirmada reconoció las bandas antigénicas de 50,1; 42,6; 38,9; 32,9; 26,3; 22,4 y 18,6 kDa; sin embargo, los sueros individuales reconocieron diferente número de bandas, siendo la de 50,1 KDa la que fue reconocida por todos ellos. Los sueros de pacientes con helmintiosis confirmadas no reconocieron la banda de 50,1 kDa; sin embargo, dieron reacción cruzada con algunas de las demás bandas, a excepción de los sueros de pacientes con cisticercosis que no reconocieron a ninguna de las bandas de estos antígenos. Se concluye que el antígeno de excreción/secreción de H. nana de 50,1 kDa es específico de este cestode por ser reconocido por todos los sueros de pacientes con himenolepiosis confirmada y no por sueros de pacientes con otras helmintiosis utilizando la técnica de “Western Blot”.

  13. The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States.

    Science.gov (United States)

    Pandori, Mark W; Westheimer, Emily; Gay, Cindy; Moss, Nicholas; Fu, Jie; Hightow-Weidman, Lisa B; Craw, Jason; Hall, Laura; Giancotti, Francesca R; Mak, Mae Ling; Madayag, Carmela; Tsoi, Benjamin; Louie, Brian; Patel, Pragna; Owen, S Michele; Peters, Philip J

    2013-12-01

    A new HIV diagnostic algorithm has been proposed which replaces the use of the HIV-1 Western blot and HIV-1 immunofluorescence assays (IFA) as the supplemental test with an HIV-1/HIV-2 antibody differentiation assay. To compare an FDA-approved HIV-1/HIV-2 antibody differentiation test (Multispot) as a confirmatory test with the HIV-1 Western blot and IFA. Participants were screened with an HIV-1/HIV-2 combination Antigen/Antibody (Ag/Ab) screening assay. Specimens with repeatedly reactive results were tested with Multispot and either Western blot or IFA. Specimens with discordant screening and confirmatory results were resolved with HIV-1 RNA testing. Individuals (37,876) were screened for HIV infection and 654 (1.7%) had a repeatedly reactive Ag/Ab assay result. On Multispot, 554 (84.7%) were HIV-1 reactive, 0 (0%) were HIV-2 reactive, 1 (0.2%) was reactive for both HIV-1 and HIV-2 (undifferentiated), 9 (1.4%) were HIV-1 indeterminate, and 90 (13.8%) were non-reactive. HIV-1 RNA was detected in 47/90 Multispot non-reactive (52.2%) specimens. Among specimens confirmed to have HIV infection (true positives), Multispot and Western blot detected HIV-1 antibody in a similar proportion of cases (93.7% vs. 94.4% respectively) while Multispot and IFA also detected HIV-1 antibody in a similar proportion of cases (84.5% vs. 83.4% respectively). In this study, Multispot confirmed HIV infections at a similar proportion to Western blot and IFA. Multispot, Western blot, and IFA, however, did not confirm all of the reactive Ag/Ab assay results and underscores the importance of HIV NAT testing to resolve discordant screening and confirmatory results. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the

  15. Identification of reference proteins for Western blot analyses in mouse model systems of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD toxicity.

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    Stephenie D Prokopec

    Full Text Available Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD.To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1 TCDD-induced fold-difference in protein content from basal levels, 2 inter- and intra- animal stability, and 3 the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2-3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed.

  16. A comparative study of Toxoplasma gondii seroprevalence in mink using a modified agglutination test, a Western blot, and enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gu, Yi; Wang, Zedong; Cai, Yufeng; Li, Xiaoxing; Wei, Feng; Shang, Limin; Li, Jiping; Liu, Quan

    2015-09-01

    Toxoplasma gondii can infect almost all warm-blooded animals, and many serological methods have been developed to detect T. gondii infection in a variety of animal species. In the present study, the seroprevalence of T. gondii infection in farmed mink in northeast China was determined using the modified agglutination test (MAT), a Western blot (WB), and 3 enzyme-linked immunosorbent assays (ELISAs) with protein A/G conjugate, using either of 2 recombinant dense granule antigens, GRA1 and GRA7, or Toxoplasma soluble antigens (TSA). There was no significant difference between the detection results of the GRA1-, GRA7-, and TSA-ELISAs and WB (McNemar chi-square, P > 0.05), but a significant difference was observed between MAT and WB (P < 0.05). A near perfect agreement (97.0%) was found between the GRA7-ELISA and WB (κ = 0.83), and a substantial agreement (92.4-93.1%) was observed in the TSA- and GRA1-ELISAs (κ = 0.68-0.73). The GRA7-ELISA showed the highest sensitivity and specificity, and the lowest false-positive and negative rates, while the MAT gave both a low sensitivity and frequent false positives in comparison to the WB. Receiver operating characteristic analysis revealed the largest area under curve of 0.85 (95% confidence interval: 0.74-0.96), and the highest relative sensitivity (72.7%) and specificity (99.0%) for a cutoff value of 0.19 in the GRA7-ELISA. These results indicate that the GRA7-ELISA is suitable for detection of T. gondii infection in mink and that MAT should be used with caution.

  17. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Science.gov (United States)

    Cánepa, Camila; Salido, Jimena; Ruggieri, Matías; Fraile, Sindy; Pataccini, Gabriela; Berini, Carolina; Biglione, Mirna

    2015-10-28

    indeterminate Western blot (WB) patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) infection, even in non-endemic areas. (a) to define the prevalence of indeterminate WB among different populations from Argentina; (b) to evaluate if low proviral load (PVL) is associated with indeterminate WB profiles; and (c) to describe mutations in LTR and tax sequence of these cases. Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3%) were WB indeterminate and of those 15 (31.3%) were PCR+. Quantitative real-time PCR (qPCR) was performed to 52 HTLV-1+ samples, classified as Group 1 (G1): 25 WB+ samples from individuals with pathologies; Group 2 (G2): 18 WB+ samples from asymptomatic carriers (AC); and Group 3 (G3): 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003) was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE) 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03). indeterminate WB results confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  18. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

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    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  19. HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

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    CARNEIRO-PROIETTI A.B.F.

    1999-01-01

    Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

  20. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  1. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

    Directory of Open Access Journals (Sweden)

    Yeda L. Nogueira

    2007-12-01

    Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

  2. Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry

    DEFF Research Database (Denmark)

    Petersen, T D; Ciofu, O; Pressler, T

    1996-01-01

    lung infection with P aeruginosa was further investigated by correlating the a beta ab IgG subclasses with pulmonary function in patients with cystic fibrosis. METHODS: Immunoglobulin G (IgG) and IgG subclass a beta ab were investigated by western blotting and quantified by laser scanning densitometry...

  3. Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.

    Science.gov (United States)

    Coyne, Cody P; Rashmir-Raven, Ann; Jones, Toni; Mochal, Cathleen; Linford, Robert L; Brashier, Michael; Eddy, Alison

    2009-01-01

    Limited research to date has characterized the potential for HRPO to function as a primary molecular probe. Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry. Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments. HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.

  4. 新孢子虫和弓形虫ELISA及western blot检测方法的建立及应用%Establishment and application of indirect ELISA assays and western blot to detect antibodies agaist Neospora caninum and Toxoplasma gondii

    Institute of Scientific and Technical Information of China (English)

    陈亮; 闫双; 刘启生; 曹雯丽; 王真; 巴音查汗

    2012-01-01

    为建立牛新孢子虫和弓形虫的免疫学检测方法,并调查新疆部分地区牛新孢子虫和弓形虫的感染情况,本研究应用纯化的新孢子虫重组蛋白SRS2 (NcSRS2)和弓形虫重组蛋白SAG2 (TgSAG2)作为包被抗原,分别建立新孢子虫和弓形虫的ELISA和western blot血清学检测方法,并进行特异性和重复性试验,以其检测662份疑似样品,并与商品化试剂盒检测结果比较验证.特异性和重复性试验结果表明,建立的方法特异性强、重复性良好.采用建立的两种ELISA方法对662份临床样品的检测结果表明,新孢子虫和弓形虫的抗体阳性率分别为13.44%(89/662)和5.29%(35/662);与IDEXX试剂盒和永辉试剂盒的符合率分别为94.11%和95.92%.此外,western blot检测的新孢子虫和弓形虫抗体阳性率分别为5.14%(34/662)和3.17%(21/662);与建立的ELISA检测方法的符合率分别为91.69%和97.89%.本研究为分析奶牛流产的原因提供了一定的依据.%Neospora caninum and Toxoplasma gondii are closely related protozoan parasites which causes of abortion and congenital disease in ruminants. To establish the methods for detection of the antibodies against N. caninum and T. gondii in bovine, The indirect ELISAs and western blot assay were developed based on purified recombinant protein SRS2 of N. cammim and SAG2 of T. gondii, respectively. The results show that these methods were highly specificity and repeatability. A total of 662 bovine serum samples were tested and the positive rates for N. caninum and T. gondii were 13.44% (89/662) and 5.29% (35/662) detected by the developed ELISAs, which were 94.11% and 95.92% agreements with TDEXX' N. caninum Antibody Test Kit and Yong hui' T. gondii Antibody Test Kit, respectively. In addition, the positive rates for N. caninum and T. gondii were 5.14% (34/662) and 3.17% (21/662) using western blot, and the agreements with the developed ELISAs were 91.69% and 97

  5. Testing UK blood donors for exposure to human parvovirus 4 using a time-resolved fluorescence immunoassay to screen sera and Western blot to confirm reactive samples.

    Science.gov (United States)

    Maple, Peter A C; Beard, Stuart; Parry, Ruth P; Brown, Kevin E

    2013-10-01

    Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed. With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken. The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA. ParV4 IgG has been found in UK blood donors and this finding needs further investigation. © 2013 American Association of Blood Banks.

  6. Diagnostic performance of ELISA, IFAT and Western blot for the detection of anti-Leishmania infantum antibodies in cats using a Bayesian analysis without a gold standard.

    Science.gov (United States)

    Persichetti, Maria Flaminia; Solano-Gallego, Laia; Vullo, Angela; Masucci, Marisa; Marty, Pierre; Delaunay, Pascal; Vitale, Fabrizio; Pennisi, Maria Grazia

    2017-03-13

    Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB.

  7. Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

    Science.gov (United States)

    Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

    2017-02-01

    Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study

  8. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

    Directory of Open Access Journals (Sweden)

    Neuza Satomi SATO

    1999-03-01

    Full Text Available Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A

  9. Analysis of glabrous canary seeds by ELISA, mass spectrometry, and Western blotting for the absence of cross-reactivity with major plant food allergens.

    Science.gov (United States)

    Boye, Joyce Irene; Achouri, Allaoua; Raymond, Nancy; Cleroux, Chantal; Weber, Dorcas; Koerner, Terence B; Hucl, Pierre; Patterson, Carol Ann

    2013-06-26

    Glabrous (hairless) canary seed belongs to the Poaceae (Gramineae) family and could serve as an alternative source of gluten-free cereal grain. In this study, allergenic cross-reactivities between hairless, dehulled canary seeds (Phalaris canariensis) and major allergenic proteins from gluten, soy, peanuts, tree nuts, sesame, and mustard were studied using commercial enzyme-linked immune sorbent assay (ELISA) kits specific for these target allergens. Mass spectrometry (MS) and immunoblotting were further used to assess for the presence of gluten-specific protein fragments. MS results revealed the likely presence of proteins homologous with rice, oat, corn, carrot, tomato, radish, beet, and chickpea. However, no presence of celiac-related gluten fragments from wheat, rye, barley, or their derivatives was found. Immunoblotting studies yielded negative results, further confirming the absence of gluten in the canary seed samples tested. No cross-reactivities were detected between canary seeds and almond, hazelnut, mustard, peanut, sesame, soy, walnut, and gluten using ELISA.

  10. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membran...

  11. Problem-Solving Test: Southwestern Blotting

    Science.gov (United States)

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  12. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  13. A novel gain of function mutant in C-kit gene and its tumorigenesis in nude mice

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Hong Liu; Qiang Xie; Fei Feng; Da-Lie Ma

    2005-01-01

    AIM: To transfect mutant C-kit cDNA at codon 579 into human embryonic kidney cell line to observe its role in the pathogenesis of gastrointestinal stromal tumor (GIST).METHODS: Eukaryotic expression vectors of pcDNA3-Kit-NW and pcDNA3-Kit-W were constructed. Then pcDNA3-Kit-NW and pcDNA3-Kit-W plasmids were transfected into human embryonic kidney cell line by Lipofectamine. The resistant done was screened by G418filtration and identified by sequencing, Western blotting,and immunocytochemical staining. Human embryonic kidney cells were divided into three groups including pcDNA3-Kit-NW, pcDNA3-Kit-W, and vector control groups. Absorbency value with a wavelength of 574 nm was detected by MTT analysis. Mice were injected with three groups of cells. Volume, mass, and histological examinations of the tumors in different groups were measured and compared.RESULTS: The C-kit gene and mutant C-kit gene were successfully cloned into the eukaryotic expression vector pcDNA3. pcDNA3-Kit-NW and pcDNA3-Kit-W were successfully transfected into human embryonic kidney cell line and showed stable expression in this cell line.Cell proliferating activity had significant differences between pcDNA3-Kit-NW and pcDNA3, pcDNA3-KitNW and pcDNA3-Kit-W (P<0.05), respectively. Tumors were only observed in nude mice implanted with cells transfected with pcDNA3-Kit-NW.CONCLUSION: Mutation of C-kit gene increases the proliferation activity of human cells and plays an important role in the malignant transformation of GIST.

  14. Comparison analysis between Western blot method and Magnetic particle Chemiluminescence method detection SLE autoantibody%免疫印迹法和磁微粒化学发光法检测SLE自身抗体的比对分析

    Institute of Scientific and Technical Information of China (English)

    周厚清

    2016-01-01

    目的:比较免疫印迹法(Western blot)和磁微粒化学发光法(Magnetic particle Chemiluminescence method)检测系统性红斑狼疮(SLE)自身抗体的一致性。方法:用免疫印迹法和磁微粒化学发光法对123份血清进行检测,其中系统性红斑狼疮阳性患者63例,健康体检者60例,用卡方检验对两种方法结果进行比较分析。结果:免疫印迹法和磁微粒化学发光法中抗Sm抗体阳性率分别为27.0%和14.3%、抗RNP/Sm阳性率分别为54.0%和42.9%、dsDNA阳性率分别为41.3%和30.2%、抗核小体抗体阳性率分别为31.7%和20.6%、抗核糖体P蛋白抗体阳性率分别为28.6%和11.1%。两种方法的准确性无显著性差异(>0.05)总符合率为80.6%,Kappa值为0.669。结论:免疫印迹法和磁微粒化学发光法检测系统性红斑狼疮自身抗体的检测结果具有可靠性,较好的符合率和准确性,磁微粒化学发光法可以作为简便、快速、经济的检测方法应用于系统性红斑狼疮自身抗体的检测。%Objective: The present study aims to compare Western blot and Magnetic particle Chemiluminescence method consistency on autoimmune antibody detection of systemic lupus erythematosus. Methods: Western blot assay and Magnetic particle Chemiluminescence method were used to detect autoimmune antibody from 123 serum samples, of which 63 patients with systemic lupus erythematosus, the others from 60 healthy persons. The results of the two methods were compared by X2 test. Results: Anti-Sm antibody positive rate of Western blot and Magnetic particle Chemiluminescence method were 27.0 and 14.3 percent respectively. Anti- RNP/Sm antibody positive rates were 39.7% and 42.9% respectively. Anti- ds- DNA antibody positive rates were 41.3% and 30.2% respectively. Anti- nucleosome antibody positive rates were 31.7% and 20.6% respectively. Anti- ribosomal protein antibody positive rates were 28.6% and 11.1% respectively. The accuracy

  15. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

  16. Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.

    Science.gov (United States)

    Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan

    2015-01-01

    Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.

  17. Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

    Science.gov (United States)

    Harpstrite, Scott E; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-10-01

    Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

  18. Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α.

    Directory of Open Access Journals (Sweden)

    Zhongpu Chen

    Full Text Available BACKGROUND: Cardiac progenitor cells (CPCs have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF, are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α could enhance the expression of c-kit. However, the mechanism is unknown. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts, c-kit(+ and c-kit(- CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+CPCs, made c-kit(-CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+ and c-kit(- CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+ and c-kit(- CPCs. CONCLUSIONS: SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+CPCs and make c-kit(-CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT

  19. Comparison of Four Commercial Kits to Detect HSV-2 IgG Antibody

    Institute of Scientific and Technical Information of China (English)

    Song Biao(宋彪); Chen Xiangsheng(陈祥生); Yin Yueping(尹跃平); Yao Xu(姚煦); Li Wengzhong(李文忠)

    2004-01-01

    Objectives: To compare the efficiency of four commercial ELISA kits in detecting type-specific HSV-2 IgG antibodies. Materials and Methods: A total of 125 subjects, including 105 with genital ulcers, and 20 controls without any history of STDs were recruited from the STD clinic for detection of type-specific HSV-2 IgG antibody with different kits. Four kinds of commercially available ELISA kits, including Quida HSV2 IgG ELISA (Aifulang Biochem Co. Ltd., Hangzhou), TORCH-HSV2 IgG (Jingmei Biotech Co. Ltd., Shanghai), CaptiaTM HSV2 IgG (Trinity biotech, USA ) and HerpeSelect TM 2 ELISA IgG (Focus technologies, USA) were used for evaluation. Western Blot assay was performed as a gold standard.Results: Compared to Western Blot results, the sensitivity and specificity of the kits (Quida HSV2, TORCHHSV2, CaptiaTM HSV2 and HerpeSelectTM 2) were 13.1% and 98.4%, 7.5% and 100%, 100% and 11.1%,87.7 % and 96.7 %, respectively. The positive predictive value (PV) and negative PV of the four kits were 88.9%and 54.3%, 100% and 55.5%, 55.6% and 100%, 96.2%and 89.2%, respectively. The areas under the ROC curve of three kits (Quida HSV2, CaptiaTM HSV2 andHerpeSelectTM 2) were 0.885 (0.822~0.948), 0.825 (0.747- 0.902), 0.974 (0.950 - 0.998), respectively Conclusion: The performance of HerpeSelectTM 2 is the best among the four kits. The results also indicate that the commercially available kits for detection of type-specific HSV2 antibody should be re-evaluated in terms of their validity.

  20. Hiding inside? Intracellular expression of non-glycosylated c-kit protein in cardiac progenitor cells.

    Science.gov (United States)

    Shi, Huilin; Drummond, Christopher A; Fan, Xiaoming; Haller, Steven T; Liu, Jiang; Malhotra, Deepak; Tian, Jiang

    2016-05-01

    Cardiac progenitor cells including c-kit(+) cells and cardiosphere-derived cells (CDCs) play important roles in cardiac repair and regeneration. CDCs were reported to contain only small subpopulations of c-kit(+) cells and recent publications suggested that depletion of the c-kit(+) subpopulation of cells has no effect on regenerative properties of CDCs. However, our current study showed that the vast majority of CDCs from murine heart actually express c-kit, albeit, in an intracellular and non-glycosylated form. Immunostaining and flow cytometry showed that the fluorescent signal indicative of c-kit immunostaining significantly increased when cell membranes were permeabilized. Western blots further demonstrated that glycosylation of c-kit was increased during endothelial differentiation in a time dependent manner. Glycosylation inhibition by 1-deoxymannojirimycin hydrochloride (1-DMM) blocked c-kit glycosylation and reduced expression of endothelial cell markers such as Flk-1 and CD31 during differentiation. Pretreatment of these cells with a c-kit kinase inhibitor (imatinib mesylate) also attenuated Flk-1 and CD31 expression. These results suggest that c-kit glycosylation and its kinase activity are likely needed for these cells to differentiate into an endothelial lineage. In vivo, we found that intracellular c-kit expressing cells are located in the wall of cardiac blood vessels in mice subjected to myocardial infarction. In summary, our work demonstrated for the first time that c-kit is not only expressed in CDCs but may also directly participate in CDC differentiation into an endothelial lineage.

  1. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and

  2. About KIT

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of interna-tional and intercultural cooperation,operating at the interface between theory and practice

  3. About KIT

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.

  4. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation. The

  5. Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Hernández-González, Ana; Noh, John; Perteguer, María Jesús; Gárate, Teresa; Handali, Sukwan

    2017-05-15

    Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA

  6. 亚洲带绦虫囊尾蚴特异性抗原筛选和鉴定%Screen and detection of specific antigens of Taenia asiatica cysticercus with two dimensional electrophoresis, western blotting and MS/MS spectrometry

    Institute of Scientific and Technical Information of China (English)

    方文; 肖靓靓; 包怀恩; 牟荣

    2011-01-01

    Objective To screen and identify specific antigens of Taenia asiatica cysticercus and to predict the function of target proteins using bioinformatics method. Methods Six three-way cross hybrid pigs were randomly divided into experimental group and control group and each experimental pig was infected with 84 000 eggs. Serum samples were collected 40 days after the infection. The total protein of Taenia asiatica cysticercus was separated by two dimensional electrophoresis and western blotting was performed to find out distinct antigens. Proteins from the two groups were identified by electrospray ion trap mass spectrometry(ESI-Trap MS). Results Totally 204 ± 11 spots were detected through Coomassie brilliant blue stained gels with relative molecular weight( Mr) of 14 400 -94 000 kDa and isoelectric point(pl) of 3. 0 - 10. 0. Western-blotting showed 6 specific antigen spots with pool sera of infected pigs. Two of the 6 antigens with known functions were ascribed to ATPase family gene 3-like protein 2 and ATP synthase beta subunit. Conclusion Two specific antigens of Taenia asiatica cysticercus have been identified.%目的 对亚洲带绦虫囊尾蚴的特异性抗原进行筛选、鉴定和生物信息学分析.方法 6头20d龄三元杂交乳猪随机分为实验组和对照组,实验组每头灌胃亚洲带绦虫虫卵悬液8.4万个;20 d后,分别制备实验组及对照组心脏血混合血清;收集、制备实验组亚洲带绦虫囊尾蚴蛋白并进行双向电泳分析,将凝胶蛋白斑点转移至聚偏氟乙烯膜,用实验组及对照组混合血清(均为1∶10)为一抗,羊抗猪辣根过氧化物酶- IgG(1∶200)为二抗,进行蛋白质印迹分析和质谱鉴定.结果 双向电泳凝胶共检测到( 204±11)个蛋白质斑点,相对分子质量为14 400 ~94 000 kPa,等电点(pI)为3.0~10.0;筛选出6个特异性抗原,经质谱鉴定出其中2个蛋白,分别为ATP酶管家基因3-类蛋白2(AT-Pase family gene 3-like protein 2)

  7. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  8. About KIT

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of internationa and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Insti-

  9. About KIT

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation, operating at the interface between theory and practice and between policy and implementation.The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  10. About KIT

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  11. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation. The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  12. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of internationaland intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Institute contributes

  13. About KIT

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  14. About KIT

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    <正>The Royal Tropical Institute ( KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation. The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  15. About KIT

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    <正>The Royal Tropical Institute (KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of internationaland intercultural cooperation,operating at the interface between theory and practice and between policy and implementation. The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  16. About KIT

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of internationaland intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Insti-tutecontributestosustainabledevelopment,poverty alleviationandculturalpreservationand exchange.

  17. About KIT

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The

  18. About KIT

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    <正>The Royal Tropical Institute(KIT)in Amsterdam is an independent centre of knowledge and expertise in the areas of international and intercultural cooperation,operating at the interface between theory and practice and between policy and implementation.The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  19. About KIT

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    <正>The Royal Tropical Institute (KIT) in Amsterdam is an independent centre of knowledge and expertise in the areas of interna-tional and intercultural cooperation,operating at the interface between theory and practice and between policy and implementa-tion. The Institute contributes to sustainable development,poverty alleviation and cultural preservation and exchange.

  20. Amplification of a novel c-Kit activating mutation Asn(822)-Lys in the Kasumi-1 cell line: a t(8;21)-Kit mutant model for acute myeloid leukemia.

    Science.gov (United States)

    Beghini, Alessandro; Magnani, Ivana; Ripamonti, Carla B; Larizza, Lidia

    2002-01-01

    A subset of AML-M2/M4Eo patients has been shown to carry c-kit mutations suggesting that myelomonoblastic leukemia cells, disrupting core binding factor through t(8;21) or inv(16) chromosomal rearrangements, have a common differentiation stage suitable to c-kit mutation. In rare core binding factor leukemia patients an increased dosage of a mutated Asp816(Tyr/Val) kit allele is achieved through nonrandom duplication of chromosome 4 where the c-kit gene is located. The c-kit gene was studied in the core binding factor leukemia cell line Kasumi-1 with t(8;21) by fluorescence in situ hybridization and mutation analysis. The dosage of Asn822(Lys) mutated allele was evaluated by fluorescence semiquantitative PCR. The correct membrane homing of KIT receptor and its activating status was analysed by immunofluorescence and Western blotting respectively. We identified in the Kasumi-1 cell line a novel Asn822(Lys) ligand-independent c-kit activating mutation and demonstrated by semiquantitative PCR that the mutated allele is about fivefold amplified compared to the normal allele. Fluorescence In Situ Hybridization analysis revealed that c-kit amplification maps to minute 4cen-q11 derived marker chromosome, often carrying duplicated signals, which are unequally distributed in the cell population. The Asn822(Lys) mutation affects a highly conserved codon within the tyrosine kinase activation loop leading, likewise the Asp(816) mutants, to constitutive ligand-independent activation of the KIT receptor. Results obtained point to the Kasumi-1 cell line as powerful in-vitro model for further investigation of altered KIT signal transduction pathways in acute myeloid leukemia with core binding factor rearrangements and a useful tool for pharmacological therapeutic targeting.

  1. Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Wei Hou; Feng Wang; Cen Qiu; Yan Zhu; Ling Huang; Jing Zhao

    2012-01-01

    AIM:To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth.METHODS:The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry,and the results were correlated with clinicopathological parameters,including the mitotic count,proliferative index (Ki-67 immunohistochemical staining),mitotic index (phospho-histone H3 immunohistochemical staining)and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling).Using primary cultured GIST cells,the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting,methyl thiazolyl tetrazolium (MTT),and apoptosis assays.RESULTS:We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples,respectively,and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis,including larger tumor size (P =0.0118),higher mitotic count (P =0.0058),higher proliferative index (P =0.0012),higher mitotic index (P =0.0282),lower apoptosis index (P =0.0484),and increased National Institutes of Health risk level (P =0.0012).We also found that the introduction of exogenous SCF potently increased KIT kinase activity,stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation,while a KIT immunoblocking antibody suppressed proliferation (P =0.01) and promoted apoptosis (P < 0.01)in cultured GIST cells.CONCLUSION:SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth.The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

  2. Bone Marrow Suppression by c-Kit Blockade Enhances Tumor Growth of Colorectal Metastases through the Action of Stromal Cell-Derived Factor-1

    Directory of Open Access Journals (Sweden)

    Kathrin Rupertus

    2012-01-01

    Full Text Available Background. Mobilization of c-Kit+ hematopoietic cells (HCs contributes to tumor vascularization. Whereas survival and proliferation of HCs are regulated by binding of the stem cell factor to its receptor c-Kit, migration of HCs is directed by stromal cell-derived factor (SDF-1. Therefore, targeting migration of HCs provides a promising new strategy of anti-tumor therapy. Methods. BALB/c mice (=16 were pretreated with an anti-c-Kit antibody followed by implantation of CT26.WT-GFP colorectal cancer cells into dorsal skinfold chambers. Animals (=8 additionally received a neutralizing anti-SDF-1 antibody. Animals (=8 treated with a control antibody served as controls. Investigations were performed using intravital fluorescence microscopy, immunohistochemistry, flow cytometry and western blot analysis. Results. Blockade of c-Kit significantly enhanced tumor cell engraftment compared to controls due to stimulation of tumor cell proliferation and invasion without markedly affecting tumor vascularization. C-Kit blockade significantly increased VEGF and CXCR4 expression within the growing tumors. Neutralization of SDF-1 completely antagonized this anti-c-Kit-associated tumor growth by suppression of tumor neovascularization, inhibition of tumor cell proliferation and reduction of muscular infiltration. Conclusion. Our study indicates that bone marrow suppression via anti-c-Kit pretreatment enhances tumor cell engraftment of colorectal metastases due to interaction with the SDF-1/CXCR4 pathway which is involved in HC-mediated tumor angiogenesis.

  3. 孕产妇HIV抗体筛查阳性与免疫印迹试验对比%Comparison of Positive of HIV Antibody Screening and Western Blot Test in Pregnant Women

    Institute of Scientific and Technical Information of China (English)

    朱厚宏; 王晨笛; 刘杨; 张祁; 孟建彤; 黄薇

    2011-01-01

    Objective To explore the relationship between the results of HIV antibody screening test and Westem Blot test ( WB) in pregnant women. Methods Thirty three positive samples from pregnant women were tested with the ELISA and Westem blot test (WB). Those with indefinite result by WB test were re-tested 3 and 6 months later, and HIV-1 viral load testing ( VL) was carried out at 6th month. The results of ELISA and the WB test were analyzed. Result The 33 samples were all positive by ELISA and 20 were HIV-1 positive by WB test. The ELISA and the WB test positive coincidence rate was 60. 61 % (20/33). Of 20 samples 0were with S/CO value more than 8, the WB test results of HIV-1 antibody were all positive. Of 13 samples were with S/CO value less than 1 , 5 were negative and 8 were indefinite in WB test. The HIV-1 viral load test result at 6th month was less than the minimum detection limit (50 IU/ml ) . Conclusion The false positive in HIV antibody screening test (ELISA) was observed. The false positive sample were in the WB test negative and indefinite ones. ELISA and the WB test positive coincidence rate increased with the S/CO value of ELISA. The HIV WB test indefinite result in pregnant women was mostly due to non-specific immune response.%目的 探讨孕产妇人类免疫缺陷病毒(HIV)抗体筛查试验阳性与免疫印迹试验(WB)结果的关系.方法 对33份孕产妇HIV抗体待复查样本,进行酶联免疫吸附试验(ELISA )及免疫印迹试验(WB),对WB试验结果不确定者进行3个月、6个月随访检测,6个月随访样本补充HIV-1病毒载量检测,分析ELISA检测与WB试验检测结果.结果 33份样本ELISA复检均呈阳性,免疫印迹试验(WB)中20份确认为HIV-1抗体阳性,ELISA检测与WB试验阳性符合率为60.61%(20/33); S/CO值>8的20份样本,WB试验结果均为HIV-1抗体阳性,I < S/CO值<8的13份样本,WB试验5份为HIV抗体阴性,8份为HIV抗体不确定;6个月随访样本HIV-1病毒载量检测,

  4. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  5. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  6. Levitation Kits Demonstrate Superconductivity.

    Science.gov (United States)

    Worthy, Ward

    1987-01-01

    Describes the "Project 1-2-3" levitation kit used to demonstrate superconductivity. Summarizes the materials included in the kit. Discusses the effect demonstrated and gives details on how to obtain kits. Gives an overview of the documentation that is included. (CW)

  7. Levitation Kits Demonstrate Superconductivity.

    Science.gov (United States)

    Worthy, Ward

    1987-01-01

    Describes the "Project 1-2-3" levitation kit used to demonstrate superconductivity. Summarizes the materials included in the kit. Discusses the effect demonstrated and gives details on how to obtain kits. Gives an overview of the documentation that is included. (CW)

  8. Necroptosis Induced by Ad-HGF Activates Endogenous C-Kit+ Cardiac Stem Cells and Promotes Cardiomyocyte Proliferation and Angiogenesis in the Infarcted Aged Heart

    Directory of Open Access Journals (Sweden)

    Jiabao Liu

    2016-12-01

    Full Text Available Background/Aims: The discovery of c-kit+ cardiac stem cells (CSCs provided us with new therapeutic targets to repair the damaged heart. However, the precise mechanisms regulating CSC proliferation and differentiation in the aged heart remained elusive. Necroptosis, a type of regulated cell death, has recently been shown to occur following myocardial infarction (MI; however, its effect on c-kit+ CSCs remains unknown. We investigated the effects of hepatocyte growth factor (HGF and necroptosis on the proliferation and differentiation of endogenous c-kit+ CSCs in aged rat hearts following MI. Methods: The c-kit+ CSCs and HGF/p-Met expression levels in neonatal, adult and aged rats were compared using immunofluorescence and Western blotting. Immediately after MI, adenovirus carrying the HGF gene (Ad-HGF was injected into the left ventricular wall surrounding the infarct areas of the aged rat heart. The proliferation and differentiation of the endogenous c-kit+ CSCs were studied using immunofluorescence. The signalling pathways were analysed via Western blotting and ELISA. Results: HGF/p-Met expression levels and c-kit+ CSC abundance gradually decreased with age. Ad-HGF promoted c-kit+ CSC differentiation into precursor cells of cardiomyocyte, endothelial and smooth muscle cell lineages and enhanced cardiomyocyte proliferation and angiogenesis in aged rats; these effects were reversed by the inhibition of necroptosis. Ad-HGF administration induced necroptosis by increasing the expression of receptor interacting protein kinase (RIP 1 and receptor interacting protein kinase (RIP 3 proteins in the infarcted heart. Moreover, Ad-HGF-induced necroptosis increased high-mobility group box 1 protein (HMGB1 levels and enhanced the abundance of c-kit+ cells in the bone marrow, which may partly account for the beneficial effect of necroptosis on the c-kit+ CSCs. Conclusion: Ad-HGF-induced necroptosis facilitated aged heart repair after MI by promoting c-kit+ CSC

  9. Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

    Science.gov (United States)

    Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

    2017-04-21

    It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway.

  10. Western blot analysis of IgG responses of sheep infected with Fasciola hepatica and F.gigantica%免疫印迹分析试验感染肝片吸虫和大片吸虫绵羊的IgG应答

    Institute of Scientific and Technical Information of China (English)

    张为宇; MOREAU Emmanuelle; 黄维义; CHAUVIN Alain

    2003-01-01

    免疫印迹(Western blot)分析试验感染肝片吸虫和大片吸虫绵羊的抗片形吸虫分泌排泄产物(FhESP和FgESP)IgG应答,结果表明,FhESP中分子质量在17.1~21.6 ku之间的4条主要蛋白带仅被肝片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别; FgESP中分子质量在19.0~71.1 ku之间的6条主要蛋白带仅被大片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别.

  11. Development of Rapid Diagnostic Kit for Identification of Hanwoo (Korean Native Cattle) Brand Meat by Detecting BIO-TAG.

    Science.gov (United States)

    Baek, Kyung Hoon; Park, Sung Kwon; Lee, Myung Hoon; Kim, Sung Il; Cho, Soo Hyun; Choi, Chang Bon

    2014-01-01

    This study was performed to develop a rapid immuno-assay kit, by using a specific antigen to detect Hanwoo brand meat. We selected a synthetic antigen specific to our target antibody, named BIO-TAG (Tyr-D-Ala-Phe), by utilizing a computer-based analysis and literature review. BIO-TAG tagged with adjuvant was subcutaneously injected in sheep and Hanwoo. The serum and meat juice of the immunized or non-immunized animal were then analyzed, to measure the titer of antibody by ELISA and Western blot. The amount of antibodies against the BIO-TAG increased (pbrand meat can be more precisely identified by our rapid diagnostic kit. This technology can deter possible fraud of counterfeit meat brands in the Korean domestic market with ease and rapidity; and offers a new tool that guarantees consumers high quality Hanwoo brand beef.

  12. Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots

    Directory of Open Access Journals (Sweden)

    Sanne M. M. Hensen

    2015-04-01

    Full Text Available Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

  13. Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Nakamura, T; Spang-Thomsen, M

    1993-01-01

    We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF......), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20...... tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF...

  14. Burn Wise Awareness Kit

    Science.gov (United States)

    Health and safety outreach materials in the form of an awareness kit. Designed specifically for state, local, and tribal air agencies working to reduce wood smoke pollution, it includes best burn tips, social media m

  15. False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

    Science.gov (United States)

    Maache, Mimoun; Komurian-Pradel, Florence; Rajoharison, Alain; Perret, Magali; Berland, Jean-Luc; Pouzol, Stéphane; Bagnaud, Audrey; Duverger, Blandine; Xu, Jianguo; Osuna, Antonio; Paranhos-Baccalà, Glaucia

    2006-01-01

    To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV. PMID:16522785

  16. Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

    Science.gov (United States)

    Che, Hui; Li, Gang; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-11-15

    Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

  17. Vietnamese Culture Kit.

    Science.gov (United States)

    Nguyen, Liem Thanh

    This booklet provides a brief description of the cultural background of the Vietnamese, the geography of the country of Vietnam, the history of the Vietnamese people, their language, beliefs, systems of values, religions, customs, feasts, and holidays. The kit is designed to provide American sponsors and teachers with meaningful information about…

  18. World Disarmament Kit.

    Science.gov (United States)

    Woito, Robert, Ed.

    This kit presents a comprehensive introduction for students to arms control and disarmament issues. Included are copies of published and unpublished articles for each topic. Section I provides a self-survey to enable students to assess their own attitudes, values, and knowledge. The survey poses questions for which students select one of several…

  19. ProjectorKit

    DEFF Research Database (Denmark)

    Weigel, Martin; Boring, Sebastian; Steimle, Jürgen

    2013-01-01

    Researchers have developed interaction concepts based on mobile projectors. Yet pursuing work in this area - particularly in building projector-based interactions techniques within an application - is cumbersome and time-consuming. To mitigate this problem, we contribute ProjectorKit, a flexible ...

  20. Build an Emergency Preparedness Kit

    Science.gov (United States)

    ... hat -kitty litter or sand for tire traction -Red or brightly- colored cloth -NOAA weather radio For more information on building emergency kits, contact your local chapter of the American Red Cross. Remember: Your emergency kit should include bottled ...

  1. ISS Expedition 09 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 09 from 04/2004-10/2004. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  2. ISS Expedition 05 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 05 from 06/2002-12/2002. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  3. ISS Expedition 10 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 10 from 10/2004-04/2005. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  4. ISS Expedition 02 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 02 from 03/2001-08/2001. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  5. ISS Expedition 37 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 37 from 05/2013-11/2013. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  6. ISS Expedition 23 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 23 from 12/2009-09/2010. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  7. ISS Expedition 24 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 24 from 04/2010-11/2010. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  8. ISS Expedition 42 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 42 from 09/2014-03/2015. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  9. ISS Expedition 34 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 34 from 12/2012-03/2013. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  10. ISS Expedition 16 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 16 from 10/2007-04/2008. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  11. ISS Expedition 03 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 03 from 08/2001-12/2001. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  12. ISS Expedition 06 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 06 from 11/2002-05/2003. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  13. ISS Expedition 11 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 11 from 04/2005-10/2005. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  14. ISS Expedition 35 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 35 from 03/2013-09/2013. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  15. ISS Expedition 38 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 38 from 09/2013-03/2014. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  16. ISS Expedition 20 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 20 from 05/2009-10/2009. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  17. ISS Expedition 08 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 08 from 10/2003-04/2004. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  18. ISS Expedition 30 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 30 from 11/2011-07/2012. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  19. ISS Expedition 39 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 39 from 11/2013-05/2014. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  20. ISS Expedition 01 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 01 from 10/2000-03/2001. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  1. ISS Expedition 36 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 36 from 03/2013-09/2013. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  2. ISS Expedition 04 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 04 from 12/2001-06/2002. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  3. ISS Expedition 32 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 32 from 05/2012-09/2012. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  4. ISS Expedition 12 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 12 from 10/2005-04/2006. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  5. ISS Expedition 41 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 41 from 05/2014-11/2014. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  6. ISS Expedition 17 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 17 from 04/2008-10/2008. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  7. ISS Expedition 26 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 26 from 10/2010-05/2011. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  8. ISS Expedition 28 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 28 from 04/2011-11/2011. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  9. ISS Expedition 40 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 40 from 03/2014-11/2014. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  10. ISS Expedition 31 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 31 from 12/2011-07/2012. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  11. ISS Expedition 18 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 18 from 10/2008-04/2009. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  12. ISS Expedition 25 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 25 from 06/2010-03/2011. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  13. ISS Expedition 14 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 14 from 09/2006-04/2007. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  14. ISS Expedition 27 Press Kit

    Data.gov (United States)

    National Aeronautics and Space Administration — Press kit for ISS mission Expedition 27 from 12/2010-09/2011. Press kits contain information about each mission overview, crew, mission timeline, benefits, and media...

  15. 49 CFR 173.161 - Chemical kits and first aid kits.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Chemical kits and First aid kits must conform to... 10 kg. (b) Chemical kits and First aid kits are excepted from the specification...

  16. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    Science.gov (United States)

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  17. Heterogeneity of PrP in Brains of Hamsters Infected with Scrapie 263K Strain and the Dynamic Detection by Western blot%羊瘙痒病263K毒株感染的仓鼠脑中PrP分子形式多样性的动态测定

    Institute of Scientific and Technical Information of China (English)

    高建梅; 高晨; 周晓勃; 张瑾; 韩俊; 张宝云; 董小平

    2003-01-01

    正常细胞的朊蛋白(PrPC)代谢和构象的改变是引发动物和人类可传播性海绵状脑病(transmissible spongiform encephalopathies,TSEs)的根本原因.将羊瘙痒病( scrapie)仓鼠适应株263K颅内接种仓鼠, 在接种后的第20、40、50、60、70、80天, 通过Western blot 动态检测仓鼠脑中PrP存在的形式.结果在接种后第40天, 在感染动物脑组织中即检测到 PrPSc分子,比临床症状出现的时间早(平均潜伏期为66.7±1.1天),且无糖基化形式的PrP分子所占百分比在接种后期增加明显.除了标准分子量大小(30kD~35kD)的 PrP分子外,在感染动物脑中存在着高分子量和低分子量形式的PrP分子 .定量分析显示,随着接种潜伏期的延长,不同形式PrP分子的含量也在增加,其中低分子量形式的PrP分子与临床症状的出现密切相关.蛋白去糖基化实验表明,在感染动物脑组织中,除了标准分子量大小的PrP蛋白外,还存在一条更小分子量的PrP条带,而正常动物脑组织仅存在标准大小的Pr P分子.低分子量形式的PrP分子具有与全长PrP分子相类似的糖基化模式.结果提示,scrapie 263K感染的仓鼠脑组织中存在不同分子形式的PrPSc,其PrP分子的代谢可能不同于正常动物.

  18. The Evaluation of Western Blotting Method for the Diagnosis of Helicobacter Pylori Infection in Children%免疫印迹试验在诊断儿童幽门螺旋杆菌感染中的应用评价

    Institute of Scientific and Technical Information of China (English)

    陈运生; 周少明; 刘彬; 孙玲玲

    2006-01-01

    目的:评价免疫印迹试验(Western Blotting)在诊断不同年龄组及不同病理表现儿童幽门螺旋杆菌(Helicobacter pylori,HP)感染中的应用价值.方法:对450例有消化道症状的患儿,进行胃镜检查并取病变处胃黏膜组织行病理学检查,以及13C-尿素呼气试验(13C-urea breath test,13C-UBT)、血清免疫印迹试验及胃液HP-DNA -PCR检查.结果:在4岁~6岁和6岁~12岁组患儿中,13C-UBT HP检出率明显高于免疫印迹试验(P<0.01);在12岁~18岁组患儿中,13C-UBT HP检出率与免疫印迹试验比较差异无显著性(P>0.05).细胞毒性基因相关蛋白(Cytotoxin-associated gene protein)阳性的HP感染在溃疡病组明显高于慢性胃炎组和正常组(P<0.05),慢性胃炎组CagA(Cytotoxin-associated gene protein,CagA) 阳性 HP显著高与正常组(P<0.05).结论:在12岁以上的少年患儿中,免疫印迹试验可作为快速诊断HP感染的可靠手段,并且能判断出是否为产毒菌株感染.在12岁以下的儿童中,免疫印迹试验最好与13C-UBT联合使用.

  19. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

    Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

  20. CRITERIA OF POSITIVITY FOR Ig ANTIBODIES IN THE METHOD OF IMMUNE BLOTTING OF LYME DISEASE

    Directory of Open Access Journals (Sweden)

    V G Barskova

    2001-01-01

    Full Text Available There are currently no accepted criteria for positive Western blots in Russian patients with Lyme borreliosis. The purpose of the current study was to develop criteria for a positive IgG westem-blot to aid particularly in the diagnosis of patients with joint manifestation of the disorder. Patients: 97 with Lyme disease, 145 - control subjects. IgG antibody responses were determined to 3 species ofB.burgdorferi sensu lato by Western blotting, using blots prepared by manufacturer. The best discriminatory ability of test criteria was chained by requiring any 3 of 11 IgG bands, a definition that could be used with B. burgdorferi sensu stricto, B.garinii and B.afzelii strains. With these 3 antigen preparation, positive IgG blots were found in 0 to 18% of patients with localized erythema migrans of < 4 weeks duration, 23 to 39% of those with disseminated infection < 20 weeks duration, and in 39 to 46% of those with late arthritis/arthralgia of >6 months duration the specificity was 93 to 99%. Thus, IgG Western blotting may bring greater specificity to serologic testing in Lyme borreliosis, but the sensitivity is limited.

  1. Molecular defects in mastocytosis: KIT and beyond KIT.

    Science.gov (United States)

    Bibi, Siham; Langenfeld, Florent; Jeanningros, Sylvie; Brenet, Fabienne; Soucie, Erinn; Hermine, Olivier; Damaj, Gandhi; Dubreuil, Patrice; Arock, Michel

    2014-05-01

    In all variants of mastocytosis, activating KIT mutations are frequently found. In adults, neoplastic mast cells (MCs) cells show the KIT mutation D816V, whereas in children, MCs invading the skin are frequently positive for non-KIT D816V mutations. The clinical course and prognosis of the disease vary among patients with systemic mastocytosis (SM). Additional KIT-independent molecular defects might cause progression. Additional oncogenic lesions have recently been identified in advanced SM. In advanced SM the presence of additional genetic lesions or altered signaling worsening the prognosis might lead to the use of alternative therapies such as combined antisignaling targeted treatments or stem cell transplantation.

  2. Analyses of the Results of HIV-1 Western Blot Confirmative Test and NAT Test in Voluntary Blood Donors%无偿献血者HIV-1抗体蛋白印迹确认与核酸检测结果分析

    Institute of Scientific and Technical Information of China (English)

    吕蓉; 王伦善; 盛琪琪; 赵阳; 蒋菲菲; 李敏; 刘忠

    2012-01-01

    Objective To explore the performance of confirmative Western blot (WB) and TMA-chemical luminescence test in detecting the reactive-samples for HIV antibody ELISA assay. Methods 117 reactive specimens detected by ELISA were repeatedly tested by two methods of primary screening assay and TMA-luminescence nucleic acid amplification testing(NAT). All samples of S/CO>0. 8 were analysed by the WB confirmative test. Serological tests participated in the external quality assessment (EQA) were surported by China CDC and Australian international CITIC as well as NAT participated in the EQA of Clinical Laboratory Center of the Ministry of Health and Australian CITIC. Results The results of all 117 tested samples were as followed, in primary screening test:S/CO>1,0. 8

  3. Windows 7 resource kit

    CERN Document Server

    Northrup, Tony; Honeycutt, Jerry; Wilson, Ed

    2009-01-01

    In-depth and comprehensive, this RESOURCE KIT delivers the information you need to administer your Windows 7 system. You get authoritative technical guidance from those who know the technology best-Microsoft Most Valuable Professionals (MVPs) and the Windows 7 product team-along with essential scripts and resources. In addition, "Direct from the Source" sidebars offer deep insights and troubleshooting tips from the Windows 7 team. Get expert guidance on how to: Use Microsoft Deployment Toolkit best practices and tools. Plan user-state migration and test application compatibility.

  4. Protein crystallography prescreen kit

    Science.gov (United States)

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  5. Mobile Probing Kit

    DEFF Research Database (Denmark)

    Larsen, Jakob Eg; Sørensen, Lene Tolstrup; Sørensen, J.K.

    2007-01-01

    characterized as being highly nomadic and thus potential users of mobile and ubiquitous technologies. The methodology has been applied in the 1ST MAGNET Beyond project in order to obtain user needs and requirements in the process of developing pilot services. We report on the initial findings from applying......Mobile Probing Kit is a low tech and low cost methodology for obtaining inspiration and insights into user needs, requirements and ideas in the early phases of a system's development process. The methodology is developed to identify user needs, requirements and ideas among knowledge workers...

  6. Education Payload Operation - Kit D

    Science.gov (United States)

    Keil, Matthew

    2009-01-01

    Education Payload Operation - Kit D (EPO-Kit D) includes education items that will be used to support the live International Space Station (ISS) education downlinks and Education Payload Operation (EPO) demonstrations onboard the ISS. The main objective of EPO-Kit D supports the National Aeronautics and Space Administration (NASA) goal of attracting students to study and seek careers in science, technology, engineering, and mathematics.

  7. Effects of BMP4/SMAD signaling pathway on mouse primordial follicle growth and survival via up-regulation of Sohlh2 and c-kit.

    Science.gov (United States)

    Ding, Xiangyun; Zhang, Xiaoli; Mu, Yulan; Li, Yi; Hao, Jing

    2013-01-01

    Bone morphogenetic protein 4 (BMP4) is essential for the development of primordial follicles, although its underlying mechanism remains largely unknown. By using cultured ovaries, the effects of BMP4 and the potential signal transduction pathways were investigated. Ovaries from 3-day-old female mouse pups were maintained in organ culture in the absence (control) or presence of BMP4 (100 ng/ml). At different culture time, the effects of BMP4 on primordial follicle growth and survival were assayed by follicle count and TUNEL labeling. The expression of phospho-SMAD1/5/8, Sohlh2, and c-kit were measured by immunohistochemistry, RT-PCR, and Western blotting. Immunohistochemistry was also performed to determine the expression pattern of BMP4, pSMAD1/5/8, Sohlh2, and c-kit in vivo during ovarian development. The results showed treatments of ovaries with BMP4 resulted in a significant (P primordial-to-primary follicle transition. The oocytes of primordial follicles treated with BMP4 were also less likely to undergo apoptosis. BMP4 enhanced the phosphorylation of SMAD1/5/8 and up-regulated the expression of Sohlh2 and c-kit in primordial follicles. During ovarian development in vivo, Sohlh2, and c-kit exhibited similar expression patterns to BMP4 and pSMAD1/5/8 in primordial follicles. The present studies suggest that BMP4/SMAD signaling pathway initiate primordial follicle growth and prevented oocyte apoptosis via up-regulation of Sohlh2 and c-kit. Copyright © 2012 Wiley Periodicals, Inc.

  8. DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

    Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

  9. Cosmic Light EDU kit

    Science.gov (United States)

    Doran, Rosa

    2015-08-01

    In 2015 we celebrate the International Year of Light, a great opportunity to promote awareness about the importance of light coming from the Cosmos and what messages it is bringing to mankind. In parallel a unique moment to attract the attention of stakeholders on the dangers of light pollution and its impact in our lives and our pursuit of more knowledge. In this presentation I want to present one of the conrnerstones of IYL2015, a partnership between the Galileo Teacher Training Program, Universe Awareness and Globe at Night, the Cosmic Light EDU kit. The aim of this project is to assemble a core set of tools and resources representing our basic knowledge pilars about the Universe and simple means to preserve our night sky.

  10. What's In Your Emergency Kit?

    Centers for Disease Control (CDC) Podcasts

    2012-12-04

    An emergency kit can help you survive during a disaster. This podcast discusses supplies to include in your kit.  Created: 12/4/2012 by Office of Public Health Preparedness and Response (OPHPR).   Date Released: 12/20/2012.

  11. Inactivation of the forkhead transcription factor FoxO3 is essential for PKB-mediated survival of hematopoietic progenitor cells by kit ligand

    DEFF Research Database (Denmark)

    Engström, Maria; Karlsson, Richard; Jönsson, Jan-Ingvar

    2003-01-01

    OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation of phosphatidylinos......OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation...... of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), which in turn inactivates forkhead transcription factors, known to be potent regulators of apoptosis. In this study, we investigated the involvement of PKB, FoxO1, FoxO3, and FoxO4 in c-kit-mediated survival. METHODS: By Western blot analysis...... of hematopoietic progenitors. Because forkhead proteins are involved in controlling apoptosis and cell-cycle progression, this may be one important mechanism by which survival of hematopoietic progenitors is mediated....

  12. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Science.gov (United States)

    Zhang, Ying-Ying; Li, Gang; Che, Hui; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-01-01

    Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  13. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Ying-Ying Zhang

    Full Text Available Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa, a voltage-gated TTX-sensitive sodium current (INa.TTX, and an inward rectifier K+ current (IKir were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  14. Evaluation of two commercially available immunological kits for the diagnosis of Helicobacter spp. in bottlenose dolphins (Tursiops truncatus).

    Science.gov (United States)

    Bernal-Guadarrama, María José; Fernández-Gallardo, Nuhacet; Zamora-Padrón, Rafael; Pacheco, Víctor; Reyes-Batlle, María; Valladares, Basilio; Lorenzo-Morales, Jacob; Martínez-Carretero, Enrique

    2015-05-01

    Helicobacter pylori is considered to be responsible for the most common gastric infections in humans worldwide. In animals, other Helicobacter species are linked to gastritis with and without the presence of ulcers in their respective hosts. Moreover, gastric ulcers have been reported for decades in wild and captive dolphins. Clinical signs include lack of appetite, anorexia, abdominal tenderness, depression, and occasional unresponsiveness. In this study, serum and stool of nine bottlenose dolphins from Loro Parque collection Tenerife, Spain were examined for the presence of Helicobacter spp. The aim of our study was to evaluate the use of two commercially available kits for the detection of H. pylori in humans: a stool antigen immunoassay (Letitest H. pylori CARD) and a Western blot assay (EUROLINE-WB H. pylori) that were adapted to identify specific Helicobacter spp. antibodies in the tested Loro Parque bottlenose dolphin collection. The utility of these diagnostic kits for their application in dolphins is demonstrated, and their use in the future for the diagnosis of Helicobacter spp. in both wild and captive dolphins is proposed in this study.

  15. FUELS IN SOIL TEST KIT: FIELD USE OF DIESEL DOG SOIL TEST KITS

    Energy Technology Data Exchange (ETDEWEB)

    Susan S. Sorini; John F. Schabron; Joseph F. Rovani, Jr.

    2002-09-30

    Western Research Institute (WRI) has developed a new commercial product ready for technology transfer, the Diesel Dog{reg_sign} Portable Soil Test Kit, for performing analysis of fuel-contaminated soils in the field. The technology consists of a method developed by WRI (U.S. Patents 5,561,065 and 5,976,883) and hardware developed by WRI that allows the method to be performed in the field (patent pending). The method is very simple and does not require the use of highly toxic reagents. The aromatic components in a soil extract are measured by absorption at 254 nm with a field-portable photometer. WRI added significant value to the technology by taking the method through the American Society for Testing and Materials (ASTM) approval and validation processes. The method is designated as ASTM Method D 5831-96, Standard Test Method for Screening Fuels in Soils. This ASTM designation allows the method to be used for federal compliance activities. In June 2001, the Diesel Dog technology won an American Chemical Society Regional Industrial Innovations Award. To gain field experience with the new technology, Diesel Dog kits have been used for a variety of site evaluation and cleanup activities. Information gained from these activities has led to improvements in hardware configurations and additional insight into correlating Diesel Dog results with results from laboratory methods. The Wyoming Department of Environmental Quality (DEQ) used Diesel Dog Soil Test Kits to guide cleanups at a variety of sites throughout the state. ENSR, of Acton, Massachusetts, used a Diesel Dog Portable Soil Test Kit to evaluate sites in the Virgin Islands and Georgia. ChemTrack and the U.S. Army Corps of Engineers successfully used a test kit to guide excavation at an abandoned FAA fuel-contaminated site near Fairbanks, Alaska. Barenco, Inc. is using a Diesel Dog Portable Soil Test Kit for site evaluations in Canada. A small spill of diesel fuel was cleaned up in Laramie, Wyoming using a Diesel

  16. Energy Management Curriculum Starter Kit

    Energy Technology Data Exchange (ETDEWEB)

    Turner, W.C.

    1987-02-01

    The Energy Management Curriculum Starter Kit was designed to help engineering educators develop and teach energy management courses. Montana State University and Oklahoma State University courses are embodied in the model curriculum given. The curricula offered at many other universities throughout the United States are also presented. The kit was designed specifically to train engineering students to be good energy managers. Courses at both the undergraduate and postgraduate level are presented.

  17. Evaluation of Survey Methods and Development of Species Distribution Models for Kit Foxes in the Great Basin Desert

    OpenAIRE

    Dempsey, Stephen J

    2013-01-01

    Historically, kit foxes (Vulpes macrotis) once occupied the desert and semi-arid regions of southwestern North America, ranging from Idaho to central Mexico. Their range-wide decline has warranted the kit fox to be listed as endangered in Colorado, threatened in California and Oregon, and designated as a state sensitive species in Idaho and Utah. Once considered the most abundant carnivore in western Utah, the kit fox has been in steep decline over the past decade, creating a demand to determ...

  18. [Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

    Science.gov (United States)

    Błachnio, Karina

    2010-01-01

    Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

  19. Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

    Science.gov (United States)

    Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

    2017-01-01

    Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

  20. Deletion of the c-kit protooncogene in the human developmental defect piebald trait

    Energy Technology Data Exchange (ETDEWEB)

    Fleischman, R.A.; Stastny, V.; Zneimer, S. (Univ. of Texas, Dallas (United States)); Saltman, D.L. (Genelabs, Inc., Redwood City, CA (United States))

    1991-12-01

    The protooncogene c-kit is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. Homozygous mutations of this gene in the mouse cause anemia, infertility, and albinism, whereas heterozygous mutant mice usually exhibit only a white forehead blaze and depigmentation of the ventral body, tail, and feet. The heterozygous mouse phenotype is very similar to human piebald trait, which is characterized by a congenital white hair forelock and ventral and extremity depigmentation. To investigate the possibility that alterations in the human c-kit gene may be a cause of piebald trait, DNA from seven unrelated affected individuals was examined by Southern blot analysis. One subject, although cytogenetically normal, has a heterozygous deletion of the c-kit protooncogene. This deletion encompasses the entire coding region for c-kit and also involves the closely linked gene for platelet-derived growth factor receptor {alpha}. These findings provide molecular evidence mapping piebald trait to the c-kit locus on chromosome 4. Although the authors cannot exclude the involvement of other closely linked genes, the demonstration of a genomic c-kit deletion in one subject with piebald trait and the marked concordance of the human and mouse phenotypes provide strong evidence for the role of c-kit in the development of human melanocytes and in the pathogenesis of piebald trait.

  1. Fast and Simple micro-RNA Northern Blots

    Directory of Open Access Journals (Sweden)

    Nham Tran

    2009-01-01

    Full Text Available RNA northern blots provide robust measurements of gene expression. The simple northern blot technique described in this report has been optimised to provide rapid, reproducible detection and analysis of mature and precursor forms of microRNAs. This protocol economises on the use of commercially available components and secondly reduces the hybridisation step to 2 hours.

  2. BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

    Science.gov (United States)

    Hubbard, Katherine; Hegarty, Peter

    2016-01-01

    Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology.

  3. The Free Universal Construction Kit

    DEFF Research Database (Denmark)

    Stephensen, Jan Løhmann; Hansen, Lone Koefoed

    2013-01-01

    With the increasing economic accessibility of 3D printers, the lessons learned and the logics cultivated on digital Web 2.0 now seems applicable to the world of material things. Released in early 2012 by the artist groups F.A.T. and Sy-lab, the Free Universal Construction Kit is a set of 3D...

  4. Active Parenting Now: Program Kit.

    Science.gov (United States)

    Popkin, Michael H.

    Based largely on the theories of Alfred Adler and Rudolf Dreikurs, this parent education curriculum is a video-based interactive learning experience that teaches a comprehensive model of parenting to parents of children ages 5 to 12 years. The kit provides parents with the skills needed to help their children develop courage, responsibility, and…

  5. The Free Universal Construction Kit

    DEFF Research Database (Denmark)

    Stephensen, Jan Løhmann; Hansen, Lone Koefoed

    2013-01-01

    With the increasing economic accessibility of 3D printers, the lessons learned and the logics cultivated on digital Web 2.0 now seems applicable to the world of material things. Released in early 2012 by the artist groups F.A.T. and Sy-lab, the Free Universal Construction Kit is a set of 3D...

  6. Natural Gas Energy Educational Kit.

    Science.gov (United States)

    American Gas Association, Arlington, VA. Educational Services.

    Prepared by energy experts and educators to introduce middle school and high school students to natural gas and its role in our society, this kit is designed to be incorporated into existing science and social studies curricula. The materials and activities focus on the origin, discovery, production, delivery, and use of natural gas. The role of…

  7. Active Parenting Now: Program Kit.

    Science.gov (United States)

    Popkin, Michael H.

    Based largely on the theories of Alfred Adler and Rudolf Dreikurs, this parent education curriculum is a video-based interactive learning experience that teaches a comprehensive model of parenting to parents of children ages 5 to 12 years. The kit provides parents with the skills needed to help their children develop courage, responsibility, and…

  8. Basic Teaching Kit on Consumer Advertising.

    Science.gov (United States)

    Proctor and Gamble Co., Cincinnati, OH.

    This advertising kit was developed by Procter and Gamble in response to requests from teachers and consumer educators who asked for materials from business about business. The kit is not intended to cover the entire field of advertising. Rather, it centers on advertising as it is known and practiced by Procter and Gamble. The purpose of the kit is…

  9. Post-staining electroblotting for efficient and reliable peptide blotting.

    Science.gov (United States)

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

    Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

  10. Lipid-binding analysis using a fat blot assay.

    NARCIS (Netherlands)

    Munnik, T.; Wierzchowiecka, M.

    2013-01-01

    Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose

  11. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    Energy Technology Data Exchange (ETDEWEB)

    Mansfield, E.S.; Worley, J.M. [Molecular Dynamics, Inc., Sunnyvale, CA (United States); Zimmerman, P.A. [Laboratory of Parasitic Diseases, Bethesda, MD (United States)] [and others

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  12. Mixed lubrication after rewetting of blotted pleural mesothelium.

    Science.gov (United States)

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Pecchiari, Matteo; Zocchi, Luciano; Agostoni, Emilio

    2013-01-15

    Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

  13. CERN’s FMC Kit

    CERN Document Server

    Cattin, M; Serrano, J; van der Bij, E; Wlostowski, T

    2014-01-01

    In the context of the renovation of controls and data acquisition electronics for accelerators the BE-CO-HT section at CERN has designed a kit based on carriers and mezzanines following the VITA FPGAMezzanine Card (FMC) standard. Carriers exist in VME64x and PCIe form factors, with a PXIe carrier underway. Mezzanines include an Analog to Digital Converter, a Time to Digital Converter, a fine delay generator and a Digital Input/Output. All of the designs are licensed under the CERN Open Hardware Licence and commercialised by companies. This paper discusses the benefits of this carrier mezzanine strategy and of the Open Hardware based commercial paradigm. It also explains the design of each layer of the FMC kit, from the hardware to the gateware and the Linux device driver. In addition, several tools to help designers developing gateware for mezzanines and new concepts such as the Self-Describing Bus (SDB) and the fmcbus are presented. Lastly, some of the plans for the future of the FMC kit and Open Hardware Re...

  14. Therapeutic Efficacy Assessment of CK6, a Monoclonal KIT Antibody, in a Panel of Gastrointestinal Stromal Tumor Xenograft Models

    Directory of Open Access Journals (Sweden)

    Thomas Van Looy

    2015-04-01

    Full Text Available We evaluated the efficacy of CK6, a KIT monoclonal antibody, in a panel of human gastrointestinal stromal tumor (GIST xenograft models. Nude mice were bilaterally transplanted with human GIST xenografts (four patient derived and two cell line derived, treated for 3 weeks, and grouped as follows: control (untreated; CK6 (40 mg/kg, 3× weekly; imatinib (50 mg/kg, twice daily; sunitinib (40 mg/kg, once daily; imatinib + CK6; sunitinib + CK6 (same doses and schedules as in the single-agent treatments. Tumor volume assessment, Western blot analysis, and histopathology were used for evaluation of efficacy. Statistical analysis was performed using Mann-Whitney U (MWU and Wilcoxon matched-pairs tests. CK6 as a single agent only reduced tumor growth rate in the UZLX-GIST3 model (P = .053, MWU compared to control, while in none of the other GIST models an effect on tumor growth rate was observed. CK6 did not result in significant anti-proliferative or pro-apoptotic effects in any of the GIST models, and moreover, CK6 did not induce a remarkable inhibition of KIT activation. Furthermore, no synergistic effect of combining CK6 with tyrosine kinase inhibitors (TKIs was observed. Conversely, in certain GIST xenografts, anti-tumor effects seemed to be inferior under combination treatment compared to single-agent TKI treatment. In the GIST xenografts tested, the anti-tumor efficacy of CK6 was limited. No synergy was observed on combination of CK6 with TKIs in these GIST models. Our findings highlight the importance of using relevant in vivo human tumor xenograft models in the preclinical assessment of drug combination strategies.

  15. Molecular Alterations of KIT Oncogene in Gliomas

    Directory of Open Access Journals (Sweden)

    Ana L. Gomes

    2007-01-01

    Full Text Available Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK, is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117 immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17 and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH and quantitative real-time PCR (qRT-PCR were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179 of cases, namely in 25% (1/4 of pilocytic astrocytomas, 25% (5/20 of diffuse astrocytomas, 20% (1/5 of anaplastic astrocytomas, 19.5% (15/77 of glioblastomas and one third (3/9 of anaplastic oligoastrocytomas. Only 5.7% (2/35 of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24 of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK

  16. Business Plans Kit For Dummies

    CERN Document Server

    Barrow, Colin

    2011-01-01

    An interactive guide to every element of the business planning processAre you an entrepreneur who wants to make it big? A manager grappling with logistics? A small business owner trying to map out a future in shaky economic times? Then look no further! Business Plans Kit For Dummies is a nuts-and-bolts guide to evaluating and invigorating your commercial strategy, no matter how big or small your business.Lay the foundations - identify your long-term goals and brainstorm your business options Develop the skills - sketch out a working model, size up competitors and fire up your marketing machine

  17. TSE strain differentiation in mice by immunohistochemical PrPSc profiles and triplex Western blot

    NARCIS (Netherlands)

    Keulen, van L.J.M.; Langeveld, J.P.M.; Dolstra, C.H.; Jacobs, J.G.; Bossers, A.; Zijderveld, van F.G.

    2015-01-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of

  18. Western Blot Analysis of the Exotoxin Components From Bacillus anthracis Separated by Isoelectric Focusing Gel Electrophoresis

    Science.gov (United States)

    2004-02-14

    adding hydroxylapatite (0.5% w/v; Calbiochem fast flow; Calbiochem, San Diego , CA). The suspensions were shaken on ice for 1–2 h, then incubated...Pellizzari, C. Recchi, G. Napolitani , M. Mock, C. Montecucco, Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine

  19. Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Marzieh ASHRAFMANSOURI

    2015-12-01

    Full Text Available Background: Cutaneous leishmaniasis (CL is a parasitic disease with a relatively wide distribution in different areas of the world, including Iran. The parasite is mainly diagnosed microscopically, but serological approaches might be useful for diagnosis as well.  This study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in Iran.Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls along with 50 sera sample from non-CL patients were collected. Native strain of Leishmania major was cultured in Schnei­der medium and soluble Leishmania antigens were prepared from amastigotes-like parasites. All of sera samples were evaluated by an immunoblot­ting system.Results: Components of 14 to 135 kDa were detectable by the sera of CL pa­tients. From 61 sera of CL patients, 59 cases (96.7% detected a 63 kDa subunit and 51 cases (83.6% recognized a 32-35 kDa component. Among all subunits, the 63 kDa band showed the highest sensitivity (96.7% and a 75 kDa band had the highest (98% specificity.Conclusion: Immunoblotting has a satisfactory performance in diagnosis of CL and this test can be used, as an aid, for proper diagnosis of CL.

  20. Indeterminate HIV-1 Western Blots: Etiology, Natural History, and Psychological Reactions

    Science.gov (United States)

    1992-09-16

    antibodies to retroviral proteins in parIents with primary Sjogren’s syndrome. Arthritis and Rheumatism 1990; 33:774-781. 51. Kunhl P, Seidl S, Holzberger...erythematosus, rheumatoid arthritis , juvenile onset diabetes, thyroiditis, and Crohn’s disease), were less likely to be tested electively for HIV, and a lower...association if both the IWB and autoantibodies were caused by another factor. Parity could reflect alloimmunization during pregnancy , producing

  1. Prehospital Cricothyrotomy Kits Used in Combat.

    Science.gov (United States)

    Schauer, Steven G; April, Michael D; Cunningham, Cord W; Long, Adrianna N; Carter, Robert

    2017-01-01

    Surgical cricothyrotomy remains the only definitive airway management modality for the tactical setting recommended by Tactical Combat Casualty Care guidelines. Some units have fielded commercial cricothyrotomy kits to assist Combat Medics with surgical cricothyrotomy. To our knowledge, no previous publications report data on the use of these kits in combat settings. This series reports the the use of two kits in four patients in the prehospital combat setting. Using the Department of Defense Trauma Registry and the Prehospital Trauma Registry, we identified four cases of patients who underwent prehospital cricothyrotomy with the use of commercial kits. In the first two cases, a Medic successfully used a North American Rescue CricKit (NARCK) to obtain a surgical airway in a Servicemember with multiple amputations from an improvised explosive device explosion. In case 3, the Medic unsuccessfully used an H&H Medical kit to attempt placement of a surgical airway in a Servicemember shot in the head by small arms fire. A second attempt to place a surgical airway using a NARCK was successful. In case 4, a Soldier sustained a gunshot wound to the chest. A Medic described fluid in the airway precluding bag-valve-mask ventilation; the Medic attempted to place a surgical airway with the H&H kit without success. Four cases of prehospital surgical airway cannulation on the battlefield demonstrated three successful uses of prehospital cricothyrotomy kits. Further research should focus on determining which kits may be most useful in the combat setting. 2017.

  2. Podcasting the Ultimate Starter Kit

    CERN Document Server

    Shipside, Steve

    2012-01-01

    Podcasting doesn't require an iPod; anyone with a computer, an MP3 player, or in some cases even a phone or a pair of shades can play podcasts. It requires very little technological know-how to set up, listen to, or even make your own programmes. Podcasting: The ultimate starter kit takes a light-hearted, friendly and refreshingly jargon-free look at eveything you need to know to get started, and with its free start-up CD it couldn't be easier. With the help of Podcasting, you can find out how to set up your software and record podcasts, where to go to find programmes on anything from religion

  3. Teen Ambassador Leadership Kit (TALK

    Directory of Open Access Journals (Sweden)

    Donna R. Gillespie

    2012-12-01

    Full Text Available Teen Ambassador Leadership Kit, (TALK, is an annual weekend retreat designed for teens interested in promoting and marketing 4-H in their communities. TALK organizers felt teens would benefit from an intensive weekend retreat focused on communication. TALK produces a network of educated and excited 4-H teens that are available to help with 4-H promotion and marketing. Participants arrive on Friday evening for team building activities, on Saturday they participate in one of the workshops offered and on Sunday morning each workshop group has the opportunity to share their completed projects and what they learned. At the conclusion of the retreat, teens are designated "County 4-H Ambassadors" and certificates of completion, professional business cards and polo shirts are presented. The TALK teen participants return home to share what they learned with their local county 4-H staff and help promote and market 4-H in their communities.

  4. KIT mutation analysis in mast cell neoplasms

    DEFF Research Database (Denmark)

    Arock, M; Sotlar, K; Akin, C;

    2015-01-01

    Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT...

  5. Culture Kits for the Elementary Classroom.

    Science.gov (United States)

    Hickey, M. Gail

    1997-01-01

    Outlines an instructional unit where students construct culture kits illustrating a specific culture. Culture kits are constructed out of realia and other material including maps, travel brochures, photographs, newspapers, souvenirs, and other items. Discusses collecting these items and possible multicultural applications. (MJP)

  6. Anatomy of a Minicourse. Audiovisual Kit.

    Science.gov (United States)

    Meyer, Rex

    This kit explains the use of minicourses at Macquarie University in New South Wales, Australia, which were designed for the inservice training of teachers. The kit contains the script of the audio commentary, a cassette tape, color slides, and a booklet entitled "Anatomy of the Minicourse." The tape briefly describes the minicourses in general and…

  7. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

  8. 100G Deployment@(DE-KIT)

    Science.gov (United States)

    Hoeft, Bruno; Petzold, Andreas

    2015-12-01

    The Steinbuch Centre for Computing (SCC) at Karlsruhe Institute of Technology (KIT) has been involved fairly early in 100GE network technology. Initiated by DFN1 (the German NREN), a first 100GE wide area network testbed over a distance of approx. 450 km was deployed between the national research organizations KIT and FZ-Jülich in 2010. Three years later in 2013. KIT joined the Caltech SuperComputing 2013 (SC132) 100GE "show floor" initiative using the transatlantic ANA-100GE link to transfer LHC data from a storage at DE-KIT (GridKa) in Europe to hard disks at the show floor of SC13 in Denver (USA). The network infrastructure of KIT as well as of the German Tier-1 installation DE-KIT (GridKa). however. is still based on 10Gbps. As highlighted in the contribution "Status and Trends in Networking at LHC Tier1 Facilities" to CHEP 2012. proactive investment is required at the Tier-1 sites. Bandwidth requirements will grow beyond current capacity and the required upgrades are expected in 2015. In close cooperation with DFN. KIT drives the upgrade from 10GE to 100GE. The process is divided into several phases. due to upgrade costs and differing requirements in different parts of the network infrastructure. The requirements of the different phases as well as the planned topology will be described. Some of the obstacles we discovered during the deployment will be discussed and solutions or workarounds presented.

  9. Multicentre evaluation of commercial kit methods

    DEFF Research Database (Denmark)

    Gram, J; Declerck, P J; Sidelmann, Johannes Jakobsen;

    1993-01-01

    in the production of data from each of the commercial kits tested. A considerable variation of PAI activity results reported from the laboratories testing the same commercial kits was observed. The range of reported results could in individual samples exceed the median value indicating an interlaboratory variation...... activity levels and a CV of about 16% for high PAI activity levels. The high imprecision of the kits in the low concentration range of PAI activity indicates that unspecific factors in plasma may interfere with determination of active PAI. This was confirmed by the evaluation of the results from one...

  10. The SCRAM tool-kit

    Science.gov (United States)

    Tamir, David; Flanigan, Lee A.; Weeks, Jack L.; Siewert, Thomas A.; Kimbrough, Andrew G.; McClure, Sidney R.

    1994-01-01

    This paper proposes a new series of on-orbit capabilities to support the near-term Hubble Space Telescope, Extended Duration Orbiter, Long Duration Orbiter, Space Station Freedom, other orbital platforms, and even the future manned Lunar/Mars missions. These proposed capabilities form a toolkit termed Space Construction, Repair, and Maintenance (SCRAM). SCRAM addresses both intra-Vehicular Activity (IVA) and Extra-Vehicular Activity (EVA) needs. SCRAM provides a variety of tools which enable welding, brazing, cutting, coating, heating, and cleaning, as well as corresponding nondestructive examination. Near-term IVA-SCRAM applications include repair and modification to fluid lines, structure, and laboratory equipment inside a shirt-sleeve environment (i.e. inside Spacelab or Space Station). Near-term EVA-SCRAM applications include construction of fluid lines and structural members, repair of punctures by orbital debris, refurbishment of surfaces eroded by contaminants. The SCRAM tool-kit also promises future EVA applications involving mass production tasks automated by robotics and artificial intelligence, for construction of large truss, aerobrake, and nuclear reactor shadow shields structures. The leading candidate tool processes for SCRAM, currently undergoing research and development, include Electron Beam, Gas Tungsten Arc, Plasma Arc, and Laser Beam. A series of strategic space flight experiments would make SCRAM available to help conquer the space frontier.

  11. Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

    Science.gov (United States)

    Cheng, Tsai-Mu; Huang, Pei-Chen; Pan, Ju-Pin; Lin, Kuan-Yu; Mao, Simon J T

    2007-04-15

    Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

  12. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

    Science.gov (United States)

    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  13. Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas.

    Science.gov (United States)

    Murakami, A; Mori, T; Sakai, H; Murakami, M; Yanai, T; Hoshino, Y; Maruo, K

    2011-09-01

    KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.

  14. Village Green Project and Air Sensor Kits

    Science.gov (United States)

    This is a presentation for the OAQPS Teachers Workshop. Will provide a background overview on the Village Green Project and our air sensor kit for outreach, then have the teachers try putting it together.

  15. Directional and Attitude Stability Control Kit

    Science.gov (United States)

    2014-07-01

    and Attitude Stability Control Kit Final Progress Report This report outlines progress on the DARPA M3 Program, project “Directional and Attitude ...2 ABSTRACT Number of Papers published in peer-reviewed journals: Number of Papers published in non peer-reviewed journals: Directional and Attitude ...Stability Control Kit Final Progress Report Report Title This report outlines progress on the DARPA M3 Program, project “Directional and Attitude

  16. The Topology ToolKit.

    Science.gov (United States)

    Tierny, Julien; Favelier, Guillaume; Levine, Joshua A; Gueunet, Charles; Michaux, Michael

    2017-08-29

    This system paper presents the Topology ToolKit (TTK), a software platform designed for the topological analysis of scalar data in scientific visualization. While topological data analysis has gained in popularity over the last two decades, it has not yet been widely adopted as a standard data analysis tool for end users or developers. TTK aims at addressing this problem by providing a unified, generic, efficient, and robust implementation of key algorithms for the topological analysis of scalar data, including: critical points, integral lines, persistence diagrams, persistence curves, merge trees, contour trees, Morse-Smale complexes, fiber surfaces, continuous scatterplots, Jacobi sets, Reeb spaces, and more. TTK is easily accessible to end users due to a tight integration with ParaView. It is also easily accessible to developers through a variety of bindings (Python, VTK/C++) for fast prototyping or through direct, dependency-free, C++, to ease integration into pre-existing complex systems. While developing TTK, we faced several algorithmic and software engineering challenges, which we document in this paper. In particular, we present an algorithm for the construction of a discrete gradient that complies to the critical points extracted in the piecewise-linear setting. This algorithm guarantees a combinatorial consistency across the topological abstractions supported by TTK, and importantly, a unified implementation of topological data simplification for multi-scale exploration and analysis. We also present a cached triangulation data structure, that supports time efficient and generic traversals, which self-adjusts its memory usage on demand for input simplicial meshes and which implicitly emulates a triangulation for regular grids with no memory overhead. Finally, we describe an original software architecture, which guarantees memory efficient and direct accesses to TTK features, while still allowing for researchers powerful and easy bindings and extensions

  17. Orbital Maneuvering Vehicle (OMV) remote servicing kit

    Science.gov (United States)

    Brown, Norman S.

    1988-01-01

    With the design and development of the Orbital Maneuvering Vehicle (OMV) progressing toward an early 1990 initial operating capability (IOC), a new era in remote space operations will evolve. The logical progression to OMV front end kits would make available in situ satellite servicing, repair, and consummables resupply to the satellite community. Several conceptual design study efforts are defining representative kits (propellant tanks, debris recovery, module servicers); additional focus must also be placed on an efficient combination module servicer and consummables resupply kit. A remote servicer kit of this type would be designed to perform many of the early maintenance/resupply tasks in both nominal and high inclination orbits. The kit would have the capability to exchange Orbital Replacement Units (ORUs), exchange propellant tanks, and/or connect fluid transfer umbilicals. Necessary transportation system functions/support could be provided by interfaces with the OMV, Shuttle (STS), or Expendable Launch Vehicle (ELV). Specific remote servicer kit designs, as well as ground and flight demonstrations of servicer technology are necessary to prepare for the potential overwhelming need. Ground test plans should adhere to the component/system/breadboard test philosophy to assure maximum capability of one-g testing. The flight demonstration(s) would most likely be a short duration, Shuttle-bay experiment to validate servicer components requiring a micro-g environment.

  18. 46 CFR 184.710 - First-aid kits.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent...

  19. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...... of virulence factors could be detected between healthy and diseased kits. By electron microscopy of faecal samples corona-, rota-, and calicivirus were demonstrated among healthy as well as diseased kits....

  20. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  1. Field Test Kit for Gun Residue Detection

    Energy Technology Data Exchange (ETDEWEB)

    WALKER, PAMELA K.; RODACY, PHILIP J.

    2002-01-01

    One of the major needs of the law enforcement field is a product that quickly, accurately, and inexpensively identifies whether a person has recently fired a gun--even if the suspect has attempted to wash the traces of gunpowder off. The Field Test Kit for Gunshot Residue Identification based on Sandia National Laboratories technology works with a wide variety of handguns and other weaponry using gunpowder. There are several organic chemicals in small arms propellants such as nitrocellulose, nitroglycerine, dinitrotoluene, and nitrites left behind after the firing of a gun that result from the incomplete combustion of the gunpowder. Sandia has developed a colorimetric shooter identification kit for in situ detection of gunshot residue (GSR) from a suspect. The test kit is the first of its kind and is small, inexpensive, and easily transported by individual law enforcement personnel requiring minimal training for effective use. It will provide immediate information identifying gunshot residue.

  2. Shuttle Kit Freezer Refrigeration Unit Conceptual Design

    Science.gov (United States)

    Copeland, R. J.

    1975-01-01

    The refrigerated food/medical sample storage compartment as a kit to the space shuttle orbiter is examined. To maintain the -10 F in the freezer kit, an active refrigeration unit is required, and an air cooled Stirling Cycle refrigerator was selected. The freezer kit contains two subsystems, the refrigeration unit, and the storage volume. The freezer must provide two basic capabilities in one unit. One requirement is to store 215 lbs of food which is consumed in a 30-day period by 7 people. The other requirement is to store 128.3 lbs of medical samples consisting of both urine and feces. The unit can be mounted on the lower deck of the shuttle cabin, and will occupy four standard payload module compartments on the forward bulkhead. The freezer contains four storage compartments.

  3. Low Cost, Advanced, Integrated Microcontroller Training Kit

    Science.gov (United States)

    Somantri, Y.; Fushshilat, I.

    2017-03-01

    This paper describes the design of an AVR microcontroller training kit with a low cost and the additional feature of an integrated downloader. The main components of this device include: Microcontroller, terminal, I/O keypad, push button, LED, seven segment display, LCD, motor stepper, and sensors. The device configuration results in low cost and ease of use; this device is suitable for laboratories with limited funding. The device can also be used as a training kit for the teaching and learning of microcontrollers.

  4. KIT safety management. Annual report 2012; KIT-Sicherheitsmanagement. Jahresbericht 2012

    Energy Technology Data Exchange (ETDEWEB)

    Frank, Gerhard (ed.)

    2013-07-01

    The KIT Safety Management Service Unit (KSM) guarantees radiological and conventional technical safety and security of Karlsruhe Institute of Technology and controls the implementation and observation of legal environmental protection requirements. KSM is responsible for - licensing procedures, - industrial safety organization, - control of environmental protection measures, - planning and implementation of emergency preparedness and response, - operation of radiological laboratories and measurement stations, - extensive radiation protection support and the - the execution of security tasks in and for all organizational units of KIT. Moreover, KSM is in charge of wastewater and environmental monitoring for all facilities and nuclear installations all over the KIT campus. KSM is headed by the Safety Commissioner of KIT, who is appointed by the Presidential Committee. Within his scope of procedure for KIT, the Safety Commissioner controls the implementation of and compliance with safety-relevant requirements. The KIT Safety Management is certified according to DIN EN ISO 9001, its industrial safety management is certified by the VBG as ''AMS-Arbeitsschutz mit System'' and, hence, fulfills the requirements of NLF / ISO-OSH 2001. KSM laboratories are accredited according to DIN EN ISO/IEC 17025. To the extent possible, KSM is committed to maintaining competence in radiation protection and to supporting research and teaching activities. The present reports lists the individual tasks of the KIT Safety Management and informs about the results achieved in 2012. Status figures in principle reflect the status at the end of the year 2012. The processes described cover the areas of competence of KSM.

  5. Effect of calcitonin gene related peptide regulated nuclear factor kappa B signal transduction on c-kit+ cardiac stem cells in hypoxia state

    Directory of Open Access Journals (Sweden)

    Xian-ping LONG

    2015-11-01

    Full Text Available Objective To investigate the effects of calcitonin gene-related peptide (CGRP on the apoptosis of c-kit+ cardiac stem cells in hypoxia. Methods Ischemia and hypoxia models of c-kit+ cardiac stem cells were reproduced in vitro. The models were divided into hypoxia+CGRP group, hypoxia+CGRP8-37 (antagonist of CGRP group, hypoxia control group, normal oxygen group, and hypoxia+BAY11-7082 [antagonist of nuclear factor kappa B (NF-κB] group. NF-κB translocation after hypoxia was detected by immunofluorescence, and NF-κB channel proteins were determined with Western blotting. The NF-κB translocation and the expression of NF-κB channel proteins after CGRP intervention were detected, and the cell apoptosis rate after intervention was determined with flow cytometry in each group. Results Under hypoxia the NF-κB signal pathway was activated, and nuclear translocation occurred in NF-κBP65 (red fluorescence. Compared with hypoxia control group, the expressions of NF-κB related proteins such as P-I-κB, NF-κBP65 and NF-κBP50 decreased obviously (P<0.05. Compared with the hypoxia+CGRP group, the expressions of NF-κB related proteins increased significantly (P<0.05 as mentioned above in hypoxia+CGRP8-37 group. Both the early and late apoptotic rates declined in hypoxia+CGRP group compared with that of hypoxia control group (P<0.05, however, the early apoptotic rate increased markedly in hypoxia+CGRP8-37 group as compared with that of hypoxia+CGRP group (P<0.05. Conclusion Under hypoxia, CGRP may regulate the NF-κB signal pathway, and at the same time suppress the apoptosis of c-kit+ cardiac stem cells. DOI: 10.11855/j.issn.0577-7402.2015.10.03

  6. Word 2013 elearning kit for dummies

    CERN Document Server

    Wempen, Faithe

    2014-01-01

    Lois Lowe is the author of several books on Microsoft Office, including Microsoft Word 2010 eLearning Kit For Dummies. She is also an online instructor who develops and teaches courses on Microsoft Office applications, computer purchase and upgrade, home office setup and emerging hardware technologies. Her courses have educated over 250,000 students for clients including Hewlett-Packard and Sony.

  7. Lifeprints ESL for Adults: Literacy. [Multimedia Kit].

    Science.gov (United States)

    Cunningham Florez, MaryAnn

    Lifeprints is a four-level program that helps adult English- as-a-Second-Language (ESL) students gain the English language skills they need to participate effectively at work and in their communities. This multimedia kit contains the first level in the series. Level one is divided into six units: "Welcome to English Class"; "Personal Information";…

  8. Countdown to Six Billion Teaching Kit.

    Science.gov (United States)

    Zero Population Growth, Inc., Washington, DC.

    This teaching kit features six activities focused on helping students understand the significance of the world population reaching six billion for our society and our environment. Featured activities include: (1) History of the World: Part Six Billion; (2) A Woman's Place; (3) Baby-O-Matic; (4) Earth: The Apple of Our Eye; (5) Needs vs. Wants; and…

  9. Teen PACK: Population Awareness Campaign Kit.

    Science.gov (United States)

    Zero Population Growth, Inc., Washington, DC.

    This packet of instructional materials is designed to teach teenagers about the effects of overpopulation on the world and on the individual. Information is presented in three related booklets. The first of the three parts of the "Teen Population Awareness Campaign Kit," illustrates overpopulation through profiles of teens living in…

  10. Numeric Data Products and Services. SPEC Kit.

    Science.gov (United States)

    Cook, Michael N., Comp.; Hernandez, John J., Comp.; Nicholson, Shawn, Comp.

    2001-01-01

    This SPEC (Systems and Procedures Exchange Center) Kit presents the results of a survey of Association of Research Libraries (ARL) member libraries. The survey addressed the following questions about numeric data (i.e., any information resource, print or non-print, with considerable numeric content) in academic libraries: (1) What relationships…

  11. Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

    Science.gov (United States)

    Colgan, D J

    1993-01-01

    The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

  12. Test/QA Plan for Verification of Microcystin Test Kits

    Science.gov (United States)

    Microcystin test kits are used to quantitatively measure total microcystin in recreational waters. These test kits are based on enzyme-linked immunosorbent assays (ELISA) with antibodies that bind specifically to microcystins or phosphate activity inhibition where the phosphatas...

  13. L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition.

    Science.gov (United States)

    Antonescu, Cristina R; Busam, Klaus J; Francone, Todd D; Wong, Grace C; Guo, Tianhua; Agaram, Narasimhan P; Besmer, Peter; Jungbluth, Achim; Gimbel, Mark; Chen, Chin-Tung; Veach, Darren; Clarkson, Bayard D; Paty, Philip B; Weiser, Martin R

    2007-07-15

    Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

  14. KIT safety management. Annual report 2013; KIT-Sicherheitsmanagement. Jahresbericht 2013

    Energy Technology Data Exchange (ETDEWEB)

    Frank, Gerhard (ed.)

    2014-07-01

    The KIT Safety Management Service Unit (KSM) guarantees radiological and conventional technical safety and security of Karlsruhe Institute of Technology and controls the implementation and observation of legal environmental protection requirements. KSM is responsible for licensing procedures, industrial safety organization, control of environmental protection measures, planning and implementation of emergency preparedness and response, operation of radiological laboratories and measurement stations, extensive radiation protection support and the execution of security tasks in and for all organizational units of KIT. Moreover, KSM is in charge of wastewater and environmental monitoring for all facilities and nuclear installations all over the KIT campus. KSM is headed by the Safety Commissioner of KIT, who is appointed by the Presidential Committee. Within his scope of procedure for KIT, the Safety Commissioner controls the implementation of and compliance with safety-relevant requirements. The KIT Safety Management is certified according to DIN EN ISO 9001, its laboratories are accredited according to DIN EN ISO/IEC 17025. To the extent possible, KSM is committed to maintaining competence in radiation protection and to supporting research and teaching activities. The present reports lists the individual tasks of the KIT Safety Management and informs about the results achieved in 2013. Status figures in principle reflect the status at the end of the year 2013. The processes described cover the areas of competence of KSM. Due to changes in the organization of the infrastructural service units in KIT, KSM has been cancelled at the end of 2013. Its tasks will mainly be covered in 2014 by the new founded service unit Safety and Environmental (Sicherheit und Umwelt, SUM). The departments Campus Security, Fire Brigade and Information Technology have been transferred to the Service Unit General Services (Allgemeine Services, ASERV).

  15. 46 CFR 121.710 - First-aid kits.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit... kits, the contents must be stowed in a suitable, watertight container that is marked “First-Aid Kit”....

  16. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...

  17. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  18. 21 CFR 880.5740 - Suction snakebite kit.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Suction snakebite kit. 880.5740 Section 880.5740... Devices § 880.5740 Suction snakebite kit. (a) Identification. A suction snakebite kit is a device consisting of a knife, suction device, and tourniquet used for first-aid treatment of snakebites by removing...

  19. 21 CFR 870.1350 - Catheter balloon repair kit.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the... effect the repair or replacement. (b) Classification. Class III (premarket approval). (c) Date PMA...

  20. 21 CFR 872.3570 - OTC denture repair kit.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false OTC denture repair kit. 872.3570 Section 872.3570...) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3570 OTC denture repair kit. (a) Identification. An OTC denture repair kit is a device consisting of a material, such as a resin monomer system...

  1. Home Pregnancy Test Kits: How Readable Are the Instructions?

    Science.gov (United States)

    Holcomb, Carol Ann

    At the conclusion of their study on home pregnancy test kits, Valinas and Perlman (1982) suggested that the instructions accompanying the kits be revised to make them easier to read. A study was undertaken to determine the readability of the printed instructions accompanying five home pregnancy test kits (Daisy II, Answer, Acu-Test, Predictor, and…

  2. 21 CFR 864.2260 - Chromosome culture kit.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Chromosome culture kit. 864.2260 Section 864.2260...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2260 Chromosome culture kit. (a) Identification. A chromosome culture kit is a device containing the necessary ingredients...

  3. 46 CFR 108.707 - First aid kit.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false First aid kit. 108.707 Section 108.707 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Miscellaneous Equipment § 108.707 First aid kit. Each unit must have a first-aid kit approved by the Mine...

  4. 33 CFR 144.01-30 - First-aid kit.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false First-aid kit. 144.01-30 Section...) OUTER CONTINENTAL SHELF ACTIVITIES LIFESAVING APPLIANCES Manned Platforms § 144.01-30 First-aid kit. On each manned platform a first-aid kit approved by the Commandant or the U.S. Bureau of Mines shall...

  5. 46 CFR 169.725 - First aid kit.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false First aid kit. 169.725 Section 169.725 Shipping COAST... Control, Miscellaneous Systems, and Equipment § 169.725 First aid kit. Each vessel must carry an approved first aid kit, constructed and fitted in accordance with subpart 160.041 of this chapter....

  6. Kit preparation of radioiodinated o-iodohippuran

    Energy Technology Data Exchange (ETDEWEB)

    Hinkle, G.H.; Basmadjian, G.P.; Kirschner, A.S.; Ice, R.D.

    1981-03-01

    The purpose of this study was to evaluate a kit preparation for radioiodinated o-iodohippuran (I). All ingredients, excluding the radionuclide, were packaged in a ready-to-use kit for easy, quick formulation. Electrophoresis was utilized to evaluate the radiochemical purity of the labeled product and indicated that the radiolabeling technique provided a product with greater than 95% radiochemical purity. Biodistribution studies in rats and rabbits provided an indication of the tissue distribution and localization of the radiopharmaceutical. Computer-generated renogram curves plotted from gamma-camera images of rabbits showed the equivalency of the 131I-labeled I and 123I-labeled I to the commercially available radiopharmaceutical.

  7. Development of Mode Conversion Waveguides at KIT

    Directory of Open Access Journals (Sweden)

    Jin Jianbo

    2015-01-01

    Full Text Available The development of mode conversion waveguides (launchers for high power gyrotrons has gone through three stages at KIT. Formerly, harmonically deformed launchers have been used in the series gyrotrons developed for the stellarator W7-X. In 2009, a numerical method for the analysis and synthesis of mirror-line launchers was developed at KIT. Such a launcher with adapted mode-converting mirrors for a 2 MW TE34,19-mode, 170GHz coaxial-cavity gyrotron has been designed and tested, and also a mirror-line launcher for the 1MW EU ITER gyrotron has been designed. Recently, based on the Helmholtz-Kirchhoff integral theorem, a novel numerical method for the synthesis of hybrid-type gyrotron launchers has been developed. As an example, TE32,9 mode launchers operating at 170GHz that have been designed using the three different methods are being compared.

  8. Western Sufism

    DEFF Research Database (Denmark)

    Sedgwick, Mark

    Western Sufism is sometimes dismissed as a relatively recent "new age" phenomenon, but in this book, Mark Sedgwick argues that it actually has very deep roots, both in the Muslim world and in the West. In fact, although the first significant Western Sufi organization was not established until 1915......, the first Western discussion of Sufism was printed in 1480, and Western interest in some of the ideas that are central to Sufi thought goes back to the thirteenth century. Sedgwick starts with the earliest origins of Western Sufism in late antique Neoplatonism and early Arab philosophy, and traces later...

  9. RNase protection assays and RNA gel blots: a direct comparison of sensitivity.

    Science.gov (United States)

    Higgs, D C; Colbert, J T

    1992-01-01

    RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

  10. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein

    Directory of Open Access Journals (Sweden)

    Taguchi Takahiro

    2010-12-01

    Full Text Available Abstract Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.

  11. Technetium-99m ceftizoxime kit preparation

    Directory of Open Access Journals (Sweden)

    Simone Odília Fernandes Diniz

    2005-10-01

    Full Text Available The aim of this work was to prepare a kit of 99mTc-ceftizoxime (99mTc-CFT, with stability and biological activity preserved, able to identify a septic focus (E. coli in the experimental infection model in rats. The preparation of the CFT kit involved the use of lyophilized solutions containing the antibiotic ceftizoxime and the sodium dithionite reducing agent (6.0 mg/mL. After lyophilization, the kit was reconstituted with 1.0 mL of sodium 99mTc-pertechnetate solution (Na99mTcO4- with an activity of 370 MBq. The solution was boiled for 10 min and filtered through a cellulose ester filter. The labeling efficiency was on the order of 92%, remaining stable for six hours and the kit remained stable for two months. The biological activity of the 99mTc-CFT was evaluated by diffusion in agar impregnated with E.coli and S. aureus. Seven Wistar rats, weighing from 200 to 250 g, were used for the development of the septic focus. After 24 hours from the induction of the infectious site (E.coli, the animals were anesthetized and 0.1 mL of 99mTc-CFT (37 MBq was injected into the tail veins of the animals. The images were obtained with a gamma camera one, two and six hours after injection and the regions of interest (ROIs were calculated. The diameters of the inhibition halos for 99mTc-CFT were 27.16 ± 0.23 and 27.17 ± 0.20 for S.aureus and E.coli, respectively, while those for the unlabeled CFT were 30.4 ± 0.33 and 29.43 ± 0.26, respectively. The results for the biodistribution of 99mTc-CFT in infected animals furnished a ratio of 1.97 ± 0.31, 2.10 ± 0.42 and 2.01 ± 0.42 for cpm-target/cpm-no target for the one, two and six-hour periods, respectively. The images showed a clear uptake of labeled antibiotic (99mTc-CFT by the infectious site during the experiment. The results attest to the viability of producing a kit with 99m technetium-labeled ceftizoxime for the investigation of infectious processes.O objetivo deste trabalho foi preparar um kit de Tc

  12. Megakaryocytes compensate for Kit insufficiency in murine arthritis

    Science.gov (United States)

    Cunin, Pierre; Penke, Loka R.; Thon, Jonathan N.; Monach, Paul A.; Jones, Tatiana; Chang, Margaret H.; Chen, Mary M.; Melki, Imene; Gurish, Michael F.; Italiano, Joseph E.; Boilard, Eric; Nigrovic, Peter A.

    2017-01-01

    The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell–deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1–dependent manner. Transfer of WT but not IL-1–deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1–driven systemic inflammatory disease. PMID:28375155

  13. Markkinointisuunnitelma : Pirttiniemen mökit

    OpenAIRE

    Hynynen, Susanna

    2010-01-01

    Tämän opinnäytetyön tarkoituksena oli luoda tehokas markkinoin-tisuunnitelma toimeksiantona maaseutumatkailuyritykselle Pirttiniemen mökit. Markkinointisuunnitelma toteutettiin toiminnallisena opinnäytetyönä joka koostuu kahdesta osasta. Teoreettisessa osuudessa tutkitaan maaseutumarkkinointia ja siihen liittyviä käsitteitä. Toiminnallinen osuus on Pirttiniemen mökeille tehty produkti eli tuotos, jossa käydään läpi perusteellisesti yrityksen markkinointisuunnitelman laatiminen käsitteellisen ...

  14. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

    Science.gov (United States)

    Aims: To compare the ability of the Italian and the U.S. bovine spongiform encephalopathy (BSE) confirmatory protocols in the detection of classical (C-) and atypical - low (L-) and high (H-) type- BSE forms. Methods and Results: Obex samples from U.S. and Italian C-type BSE cases, a U.S. H-type and...

  15. [Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody].

    Science.gov (United States)

    Yao, P P; Xu, F; Sun, Y S; Yang, Z R; Zhang, Y; Yue, M; Zhu, H P

    2017-04-10

    Objective: S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology. The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum. Methods: Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z10S-TN was constructed by using the routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP. WB assay was established to detect the serum samples from 95 confirmed HFRS patients. Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method. Results: rBAC-Z10S-TN was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV IgG could efficiently recognize rNP and hybridize with the recombinant protein. 97.67% of the serum samples from the HFRS patients were positive confirmed by WB. Conclusions: We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10. WB assay which was established in this study could be used as a new serological test for HFRS diagnosis, thanks to the simplicity, safety, sensitivity and specificity of this method.

  16. Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays

    Directory of Open Access Journals (Sweden)

    Chandra Kirana

    2012-01-01

    Full Text Available Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC remains disappointing with some 40–50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF area and liver metastasis (LM than those from main tumour body (MTB. Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

  17. Western-blot detection of PrP**sc in archived paraffin-embedded brainstem from scrapie-affected sheep

    Science.gov (United States)

    Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies or prion diseases. Immunoassays that identify disease-associated prion protein (PrP**Sc) are integral to the diagnosis o...

  18. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation

    OpenAIRE

    Badr, Haitham A.; Dina M. M. AlSadek; Mohit P. Mathew; Chen-Zhong Li; Djansugurova, Leyla B.; Yarema, Kevin J.; Hafiz Ahmed

    2015-01-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show s...

  19. Characterization of Polyclonal Antibody Induced by Autoantibody TPO (Thyroidperoxidase) From Autoimmune Thyroid Disease (AITD) Serum with ELISA and Western Blotting

    OpenAIRE

    Maulidya Aulia Fiqriyana; Aulanni'am Aulanni'am; Anna Roosdiana

    2013-01-01

    Autoantibody TPO is a potential marker for early detection of autoimmune thyroid disease (AITD). Autoantibody TPO has a specifity and a sensitivity ranging from 82% to100% in comparison to other AITD serology markers. Concentration of autoantibody TPO in sera had a positive correlation with activities of chronic AITD. This research have been conducted to investigate the characteristic of polyclonal antibody TPO induced by autoantibody TPO from serum of AITD patients. The autoantibody TPO was ...

  20. Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

    Science.gov (United States)

    Oldal, Miklós; Németh, Viktória; Madai, Mónika; Kemenesi, Gábor; Dallos, Bianka; Péterfi, Zoltán; Sebők, Judit; Wittmann, István; Bányai, Krisztián; Jakab, Ferenc

    2014-06-01

    Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

  1. 14 CFR Appendix A to Part 121 - First Aid Kits and Emergency Medical Kits

    Science.gov (United States)

    2010-01-01

    ... administer required drugs) 4 Needles (sizes necessary to administer required drugs) 6 50% Dextrose injection... dose ampule or equivalent 2 Nitroglycerin tablets 10 Basic instructions for use of the drugs in the kit... administer required medications) 4 Analgesic, non-narcotic, tablets, 325 mg 4 Antihistamine tablets, 25 mg...

  2. To Kit or Not to Kit? Evaluating and Implementing Science Materials and Resources

    Science.gov (United States)

    Schiller, Ellen; Melin, Jacque; Bair, Mary

    2016-01-01

    With the release of the "Next Generation Science Standards," many schools are reexamining the science materials they are using. Textbook companies and kit developers are eager to meet the demand for "NGSS"-aligned teaching materials. Teacher may have been asked to serve on a science curriculum committee, or to evaluate current…

  3. 21 CFR 880.5960 - Lice removal kit.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lice removal kit. 880.5960 Section 880.5960 Food... § 880.5960 Lice removal kit. (a) Identification. The lice removal kit is a comb or comb-like device intended to remove and/or kill lice and nits from head and body hair. It may or may not be battery...

  4. Data Center Energy Efficiency Measurement Assessment Kit Guide and Specification

    Energy Technology Data Exchange (ETDEWEB)

    None

    2012-10-26

    A portable and temporary wireless mesh assessment kit can be used to speed up and reduce the costs of a data center energy use assessment and overcome the issues with respect to shutdowns. The assessment kit is comprised of temperature, relative humidity, and pressure sensors. Also included are power meters that can be installed on computer room air conditioners (CRACs) without intrusive interruption of data center operations. The assessment kit produces data required for a detailed energy assessment of the data center.

  5. The Virtual Physiological Human ToolKit.

    Science.gov (United States)

    Cooper, Jonathan; Cervenansky, Frederic; De Fabritiis, Gianni; Fenner, John; Friboulet, Denis; Giorgino, Toni; Manos, Steven; Martelli, Yves; Villà-Freixa, Jordi; Zasada, Stefan; Lloyd, Sharon; McCormack, Keith; Coveney, Peter V

    2010-08-28

    The Virtual Physiological Human (VPH) is a major European e-Science initiative intended to support the development of patient-specific computer models and their application in personalized and predictive healthcare. The VPH Network of Excellence (VPH-NoE) project is tasked with facilitating interaction between the various VPH projects and addressing issues of common concern. A key deliverable is the 'VPH ToolKit'--a collection of tools, methodologies and services to support and enable VPH research, integrating and extending existing work across Europe towards greater interoperability and sustainability. Owing to the diverse nature of the field, a single monolithic 'toolkit' is incapable of addressing the needs of the VPH. Rather, the VPH ToolKit should be considered more as a 'toolbox' of relevant technologies, interacting around a common set of standards. The latter apply to the information used by tools, including any data and the VPH models themselves, and also to the naming and categorizing of entities and concepts involved. Furthermore, the technologies and methodologies available need to be widely disseminated, and relevant tools and services easily found by researchers. The VPH-NoE has thus created an online resource for the VPH community to meet this need. It consists of a database of tools, methods and services for VPH research, with a Web front-end. This has facilities for searching the database, for adding or updating entries, and for providing user feedback on entries. Anyone is welcome to contribute.

  6. Phase II Study of Nilotinib in Melanoma Harboring KIT Alterations Following Progression to Prior KIT Inhibition.

    Science.gov (United States)

    Carvajal, Richard D; Lawrence, Donald P; Weber, Jeffrey S; Gajewski, Thomas F; Gonzalez, Rene; Lutzky, Jose; O'Day, Steven J; Hamid, Omid; Wolchok, Jedd D; Chapman, Paul B; Sullivan, Ryan J; Teitcher, Jerrold B; Ramaiya, Nikhil; Giobbie-Hurder, Anita; Antonescu, Cristina R; Heinrich, Michael C; Bastian, Boris C; Corless, Christopher L; Fletcher, Jonathan A; Hodi, F Stephen

    2015-05-15

    Although durable responses can be achieved with tyrosine kinase inhibitors such as imatinib in melanomas harboring KIT mutations, the efficacy of alternative inhibitors after progression to imatinib and the activity of these agents on brain metastases are unknown. We conducted a phase II study of nilotinib 400 mg twice a day in two cohorts of patients with melanomas harboring KIT mutations or amplification: (A) those refractory or intolerant to a prior KIT inhibitor; and (B) those with brain metastases. The primary endpoint was 4-month disease control rate. Secondary endpoints included response rate, time-to-progression (TTP), and overall survival (OS). A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in cohorts A and B, respectively. Twenty patients were enrolled and 19 treated (11 in cohort A; 8 in cohort B). Three patients on cohort A [27%; 95% confidence interval (CI), 8%-56%] and 1 on cohort B (12.5%; 90% CI, 0.6%-47%) achieved the primary endpoint. Two partial responses were observed in cohort A (18.2%; 90% CI, 3%-47%); none were observed in cohort B. The median TTP and OS was 3.3 (90% CI, 2.1-3.9 months) and 9.1 months (90% CI, 4.3-14.2 months), respectively, in all treated patients. Nilotinib may achieve disease control in patients with melanoma harboring KIT alterations and whose disease progressed after imatinib therapy. The efficacy of this agent in KIT-altered melanoma with brain metastasis is limited. ©2015 American Association for Cancer Research.

  7. PEMANFAATAN KIT OPTIK SEBAGAI WAHANA DALAM PENINGKATAN SIKAP ILMIAH SISWA

    Directory of Open Access Journals (Sweden)

    - Wahyudi

    2012-01-01

    Full Text Available Penelitian ini mengkaji bagaimana bentuk kegiatan pemanfaatan kit yang dapat meningkatkan sikap ilmiah siswa, serta sikapsikapilmiah apa saja yang dapat dikembangkan melalui pemanfaatan kit dalam pembelajaran. Penelitian tindakan kelas inidilaksanakan dalam empat siklus dengan memanfaatkan kit optik pada pokok bahasan optika geometri. Hasil penelitianmenunjukkan bahwa peningkatan sikap ilmiah dapat dilakukan dengan mengembangkan perangkat kegiatan pemanfaatan kitOptik dalam pembelajaran, meliputi: rencana pelaksanaan pembelajaran (RPP yang berorientasi pada aktivitas belajar siswa,berupa pembiasaan bersikap ilmiah dengan melakukan kerja ilmiah, lembar kerja siswa (LKS yang dirancang dengan pendekataninquiry yang menjadi arah serta panduan bagi siswa dalam bekerja ilmiah. Sikap ilmiah yang dapat dikembangkan dalam penelitianini adalah objektif, kritis, ulet, rendah hati, dapat bekerjasama dengan orang lain dan pandangan positif terhadap kegagalan. This Classroom action research discusses how activities of kit optic utilization can improve students' scientific attitudes and whatscientific attitudes can be developed in their learning. This research is conducted in four cycles that applies kit optic as the maintopic in optical geometry. The result shows that students' scientific attitudes can be improved by implementing specific units ofactivity in kit optic utilization in students' learning. They consist of RPP (The Plan for Teaching Implementation with the orientationof students' learning activities in the forms of developing students' habit of scientific attitude in scientific project and students' paperwork through inquiry approach. This research suggests that kit optic utilization improves students' scientific attitudes of beingobjective, critical, perseverance, modest, cooperative, and optimistic toward failures.Keywords: kit optic, scientific attitudes, optical geometry, scientific attitude

  8. Desensitized Optimal Filtering and Sensor Fusion Tool Kit Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Research on desensitized optimal filtering techniques and a navigation and sensor fusion tool kit using advanced filtering techniques is proposed. Research focuses...

  9. StochKit-FF: Efficient Systems Biology on Multicore Architectures

    CERN Document Server

    Aldinucci, Marco; Liò, Pietro; Sorathiya, Anil; Torquati, Massimo

    2010-01-01

    The stochastic modelling of biological systems is an informative, and in some cases, very adequate technique, which may however result in being more expensive than other modelling approaches, such as differential equations. We present StochKit-FF, a parallel version of StochKit, a reference toolkit for stochastic simulations. StochKit-FF is based on the FastFlow programming toolkit for multicores and exploits the novel concept of selective memory. We experiment StochKit-FF on a model of HIV infection dynamics, with the aim of extracting information from efficiently run experiments, here in terms of average and variance and, on a longer term, of more structured data.

  10. Autometallography: tissue metals demonstrated by a silver enhancement kit

    DEFF Research Database (Denmark)

    Danscher, G; Nørgaard, J O; Baatrup, E

    1987-01-01

    In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit...... silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium...

  11. Use of AffiProbe HPV test kit for detection of human papillomavirus DNA in genital scrapes.

    OpenAIRE

    Ranki, M; Leinonen, A W; Jalava, T. (Tiina); Nieminen, P.; Soares, V R; Paavonen, J; Kallio, A

    1990-01-01

    The presence of human papillomavirus (HPV) DNA in cervical and vaginal scrapes was analyzed by the AffiProbe HPV test kit (Orion Corp., Orion Pharmaceutica, Helsinki, Finland), which is a 1-day solution hybridization test for HPV type 6/11, 16, or 18. The AffiProbe test was compared with a commercially available dot blot test (ViraPap and ViraType tests; Life Technologies Inc., Gaithersburg, Md.). The study group consisted of 178 patients seen in a gynecological outpatient clinic. Altogether,...

  12. [Application of the QIAamp DNA Investigator Kit and Prepfiler Forensic DNA Extraction Kit in genomic DNA extraction from skeletal remains].

    Science.gov (United States)

    Ludwikowska-Pawłowska, Małgorzata; Jacewicz, Renata; Jedrzejczyk, Maciej; Prośniak, Adam; Berent, Jarosław

    2009-01-01

    The report presents an application of the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit in genomic DNA extraction from post-mortem highly degraded skeletal remains. The analysis included 25 bone samples collected on autopsy. DNA extraction was performed in accordance with the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit manufacturer's isolation protocols. Amplification was performed on a Biometra termocycler using the AmpFISTR Identifiler PCR Amplification Kit according to the manufacturer's protocol. Typing of PCR products was carried out on an ABI Prism 377 DNA sequencer. The recommended parameters for GeneScan analysis and Genotyper software were followed. The authors demonstrated that the QIAamp DNA Investigator Kit was more effective, convenient and statistically significantly better method which may be employed in DNA extraction from bone specimens.

  13. GTP-blot analysis of small GTP-binding proteins. The C-terminus is involved in renaturation of blotted proteins.

    Science.gov (United States)

    Klinz, F J

    1994-10-01

    Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [alpha-32P]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-32P]GTP was decreased many-fold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [alpha-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha-32P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

  14. Dr. E. Kits van Waveren (1906—1995)

    NARCIS (Netherlands)

    Bas, C.

    1996-01-01

    With the death on 3 September 1995 of Dr. E. Kits van Waveren, the Dutch mycologists lost one of their most prominent, internationally known amateurs, a specialist on the taxonomy of the genus Psathyrella and an ardent collector. Dr. Kits van Waveren, honorary staff member of the Rijksherbarium at L

  15. Somatic mutations of KIT in familial testicular germ cell tumours

    NARCIS (Netherlands)

    Rapley, EA; Hockley, S; Warren, W; Johnson, L; Huddart, R; Crockford, G; Forman, D; Leahy, MG; Oliver, DT; Tucker, K; Friedlander, M; Phillips, KA; Hogg, D; Jewett, MAS; Lohynska, R; Daugaard, G; Richard, S; Heidenreich, A; Geczi, L; Bodrogi, [No Value; Olah, E; Ormiston, WJ; Daly, PA; Looijenga, LHJ; Guilford, P; Aass, N; Fossa, SD; Heimdal, K; Tjulandin, SA; Liubchenko, L; Stoll, H; Weber, W; Einhorn, L; Weber, BL; McMaster, M; Greene, MH; Bishop, DT; Easton, D; Stratton, M

    2004-01-01

    Somatic mutations of the KIT gene have been reported in mast cell diseases and gastrointestinal stromal tumours. Recently, they have also been found in mediastinal and testicular germ cell tumours (TGCTs), particularly in cases with bilateral disease. We screened the KIT coding sequence ( except exo

  16. Energy Education Incentives: Evaluating the Impact of Consumer Energy Kits

    Science.gov (United States)

    Kirby, Sarah D.; Guin, Autumn; Langham, Laura

    2015-01-01

    Measuring the energy and environmental impact of residential energy education efforts is difficult. The E-Conservation residential energy management program uses consumer energy kits to document the impact of energy-efficient improvements. The consumer energy kit provides an incentive for individuals attending energy education workshop, helps…

  17. Factory packed and expired - about emergency insect sting kits.

    Science.gov (United States)

    Fischer, Jörg; Knaudt, Björn; Caroli, Ulrich M; Biedermann, Tilo

    2008-09-01

    Patients allergic to insect venom are instructed to always carry emergency medication-an emergency kit. According to the current guidelines, these emergency kits should contain a H1-receptor-blocking antihistamine and corticosteroid for oral use, as well as an epinephrine inhaler and in particular situations an epinephrine auto-injector. For quality management reasons patients with wasp venom allergy who presented for sting challenge provocation test were told to demonstrate their emergency kits. Concomitantly, constituents of the patient's emergency kits were checked for date of expiry of the medication and the patients were interviewed on storage and use of the emergency kits. In total 42 patients with a median duration time of systemic immunotherapy of 2.5 year were evaluated. Medication post date of expiration was found in 54% of the kits (n = 39). Only 31% of the patients could demonstrate how to use the kits correctly. Problems were especially evident concerning for using the application of the inhaler and auto-injector. 50% of the patients demonstrated using epinephrine auto-injectors in such way that an accidental injection into the fingers would have resulted. To assure safe and effective handling of all components of the emergency kit, continuous training and repeated supervised practice is necessary.

  18. Energy Education Incentives: Evaluating the Impact of Consumer Energy Kits

    Science.gov (United States)

    Kirby, Sarah D.; Guin, Autumn; Langham, Laura

    2015-01-01

    Measuring the energy and environmental impact of residential energy education efforts is difficult. The E-Conservation residential energy management program uses consumer energy kits to document the impact of energy-efficient improvements. The consumer energy kit provides an incentive for individuals attending energy education workshop, helps…

  19. School-to-Work Jump-Start Equity Kit.

    Science.gov (United States)

    Education Development Center, Inc., Newton, MA. Women's Educational Equity Act Dissemination Center.

    This starter kit is a resource for state and local school-to-work (STW) directors, educators, parents, students, business and community, and economic development organizations serving all students through STW. The kit begins with four articles: "STW and Gender Equity: Opportunity for or Barrier to Economic Parity?" (Katherine Hanson, Joyce…

  20. Marshall Space Flight Center Telescience Resource Kit

    Science.gov (United States)

    Wade, Gina

    2016-01-01

    Telescience Resource Kit (TReK) is a suite of software applications that can be used to monitor and control assets in space or on the ground. The Telescience Resource Kit was originally developed for the International Space Station program. Since then it has been used to support a variety of NASA programs and projects including the WB-57 Ascent Vehicle Experiment (WAVE) project, the Fast Affordable Science and Technology Satellite (FASTSAT) project, and the Constellation Program. The Payloads Operations Center (POC), also known as the Payload Operations Integration Center (POIC), provides the capability for payload users to operate their payloads at their home sites. In this environment, TReK provides local ground support system services and an interface to utilize remote services provided by the POC. TReK provides ground system services for local and remote payload user sites including International Partner sites, Telescience Support Centers, and U.S. Investigator sites in over 40 locations worldwide. General Capabilities: Support for various data interfaces such as User Datagram Protocol, Transmission Control Protocol, and Serial interfaces. Data Services - retrieve, process, record, playback, forward, and display data (ground based data or telemetry data). Command - create, modify, send, and track commands. Command Management - Configure one TReK system to serve as a command server/filter for other TReK systems. Database - databases are used to store telemetry and command definition information. Application Programming Interface (API) - ANSI C interface compatible with commercial products such as Visual C++, Visual Basic, LabVIEW, Borland C++, etc. The TReK API provides a bridge for users to develop software to access and extend TReK services. Environments - development, test, simulations, training, and flight. Includes standalone training simulators.

  1. Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

    Science.gov (United States)

    Lotinun, Sutada; Krishnamra, Nateetip

    2016-08-16

    c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

  2. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    OpenAIRE

    Choi, Yeonim; Wang, Hye-young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae; Lee, Hyeyoung

    2013-01-01

    Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specifi...

  3. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    Science.gov (United States)

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  4. Education kits for fiber optics, optoelectronics, and optical communications

    Science.gov (United States)

    Hájek, Martin; Švrček, Miroslav

    2007-04-01

    Our company MIKROKOM, s.r.o. is engaged for many years in development of education equipment and kits for fiber optics, optoelectronics and optical communications. We would like to inform competitors of conference about results of this long-time development. Requirements on education kits and equipment in a modern and dynamic area as is optical communications and fiber optics are quite difficult. The education kits should to clearly introduce students to given issue - the most important physical principles and technical approaches, but it should to introduce also to new and modern technologies, which are quickly changing and developing. On the other hand should be these tools and kits reasonable for the schools. In our paper we would like to describe possible ways of development of this education kits and equipment and present our results of long-time work, which covers very wide range. On the one hand we developed equipment and kits for clear demonstration of physical effects using plastic optical fibers POF, next we prepare kits with a glass fibers, which are the most used fibers in practice and after as much as the kits, which covers broad range of passive and active elements of the optical networks and systems and which makes possible to create complex optical transmission connection. This kind of systems with using corresponding tools and equipment introduce the students to properties, manipulation, measurement and usage of optical fibers, traces and many active and passive components. Furthermore, with using different sorts of optical sources, photodetectors, fiber optics couplers etc., students can get acquainted with all optoelectronics transmission system, which uses different sorts of signals. Special part will be devoted also to effort mentioned before - to implement modern technologies such as e.g. Wavelength Division Multiplex (WDM) into the education kits. Our presentation will inform auditors about development of mentioned education kits and

  5. Oncogenic c-kit transcript is a target for binase.

    Science.gov (United States)

    Mitkevich, Vladimir A; Petrushanko, Irina Y; Kretova, Olga V; Zelenikhin, Pavel V; Prassolov, Vladimir S; Tchurikov, Nickolai A; Ilinskaya, Olga N; Makarov, Alexander A

    2010-07-01

    Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase--RNase from Bacillus intermedius--on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.

  6. Effects of glial cell line-derived neurotrophic factor on the proliferation and differentiation of PLZF and c-Kit of spermatogonial stem cells of rats in vitro%胶质细胞源性神经营养因子对体外培养大鼠精原干细胞PLZF和c-Kit表达的影响

    Institute of Scientific and Technical Information of China (English)

    夏伟; 曾甫清; 叶哲伟; 白杨

    2010-01-01

    Objective To investigate the effects of glial cell line-derived neurotrophic factor(GDNF)on the gene transcriptions and expressions of promyelocytic leukaemia zinc finger(PLZF)and c-Kit in spermatogonial stem cells(SSC).Methods SSC was cultured in DMEM plus different concentrations of GDNF(0,10,50,100 ng/ml).Quantitative reverse transcription PCR was used to detect the mRNA of PLZF and c-Kit.The expressions of PLZF and c-Kit protein were detected by Western blot assay.Results With the control group and 10ng/ml of GDNF group cell growth has no obvious influence,but with 50 ng/ml of GDNF group and 100 ng/ml of GDNF group cell growth was enhanced significantly.In the control group,the PLZF mRNA was 0.28±0.13 and c-Kit mRNA was 0.65±0.21.In the 10 ng/ml of GDNF group,PLZF mRNA was 0.27±0.14 and c-Kit tuRNA was 0.62±0.19.Compared with the control group,10 ng/ml of GDNF group had not influence on PLZF and c-Kit mRNA.In the 50 ng/ml of GDNF group PLZF mRNA was 0.64±0.28 and c-Kit mRNA was 0.34±0.15.Compared with the control group,50 ng/ml of GDNF enhanced the transcription of PLZF mRNA(P<0.05),decreased the transeription of c-Kit mRNA (P<0.05).In the 100 ng/ml of GDNF gmup,PLZF mRNA was 0.68±0.27 and c-Kit mRNA was 0.28±0.18.In the 100 ng/ml of GDNF group,there was no difference compared with 50 ng/ml of GDNF.In the control group,PLZF protein was 0.34±0.13 and c-Kit protein was 0.72±0.27.In 10 ng/ml of GDNF group,PLZF protein was 0.38±0.18 and c-Kit protein was 0.69±0.26.Compared with the control group,10 ng/ml of GDNF had no influence on PLZF and c-Kit protein.In 50 ng/ml of GDNF group,PLZF protein was 0.68±0.26 and c-Kit protein was 0.35±0.15.50 ng/ml of GDNF enhanced the expression of PLZF protein(P<0.05),decreased the expression of c-Kit protein(P<0.05).In 100 ng/ml of GDNF group PLZF protein was 0.70±0.27 and c-Kit protein Was 0.32±0.11,100 ng/ml of GDNF had no influence on PLZF and c-Kit protein compared with 50 ng/ml of GDNF

  7. Detection of hazelnuts and almonds using commercial ELISA test kits.

    Science.gov (United States)

    Garber, Eric A E; Perry, Jesse

    2010-03-01

    Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.

  8. Loss of c-KIT expression in thyroid cancer cells.

    Science.gov (United States)

    Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

    2017-01-01

    Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

  9. Dumpster Optics: teaching and learning optics without a kit

    Science.gov (United States)

    Donnelly, Judy; Magnani, Nancy; Robinson, Kathleen

    2016-09-01

    The Next Generation Science Standards (NGSS) and renewed emphasis on STEM education in the U.S. have resulted in the development of many educational kits for teaching science in general and optics in particular. Many teachers do not have funding to purchase kits and practical experience has shown that even costly kits can have poorly written and misleading instructions and may include experiments that would not work in a classroom. Dumpster Optics lessons are designed to use inexpensive, commonly found materials. All lessons have been field-tested with students. We will describe the development of the lessons, provide examples of field testing experiences and outline possible future activities.

  10. Development of a Backpack Survival Kit for Ejection Seats.

    Science.gov (United States)

    1982-02-08

    7 AD-A113 653 NAVAL AIR DEVELOPMENT CENTER WARMINSTER PA AIRCRAFT -ETC F/6 6/7 DEVELOP04ENT OF A BACKPACK SURVIVAL KIT FOR EJECTION SEATS. (U) FEB...82 T J ZENOBI, 6 F WHITMAN UNCLASSIFIED NADC 22216 NL EEEEEE -EuJ REPORT NO. NADC-82024.60 DEVELOPMENT OF A BACKPACK SURVIVAL KIT FOR EJECTION SEATS...RECIPIENT’S CATALOG NUMBER NADC-82024-60 - I" J 4. TITLE (and Subtitle) S. TYPE OF REPORT A PERIOD COVERED Development of a Backpack Survival Kit Phase Report

  11. Clinical usefulness of trypsin radioimmunoassay kit

    Energy Technology Data Exchange (ETDEWEB)

    Kanamori, Isao; Nakano, Satoshi; Hurukawa, Masakazu; Okumura, Yasuki; Higuchi, Chizuko; Yanase, Mikiko; Onogi, Michiteru

    1988-08-01

    From the clinical use of RIA-gnost trypsin kit, the following results were obtained. 1. Standard curve showed a steep and good curve was shown. 2. Incubation: The condition for the first incubation was set at the room temperature for 10-24 hours and that for the second incubation at the room temperature for 3-5 hours. With these settings, satisfactory results were obtained. 3. Reproducibility and recovery: The C.V. of the reproducibility and the recovery were considered superior, and the values were below 10 % and +-3 %, respectively. 4. Correlation between trypsin and serum elestase-1: An excellent positive correlation (coefficient of correlation r = 0.889) was shown. 5. Serum trypsin concentration of normal and pancreatic diseases: The normal range was from 100 to 500 ng/ml. Acute pancreatitis rose obviously. Diabetes mellitus and chronic pancreatitis was below 500 ng/ml and the pancreatic cancer showed a tendency to scatter in the range of 50-1 250 ng/ml. The above results indicated that serum trypsin can be easily measured with high precision by using this method. Thus the method is considered useful for the diagnosis of pancreatic diseases.

  12. Math Description Engine Software Development Kit

    Science.gov (United States)

    Shelton, Robert O.; Smith, Stephanie L.; Dexter, Dan E.; Hodgson, Terry R.

    2010-01-01

    The Math Description Engine Software Development Kit (MDE SDK) can be used by software developers to make computer-rendered graphs more accessible to blind and visually-impaired users. The MDE SDK generates alternative graph descriptions in two forms: textual descriptions and non-verbal sound renderings, or sonification. It also enables display of an animated trace of a graph sonification on a visual graph component, with color and line-thickness options for users having low vision or color-related impairments. A set of accessible graphical user interface widgets is provided for operation by end users and for control of accessible graph displays. Version 1.0 of the MDE SDK generates text descriptions for 2D graphs commonly seen in math and science curriculum (and practice). The mathematically rich text descriptions can also serve as a virtual math and science assistant for blind and sighted users, making graphs more accessible for everyone. The MDE SDK has a simple application programming interface (API) that makes it easy for programmers and Web-site developers to make graphs accessible with just a few lines of code. The source code is written in Java for cross-platform compatibility and to take advantage of Java s built-in support for building accessible software application interfaces. Compiled-library and NASA Open Source versions are available with API documentation and Programmer s Guide at http:/ / prim e.jsc.n asa. gov.

  13. Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells.

    Science.gov (United States)

    Miyoshi, Tomoko; Otsuka, Fumio; Nakamura, Eri; Inagaki, Kenichi; Ogura-Ochi, Kanako; Tsukamoto, Naoko; Takeda, Masaya; Makino, Hirofumi

    2012-07-06

    Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.

  14. The effect of nest box temperature on kit growth rate and survival in the American mink (Neovison vison)

    DEFF Research Database (Denmark)

    Schou, Toke Munk; Malmkvist, Jens

    2015-01-01

    dying and kits growth. Instead parameters concerning litter composition did have significant effects on kit growth and survival. Litters with high number of Totborn and kit AliveD1 affected kit growth and kit viability negatively (increased number of live born kits dying), which indicates that factors...

  15. 建立我国HIV阳转血浆盘并评价HIV酶免血筛试剂窗口期%Evaluation of HIV diagnosis kits used for blood screening with NIFDC-established HIV-1 seroconver-sion panels

    Institute of Scientific and Technical Information of China (English)

    黄维金; 宋爱京; 许四宏; 王佑春

    2016-01-01

    目的:建立我国HIV系列阳转血清盘并用其评价HIV酶免血筛诊断试剂的窗口期。方法收集HIV感染者的系列血清,利用Western blot确认试剂、HIV抗原定量检测试剂、核酸定量检测试剂等检测系列样品,RT-PCR的方法扩增HIV env序列并测序和分型,以确认HIV感染。对国内外5种第四代和14种第三代酶免HIV血液筛查诊断试剂的检测窗口期进行评价。结果共收集确认8套HIV感染者的阳转系列血浆,AE亚型6套,B亚型1套,未分型1套,共含有36份样品。用HIV系列阳转血清盘评价发现四代抗原抗体联合检测试剂较三代抗体检测试剂具有更早的窗口期检测能力,前者评分9.4分,后者3.6分,差异具有统计学意义(t=8.547,P<0.01);部分国产四代试剂质量已经达到进口试剂的水平;核酸试剂的窗口期检测能力高于酶免试剂,在3套血清盘中至少提高2周以上;确认试剂的灵敏度低于血筛试剂的灵敏度;4/5四代试剂在检测感染早期样品时会出现检测信号值/界值( S/CO)下降的情况,存在第二窗口期漏检风险。结论本研究建立了8套我国的HIV系列阳转血清盘,评价发现我国HIV血液筛查酶免诊断试剂窗口期检测能力存在差异。%Objective To establish HIV-1 seroconversion panels with the samples collected by National Institutes for Food and Drug Control ( NIFDC) and to evaluate the window periods of HIV enzyme immunoassay ( EIA) diagnostic kits used for blood screening with them. Methods Serum specimens were collected from different plasma donation stations in China. All suspected HIV infection specimens were screened for HIV by using the nucleic acid amplification testing (NAT), Western blot confirmatory assay and P24 quantitative detection assay. The HIV env gene sequences were amplified by RT-PCR for further confirmation of HIV infection. The PCR products were sequenced and genotyped. The confirmed seroconver-sion panels were used to

  16. Applications of homemade kit in mutation detection of genes

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunxia; XU Guowang; SHI Xianzhe; MA Jianmei; ZHANG Yan; L(U) Shen; YANG Qing

    2004-01-01

    Several methods of mutation detection, such as single-strand conformation polymorphism (SSCP), tandem SSCP/heteroduplex analysis and SNaPshot analysis were developed using homemade kit on ABI 310 genetic analyzer, and were successfully applied to mutation detection of 31 colorectal tumor samples. The sieving capability of homemade kit and commercial kit were compared, results demonstrate that homemade kit has higher resolution and shorter analysis time. In clinical tumor samples, 26% K-ras (exon 1) and 24% p53 (exons 7-8) were found to have mutations, and all mutations were single point variations. A majority of mutations occurred in one gene, only 1 tumor contained alterations in the two genes, which indicates that development of colorectal cancer lies on alternate pathways, and may correlate with different gene mutations.

  17. Desensitized Optimal Filtering and Sensor Fusion Tool Kit Project

    Data.gov (United States)

    National Aeronautics and Space Administration — It is proposed to develop desensitized optimal filtering techniques and to implement these algorithms in a navigation and sensor fusion tool kit. These proposed...

  18. 21 CFR 872.3600 - Partially fabricated denture kit.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3600 Partially fabricated denture kit. (a... mold, by partially polymerizing the resin denture base materials while the materials are in...

  19. HOUSE DUST MITE ALLERGEN (Derp1 AND Blot5) LEVELS IN ASTHMATICS' HOME IN HONGKONG

    Institute of Scientific and Technical Information of China (English)

    Bao-qing Sun; Adrian Wu; Albert Chan; Stanley Chik; Dorothy Wong; Nan-shan Zhong

    2004-01-01

    Objective To measure Derpl and Blot5 allergen levels in asthmatics' homes in Hongkong.Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients' house: bed and floor. Derpl and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique.Results The levels of Derpl allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g)and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P=0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derpl and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P< 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 =0 μg/g of dust as compared with Derpl in bed and on the floor (4.3% and 14.5% respectively, P< 0.05).Conclusions Derpl and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus (Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hongkong.

  20. Late development of homoeothermy in mink (Mustela vison) kits - a strategy for maximum survival rate

    DEFF Research Database (Denmark)

    Tauson, A-H; Chwalibog, André; Tygesen, M P

    2006-01-01

    of heat production (HE) by means of indirect calorimetry lasting 3 h were performed on neonatal kits and kits from 1 to 54 days of age. Both single kits and groups of 4-5 huddling kits were kept at 15 degrees C (L) or 30 degrees C (H) [from 35 days onwards at 25 degrees C (H)]. Animals were weighed before...

  1. PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

    Science.gov (United States)

    Lezmi, Stéphane; Bencsik, Anna; Baron, Thierry

    2006-10-01

    Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.

  2. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

    Directory of Open Access Journals (Sweden)

    Yadetie Fekadu

    2004-06-01

    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  3. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...

  4. Field soil aggregate stability kit for soil quality and rangeland health evaluations

    Science.gov (United States)

    Herrick, J.E.; Whitford, W.G.; de Soyza, A. G.; Van Zee, J. W.; Havstad, K.M.; Seybold, C.A.; Walton, M.

    2001-01-01

    Soil aggregate stability is widely recognized as a key indicator of soil quality and rangeland health. However, few standard methods exist for quantifying soil stability in the field. A stability kit is described which can be inexpensively and easily assembled with minimal tools. It permits up to 18 samples to be evaluated in less than 10 min and eliminates the need for transportation, minimizing damage to soil structure. The kit consists of two 21??10.5??3.5 cm plastic boxes divided into eighteen 3.5??3.5 cm sections, eighteen 2.5-cm diameter sieves with 1.5-mm distance openings and a small spatula used for soil sampling. Soil samples are rated on a scale from one to six based on a combination of ocular observations of slaking during the first 5 min following immersion in distilled water, and the percent remaining on a 1.5-mm sieve after five dipping cycles at the end of the 5-min period. A laboratory comparison yielded a correlation between the stability class and percent aggregate stability based on oven dry weight remaining after treatment using a mechanical sieve. We have applied the method in a wide variety of agricultural and natural ecosystems throughout western North America, including northern Mexico, and have found that it is highly sensitive to differences in management and plant community composition. Although the field kit cannot replace the careful laboratory-based measurements of soil aggregate stability, it can clearly provide valuable information when these more intensive procedures are not possible.

  5. Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

    Directory of Open Access Journals (Sweden)

    Erin Curry

    2008-01-01

    Full Text Available A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR, imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY and a multicopy gene (β-actin. Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1–4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.

  6. Biofilm detection by wound blotting can predict slough development in pressure ulcers: A prospective observational study.

    Science.gov (United States)

    Nakagami, Gojiro; Schultz, Gregory; Gibson, Daniel J; Phillips, Priscilla; Kitamura, Aya; Minematsu, Takeo; Miyagaki, Tomomitsu; Hayashi, Akitatsu; Sasaki, Sanae; Sugama, Junko; Sanada, Hiromi

    2016-12-26

    Bacteria have been found to form multicellular aggregates which have collectively been termed "biofilms." It is hypothesized that biofilm formation is a means to protect bacterial cells including protection form the immune response of humans. This protective mechanism is believed to explain persistent chronic wound infections. At times, the biofilms are abundant enough to see, and remove by simple wiping. However, recent evidence has shown that the removal of these visible portions are not sufficient, and that biofilms can continue to form even with daily wiping. In this work, we tested an approach to detect the biofilms which are present after clinically wiping or sharp wound debridement. Our method is based on a variation of impression cytology in which a nitrocellulose membrane was used to collect surface biofilm components, which were then differentially stained. In this prospective study, members of an interdisciplinary pressure ulcer team at a university hospital tested our method's ability to predict the generation of wound slough in the week that followed each blotting. A total of 70 blots collected from 23 pressure ulcers produced 27 wounds negative for staining and 43 positive. In the negative blots 55.6% were found to have decreased wound slough, while 81.4% with positive staining had either increase or unchanged wound slough generation. These results lead to an odds ratio of positive blotting cases of 9.37 (95% confidence intervals: 2.47-35.5, p = 0.001) for slough formation; suggesting that the changes in wound slough formation can be predicted clinically using a non-invasive wound blotting method.

  7. A flavor kit for BSM models

    Energy Technology Data Exchange (ETDEWEB)

    Porod, Werner [Universitaet Wuerzburg, Institut fuer Theoretische Physik und Astronomie, Wuerzburg (Germany); Staub, Florian [BCTP und Physikalisches Institut, Universitaet Bonn, Bonn (Germany); Vicente, Avelino [Universite de Liege, IFPA, Liege (Belgium)

    2014-08-15

    We present a new kit for the study of flavor observables in models beyond the standard model. The setup is based on the public codes SARAH and SPheno and allows for an easy implementation of new observables. The Wilson coefficients of the corresponding operators in the effective lagrangian are computed by SPheno modules written by SARAH. New operators can also be added by the user in a modular way. For this purpose a handy Mathematica package called thePreSARAHhas been developed. This uses FeynArts and FormCalc to derive generic form factors at tree- and 1-loop levels and to generate the necessary input files for SARAH. This framework has been used to implement BR(l{sub α} → l{sub β}γ), BR(l{sub α} → 3l{sub β}), CR(μ - e, A), BR(τ → P l), BR(h → l{sub α}l{sub β}), BR(Z → l{sub α}l{sub β}), BR(B{sup 0}{sub s,d} → l anti l), BR(anti B → X{sub s}γ), BR(anti B → X{sub s}l anti l), BR(anti B → X{sub d,s}νanti ν), BR(K{sup +} → π{sup +}νanti ν), BR(K{sub L} → π{sup 0}νanti ν), ΔM{sub B{sub s,B{sub d}}}, ΔM{sub K}, ε{sub K}, BR(B → Kμanti μ), BR(B → lν), BR(D{sub s} → lν) and BR(K → lν) in SARAH. Predictions for these observables can now be obtained in a wide range of SUSY and non-SUSY models. Finally, the user can use the same approach to easily compute additional observables. (orig.)

  8. Telescience Resource Kit (TReK)

    Science.gov (United States)

    Lippincott, Jeff

    2015-01-01

    Telescience Resource Kit (TReK) is one of the Huntsville Operations Support Center (HOSC) remote operations solutions. It can be used to monitor and control International Space Station (ISS) payloads from anywhere in the world. It is comprised of a suite of software applications and libraries that provide generic data system capabilities and access to HOSC services. The TReK Software has been operational since 2000. A new cross-platform version of TReK is under development. The new software is being released in phases during the 2014-2016 timeframe. The TReK Release 3.x series of software is the original TReK software that has been operational since 2000. This software runs on Windows. It contains capabilities to support traditional telemetry and commanding using CCSDS (Consultative Committee for Space Data Systems) packets. The TReK Release 4.x series of software is the new cross platform software. It runs on Windows and Linux. The new TReK software will support communication using standard IP protocols and traditional telemetry and commanding. All the software listed above is compatible and can be installed and run together on Windows. The new TReK software contains a suite of software that can be used by payload developers on the ground and onboard (TReK Toolkit). TReK Toolkit is a suite of lightweight libraries and utility applications for use onboard and on the ground. TReK Desktop is the full suite of TReK software -most useful on the ground. When TReK Desktop is released, the TReK installation program will provide the option to choose just the TReK Toolkit portion of the software or the full TReK Desktop suite. The ISS program is providing the TReK Toolkit software as a generic flight software capability offered as a standard service to payloads. TReK Software Verification was conducted during the April/May 2015 timeframe. Payload teams using the TReK software onboard can reference the TReK software verification. TReK will be demonstrated on-orbit running on

  9. LocoKit - A Construction Kit for Exploration of Morphology of Legged Robots

    DEFF Research Database (Denmark)

    Larsen, Jørgen Christian; Støy, Kasper

    2011-01-01

    Producing steady stable and energy efficient locomotion in legged robots with the ability to walk in unknown terrain is a big challenge in robotics. In addressing this challenge, it is often desirable to experiment with different morphologies and see how they influence on the way the robot walks....... This is however not always easy, since robots are often built as a fixed system with a limited possibility of changing the morphology without redesign a significant part of the robot. This work is focusing on the creation of a robotic construction kit specifically aimed at easing the process of constructing...... legged robots. This is accomplished by giving the creator the possibility to easily do morphological changes to the robot even after it have been build, to see how it effects the robot’s ability to walk in unknown terrain....

  10. LocoKit - A Construction Kit for Exploration of Morphology of Legged Robots

    DEFF Research Database (Denmark)

    Larsen, Jørgen Christian; Støy, Kasper

    2011-01-01

    Producing steady stable and energy efficient locomotion in legged robots with the ability to walk in unknown terrain is a big challenge in robotics. In addressing this challenge, it is often desirable to experiment with different morphologies and see how they influence on the way the robot walks....... This is however not always easy, since robots are often built as a fixed system with a limited possibility of changing the morphology without redesign a significant part of the robot. This work is focusing on the creation of a robotic construction kit specifically aimed at easing the process of constructing...... legged robots. This is accomplished by giving the creator the possibility to easily do morphological changes to the robot even after it have been build, to see how it effects the robot?s ability to walk in unknown terrain....

  11. The Role of c-KIT in Tumorigenesis: Evaluation in Canine Cutaneous Mast Cell Tumors

    Directory of Open Access Journals (Sweden)

    Joshua D. Webster

    2006-02-01

    Full Text Available The c-KIT proto-oncogene has been implicated in the pathogenesis of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and mast cell tumors (MCTs in canines. Cutaneous MCTs are common neoplasms in dogs and have a variable biologic behavior. The goal of this study was to define the prognostic significance of c-KIT mutations identified in canine MCTs and the associations between c-KIT mutations, KIT localization, and KIT expression levels. Microdissection and polymerase chain reaction were performed on 60 MCTs to identify c-KIT mutations. Anti-KIT antibodies were used for immunohistochemical evaluation of KIT localization. Forty-two MCTs were included in a tissue microarray, and KIT expression was quantified using immunofluorescence. Canine MCTs with c-KIT mutations were significantly associated with an increased incidence of recurrent disease and death. c-KIT mutations were also significantly associated with aberrant protein localization; however, the level of KIT expression did not correlate with either c-KIT mutations or changes in protein localization. Considering the high prevalence of canine MCTs and the central role of c-KIT in the tumorigenesis of certain tumors, canine MCTs are an excellent model for characterizing the role of c-KIT in neoplastic diseases and is a potential target for novel therapeutic agents in clinical trials.

  12. Uji Stabilitas Kit Cair Tetrofosmin pada Berbagai Kondisi Penyimpanan

    Directory of Open Access Journals (Sweden)

    Yunilda Yunilda

    2016-09-01

    Full Text Available Tetrofosmin radiopharmaceutical compound that is being developed by PTRR BATAN for use as myocardial perfusion imaging agent and cancer diagnostic agent in nuclear medicine. The aim of this study is observe the stability of this liquid kits after stored in various condition to determine its expire date various condition. The stability test was done of 1 hour, 3 hours, 5 hours and 7 hours after labeling. The radiochemical was determined its radiochemical purity has to be ≥ 90 %. Analysis of radiochemical purity was carried out by separation method which using Sep-Pak C18 cartride. Storage condition of tetrofosmin kit was carried out at various temperatures as in the deep freezer (-800C, freezer (-180C, refrigerator (2-60C and cool box (2-60C. The result showed that the liquid tetrofosmin kits stored in deep freezer, freezer, refrigerator were stable up to 23 months, 8 weeks and 4 days respectively. Simulation of stability  test after stored in a cool box was done by observing the temperature of cool box, and the result showed that the temperature in the cool box was constant as in refrigerator for up to 24 hours. Tetrofosmin which has been labeled with technetium-99m can with stand up 7 hours. It is concluded that expiry date of liquid tetrofosmin kit related with storage temperature, the lower the temperature the longer the expiry date of the kits.

  13. Uji Stabilitas Kit Cair Tetrofosmin pada Berbagai Kondisi Penyimpanan

    Directory of Open Access Journals (Sweden)

    Yunilda

    2016-02-01

    Full Text Available Tetrofosmin radiopharmaceutical compound that is being developed by PTRR BATAN for use as myocardial perfusion imaging agent and cancer diagnostic agent in nuclear medicine. The aim of this study is observe the stability of this liquid kits after stored in various condition to determine its expire date various condition. The stability test was done of 1 hour, 3 hours, 5 hours and 7 hours after labeling. The radiochemical was determined its radiochemical purity has to be ≥ 90 %. Analysis of radiochemical purity was carried out by separation method which using Sep-Pak C18 cartride. Storage condition of tetrofosmin kit was carried out at various temperatures as in the deep freezer (-800C, freezer (-180C, refrigerator (2-60C and cool box (2-60C. The result showed that the liquid tetrofosmin kits stored in deep freezer, freezer, refrigerator were stable up to 23 months, 8 weeks and 4 days respectively. Simulation of stability test after stored in a cool box was done by observing the temperature of cool box, and the result showed that the temperature in the cool box was constant as in refrigerator for up to 24 hours. Tetrofosmin which has been labeled with technetium-99m can with stand up 7 hours. It is concluded that expiry date of liquid tetrofosmin kit related with storage temperature, the lower the temperature the longer the expiry date of the kits.

  14. KiT: a MATLAB package for kinetochore tracking.

    Science.gov (United States)

    Armond, Jonathan W; Vladimirou, Elina; McAinsh, Andrew D; Burroughs, Nigel J

    2016-06-15

    During mitosis, chromosomes are attached to the mitotic spindle via large protein complexes called kinetochores. The motion of kinetochores throughout mitosis is intricate and automated quantitative tracking of their motion has already revealed many surprising facets of their behaviour. Here, we present 'KiT' (Kinetochore Tracking)-an easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies. KiT supports 2D, 3D and multi-colour movies, quantification of fluorescence, integrated deconvolution, parallel execution and multiple algorithms for particle localization. KiT is free, open-source software implemented in MATLAB and runs on all MATLAB supported platforms. KiT can be downloaded as a package from http://www.mechanochemistry.org/mcainsh/software.php The source repository is available at https://bitbucket.org/jarmond/kit and under continuing development. Supplementary data are available at Bioinformatics online. jonathan.armond@warwick.ac.uk. © The Author 2016. Published by Oxford University Press.

  15. DIFFERENTIATION OF PSEUDOCONDYLOMA OF VULVA AND CONDYLOMA ACUMINATA BY DOT BLOT HYBRIDIZATION AND POLYMERASE CHAIN REACTION

    Institute of Scientific and Technical Information of China (English)

    刘跃华; 王家璧; 司静懿

    1996-01-01

    This study differentiated pseudocondyloma of vulva from condyloma acunainata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases o{ pseudocondyloma of vulva and 65 cases of condyloma acuminata were selected for the sttldy. The genital lesions were examined clinically and were biopsled. Each biopsy v-as subjected to histological examination and HPV DNA analysis by dot blot hybridization and PCR. Dot blot analysis detected HPV DNA in 19(82.6%) out of 23 cases of condyloma acuminata and 2(25%) out of 8 cases pseudocondyloma of vulvae(P<0. 05). PCR detected HPV DNA in 51(92.7%) our of 55 cases of eondyloma acuminata, compared with none in 23 cases of pseudocondylorna(P<0. 001). HPV DNA was present in the majority of condyloma acuminata specimens, HPV 6 and 11 were the predominant types. Peudocondyloma is probably not associated with HPV. PCR was the most sensitive and useful techntque for HPV DNA detection.

  16. Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2013-01-01

    Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

  17. Evaluation and selection of indirect ELISA and sandwich ELISA kits for anti-HCV detection

    Directory of Open Access Journals (Sweden)

    LI Yongzhen

    2014-10-01

    Full Text Available ObjectiveTo compare the sensitivity and specificity between indirect enzyme-linked immunosorbent assay (ELISA and double-antibody sandwich ELISA kits produced in China and to select the best ELISA kit. MethodsSamples for evaluation included 60 serum plates and 40 serum samples positive or weakly positive for antibody to hepatitis C virus (anti-HCV which were confirmed by recombinant immunoblot assay. These samples were tested with a sandwich ELISA kit and three indirect ELISA kits, all of which were produced in China. Comparison between ELISA kits was made by paired chi-square test; comparison of false negative rate was made by R×C contingency table test. ResultsThe sensitivities of three indirect ELISA kits and a sandwich ELISA kit were 90.2%, 78.0%, 95.1%, and 97.6%, respectively, and the specificities were 78.1%, 72.6%, 94.1%, and 100%, respectively. The sandwich ELISA kit had a 4-8 times higher sensitivity than indirect ELISA kits. The R×C contingency table test revealed significant differences in false negative rate between ELISA kits and combinations of ELISA kits (χ2=29.898, P<0.05. ConclusionSandwich ELISA kit has higher sensitivity and specificity than indirect ELISA kits. Combined use of sandwich ELISA and indirect ELISA kits can significantly reduce the false negative rate and effectively prevent missed anti-HCV detection.

  18. Mass and Volume Optimization of Space Flight Medical Kits

    Science.gov (United States)

    Keenan, A. B.; Foy, Millennia Hope; Myers, Jerry

    2014-01-01

    Resource allocation is a critical aspect of space mission planning. All resources, including medical resources, are subject to a number of mission constraints such a maximum mass and volume. However, unlike many resources, there is often limited understanding in how to optimize medical resources for a mission. The Integrated Medical Model (IMM) is a probabilistic model that estimates medical event occurrences and mission outcomes for different mission profiles. IMM simulates outcomes and describes the impact of medical events in terms of lost crew time, medical resource usage, and the potential for medically required evacuation. Previously published work describes an approach that uses the IMM to generate optimized medical kits that maximize benefit to the crew subject to mass and volume constraints. We improve upon the results obtained previously and extend our approach to minimize mass and volume while meeting some benefit threshold. METHODS We frame the medical kit optimization problem as a modified knapsack problem and implement an algorithm utilizing dynamic programming. Using this algorithm, optimized medical kits were generated for 3 mission scenarios with the goal of minimizing the medical kit mass and volume for a specified likelihood of evacuation or Crew Health Index (CHI) threshold. The algorithm was expanded to generate medical kits that maximize likelihood of evacuation or CHI subject to mass and volume constraints. RESULTS AND CONCLUSIONS In maximizing benefit to crew health subject to certain constraints, our algorithm generates medical kits that more closely resemble the unlimited-resource scenario than previous approaches which leverage medical risk information generated by the IMM. Our work here demonstrates that this algorithm provides an efficient and effective means to objectively allocate medical resources for spaceflight missions and provides an effective means of addressing tradeoffs in medical resource allocations and crew mission success

  19. Next-Generation STR Genotyping Kits for Forensic Applications.

    Science.gov (United States)

    Mulero, J J; Hennessy, L K

    2012-01-01

    Forensic DNA typing has been a constantly evolving field driven by innovations from academic laboratories as well as kit manufacturers. Central to these technological advances has been the transition from multilocus-probe restriction fragment length polymorphism (RFLP) methods to short tandem repeat (STR) PCR-based assays. STRs are now the markers of choice for forensic DNA typing and a wide variety of commercial STR kits have been designed to meet the various needs of a forensic lab. This review provides an overview of the commercial STR kits made available since the year 2000 and explains the rationale for creating these kits. Substantial progress has been made in key areas such as sample throughput, speed, and sensitivity. For example, a significant advancement for databasing labs was the capability of direct amplification from a blood or buccal sample without need for DNA extraction or purification, enabling increased throughput. Other key improvements are greater tolerance for inhibitors (e.g., humic acid, hematin, and tannic acid) present in evidence samples, PCR cycling times decreased by 1-1.5 h, and greater sensitivity with improved buffer components and thermal cycling conditions. These improvements that have been made over the last 11 years have enhanced the ability of forensic laboratories to obtain a DNA profile from more challenging samples. However, with the proliferation of kits from different vendors the primer binding sequences of the loci vary, which could result in discordant events that would need to be resolved either via a database-driven software solution or simply by evaluating discordant samples with multiple kits.

  20. The Quality Lighting Teaching Kit: enlightening our future

    Science.gov (United States)

    Walker, Constance E.; Pompea, Stephen M.

    2016-09-01

    The U.S. National Optical Astronomy Observatory's Education and Public Outreach group has produced a Quality Lighting Teaching (QLT) Kit, as an outcome of the International Year of Light 2015. The kits are designed around problem-based learning scenarios. The kit's six activities allow students to address real lighting problems that relate to wildlife, sky glow, aging eyes, energy consumption, safety, and light trespass. The activities are optimized for 11-14 year olds but can be expanded to younger and older. Most of the activities can be done within in a few minutes with the exception of the Energy Activity. The activities can be done during class or afterschool and as stations (that the students rotate through) or as stand-alones (one at a time). All aspects of the program are as ready-for-use. Everything you need for the six activities is included in the kit. Tutorial videos (on the program's webpage) have been created on how to do the activities. They can be found on the webpage, www.noao.edu/education/qltkit.php. Fourteen Google+ Hangouts on Air have been offered, addressing questions on the activities and logistics. Assessments (in the form of pre- and post-surveys for the students and as post-surveys for the instructors) provide learning outcomes and improvements. Eighty-nine out of 100 kits have been distributed to SPIE, OSA, CIE, IDA and the IAU in 31 countries. The QLT Kit is a stepping-stone to bring awareness to the (younger) public on how quality lighting locally can redress issues like light pollution globally.

  1. Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

    Science.gov (United States)

    Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

    2017-02-13

    Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.Oncogene advance online publication, 13 February 2017; doi:10.1038/onc.2016.519.

  2. The Basics of Line Balancing and JIT Kitting

    CERN Document Server

    Townsend, Beverly

    2012-01-01

    Accessible to the Lean novice and shop floor employee, The Basics of Line Balancing and JIT Kitting explores line balancing and the pre-assembly of components into a finished product in a just-in-time fashion (JIT Kitting). It explains how to use time studies, develop yamazumi charts, discover and eliminate waste, balance your line, and create new standard work content for the shop floor. The book facilitates a clear understanding of the seven deadly wastes (muda) as well as what you can do to eliminate them from your facility. Describing the purpose and use of standard work, it explains how t

  3. Emergency medical kit for commercial airlines: an update.

    Science.gov (United States)

    Thibeault, Claude; Evans, Anthony

    2007-12-01

    In 1998, the Air Transport Medicine (ATM) Committee of the Aerospace Medical Association (AsMA) made its first recommendations concerning medical kits for commercial airlines. These were updated in 2002 and the ATM has continued to monitor medical kit usage, as well as pharmaceutical developments, and a further revision is now needed. This has taken into account ongoing work of the International Civil Aviation Organization and recommendations of the International Air Transport Association in the field of passenger and crew health. Based on the above, the Committee proposes the following update to its 2002 recommendations.

  4. Kit de pràctiques basat en microcontrolador PIC

    OpenAIRE

    Verdaguer Estarlich, Meritxell

    2013-01-01

    Aquest treball consisteix en el disseny i prototipat d‟un kit de pràctiques com a suport a la docència, basat en l‟arquitectura PIC. El microcontrolador escollit per a la implementació és el PIC18F4550, el qual reuneix les característiques necessàries per a la necessitat requerida. El kit compta amb diversos dispositius, els de sortida com són díodes LED, visualitzador 7 segments, display LCD; i d‟altres d‟entrada: teclat hexadecimal, interruptors, optoacobladors i un convertidor analòg...

  5. GossipKit: A Framework of Gossip Protocol Family

    OpenAIRE

    Lin, Shen; Taiani, Francois; Blair, Gordon S.; European Science Foundation

    2007-01-01

    A large number of gossip protocols have been developed in the last few years to address a wide range of functionalities. So far, however, very few software frameworks have been proposed to ease the development and deployment of these gossip protocols. To address this issue, this paper presents GossipKit, an event-driven framework that provides a generic and extensible architecture for the development of (re)configurable gossip-oriented middleware. GossipKit is based on a generic interaction m...

  6. Kit de pràctiques basat en microcontrolador PIC

    OpenAIRE

    Verdaguer Estarlich, Meritxell

    2013-01-01

    Aquest treball consisteix en el disseny i prototipat d‟un kit de pràctiques com a suport a la docència, basat en l‟arquitectura PIC. El microcontrolador escollit per a la implementació és el PIC18F4550, el qual reuneix les característiques necessàries per a la necessitat requerida. El kit compta amb diversos dispositius, els de sortida com són díodes LED, visualitzador 7 segments, display LCD; i d‟altres d‟entrada: teclat hexadecimal, interruptors, optoacobladors i un convertidor analòg...

  7. The effect of nest box temperature on kit growth rate and survival in the American mink (Neovison vison)

    DEFF Research Database (Denmark)

    Schou, Toke Munk; Malmkvist, Jens

    2015-01-01

    growth. Instead parameters concerning litter composition did have significant effects on kit growth and survival. Litters with high number of Totborn and kit AliveD1 affected kit growth and kit viability negatively (increased number of live born kits dying), which indicates that factors acting...

  8. Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation

    DEFF Research Database (Denmark)

    Meerson, Ari; Ploug, Thorkil

    2016-01-01

    of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could...

  9. Developing Save Your Food Kit (Sayofu Kit) to Support Inquiry, Improve Student Learning Outcomes at SMP Plus Hidayatul Mubtadiin and Public Awareness on Food Additives

    Science.gov (United States)

    Astutik, J.

    2017-02-01

    Food additives are materials that can not be separated from the lives of students and the community. Based on the preliminary questionnaire, it indicates the lack of kit supporting material additives in some schools and communities. The research objectives of this development are (1) to develop Kit experiment (SAYOFU KIT) and supplementary books to improve student learning outcomes in the classroom and public awareness on food additives (2) to describe the feasibility and potential effectiveness of SAYOFU KIT developed (3) to analyze the practice of SAYOFU KIT and benefits for students and the community. This development study uses 4-D models Thiagarajan, et al (1974). Through some stages, they are: defining, designing, developing and disseminating which involes the students and community. The developed SAYOFU KIT includes additives sample kit, borax test kit, curcumin test kit, formaldehyde test kit, modification heater to the identification of dyes and dye test paper. The study is conducted at SMP Plus Hidayatul Mubtadiin, and TKIT Al Uswah. The products are validated by experts and education practitioners. Qualitative data processing uses descriptive method, whereas quantitative data by using the N-gain. The average yield of expert validation of SAYOFU KIT with supplementary books 76.50% teacher’s book and 76.30% student’s book are eligible. The average yield of 96.81% validation of educational practitioners criteria, piloting a small group of 83.15%, and 82.89% field trials are very decent. The average yield on the student questionnaire responses SAYOFU kit and supplementary book is 87.6% with the criteria very well worth it. N-Gain 0:56 cognitive achievement with the criteria enough. The results of the public poll showed 95% feel the benefits SAYOFU kits for testing food. Based from description indicates that SAYOFU Kit developed feasible, practical, useful to support inquiry learning and improve student learning outcomes as well as public awareness of

  10. Genoviz Software Development Kit: Java tool kit for building genomics visualization applications

    Directory of Open Access Journals (Sweden)

    Chervitz Stephen A

    2009-08-01

    Full Text Available Abstract Background Visualization software can expose previously undiscovered patterns in genomic data and advance biological science. Results The Genoviz Software Development Kit (SDK is an open source, Java-based framework designed for rapid assembly of visualization software applications for genomics. The Genoviz SDK framework provides a mechanism for incorporating adaptive, dynamic zooming into applications, a desirable feature of genome viewers. Visualization capabilities of the Genoviz SDK include automated layout of features along genetic or genomic axes; support for user interactions with graphical elements (Glyphs in a map; a variety of Glyph sub-classes that promote experimentation with new ways of representing data in graphical formats; and support for adaptive, semantic zooming, whereby objects change their appearance depending on zoom level and zooming rate adapts to the current scale. Freely available demonstration and production quality applications, including the Integrated Genome Browser, illustrate Genoviz SDK capabilities. Conclusion Separation between graphics components and genomic data models makes it easy for developers to add visualization capability to pre-existing applications or build new applications using third-party data models. Source code, documentation, sample applications, and tutorials are available at http://genoviz.sourceforge.net/.

  11. KIT competence center for decommissioning. Innovation and promotion of trainees; Kompetenzzentrum Rueckbau am KIT. Nachwuchsfoerderung und Innovationen fuer den Rueckbau

    Energy Technology Data Exchange (ETDEWEB)

    Gentes, Sascha [Karlsruher Institut fuer Technologie (KIT), Karlsruhe (Germany). Inst. fuer Technologie und Management im Baubetrieb

    2016-03-15

    The safe decommissioning of nuclear installations is technically feasible, but is also still a challenge for science, technology and industry. The expertise and know how for decommissioning must be ensured because it will be needed for further decades. Already in 2008 the Karlsruhe Institute of Technology (KIT) had identified this challenge that later emerged through the closure of nuclear power plants in Germany. The KIT opened the professorship Technology and Management of the Decommissioning of Nuclear Installations. In 2014, this section was extended through the dismantling of conventional installations.

  12. Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay.

    Science.gov (United States)

    Villalta, Danilo; Sorrentino, Maria Concetta; Girolami, Elia; Tampoia, Marilina; Alessio, Maria Grazia; Brusca, Ignazio; Daves, Massimo; Porcelli, Brunetta; Barberio, Giuseppina; Bizzaro, Nicola

    2015-01-01

    To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases. Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins. With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively. The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors

    Directory of Open Access Journals (Sweden)

    Kiupel Matti

    2006-04-01

    Full Text Available Abstract Background Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. Methods In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16–20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. Results No mutations or polymorphisms were identified in exons 16–20 of any of the MCTs examined. Conclusion In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs.

  14. Src-like-adaptor protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling.

    Science.gov (United States)

    Kazi, Julhash U; Agarwal, Shruti; Sun, Jianmin; Bracco, Enrico; Rönnstrand, Lars

    2014-02-01

    The Src-like-adaptor protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in a variety of cells and regulates receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves the SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitylation which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on residues Y120, Y258 and Y273. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine-phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.

  15. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

    OpenAIRE

    Rozier, Claude; Mache, Régis

    1984-01-01

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloropl...

  16. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific...... that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting...

  17. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus (BWYV

  18. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus

  19. Language Coordinators Resource Kit. Section Two: Teacher Supervision.

    Science.gov (United States)

    Peace Corps, Washington, DC. Information Collection and Exchange Div.

    The guide is one section of a resource kit designed to assist Peace Corps language instruction coordinators in countries around the world in understanding the principles underlying second language learning and teaching and in organizing instructional programs. This section focuses on selection and supervision of language teachers. An introductory…

  20. National High Blood Pressure 12-Month Kit. May 1988.

    Science.gov (United States)

    National Heart and Lung Inst. (DHHS/NIH), Bethesda, MD. National High Blood Pressure Education Program.

    Part I of this kit provides information for program planners and health professionals on ways to overcome barriers to health care among the medically underserved, promote high blood pressure control through the media and other community channels, and improve adherence to treatment among hypertensive patients. It lists additional resources for…

  1. Sex Fairness in Career Guidance: A Learning Kit.

    Science.gov (United States)

    Stebbins, Linda B.; And Others

    This learning kit presents self-administered curriculum materials which can be used by counselors and counselor educators to aid in the elimination of sex-role stereotyping and sex bias in career choice. Curriculum materials are organized into four chapters: (1) "Orientation to Sex Fairness" introduces the dual role system, discusses traditional…

  2. Programming with the KIBO Robotics Kit in Preschool Classrooms

    Science.gov (United States)

    Elkin, Mollie; Sullivan, Amanda; Bers, Marina Umaschi

    2016-01-01

    KIBO is a developmentally appropriate robotics kit for young children that is programmed using interlocking wooden blocks; no screens or keyboards are required. This study describes a pilot KIBO robotics curriculum at an urban public preschool in Rhode Island and presents data collected on children's knowledge of foundational programming concepts…

  3. [Kit of Materials for Needs Assessment and Evaluation.

    Science.gov (United States)

    California State Dept. of Education, Sacramento. Bureau of Intergroup Relations.

    The items included in this kit represent a variety of needs assessment instruments and evaluation designs and methods offered by school districts in their plans for implementation of Article 3.3, Education Code Sections 13344-13344.4, school staff preparation in the history, culture, and current problems of racial and ethnic minorities. Some are…

  4. World War II Spy Kit: "The Great Nazi Intelligence Coup."

    Science.gov (United States)

    Haight, David

    This instructional packet is designed to introduce students to primary source material by having them participate in an historical "what might have been." Students engage in critical thinking and document analysis, and through the process learn about Operation OVERLORD and World War II in general. This spy kit centers on Operation…

  5. Documentation for MeshKit - Reactor Geometry (&mesh) Generator

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Rajeev [Argonne National Lab. (ANL), Argonne, IL (United States); Mahadevan, Vijay [Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-09-30

    This report gives documentation for using MeshKit’s Reactor Geometry (and mesh) Generator (RGG) GUI and also briefly documents other algorithms and tools available in MeshKit. RGG is a program designed to aid in modeling and meshing of complex/large hexagonal and rectilinear reactor cores. RGG uses Argonne’s SIGMA interfaces, Qt and VTK to produce an intuitive user interface. By integrating a 3D view of the reactor with the meshing tools and combining them into one user interface, RGG streamlines the task of preparing a simulation mesh and enables real-time feedback that reduces accidental scripting mistakes that could waste hours of meshing. RGG interfaces with MeshKit tools to consolidate the meshing process, meaning that going from model to mesh is as easy as a button click. This report is designed to explain RGG v 2.0 interface and provide users with the knowledge and skills to pilot RGG successfully. Brief documentation of MeshKit source code, tools and other algorithms available are also presented for developers to extend and add new algorithms to MeshKit. RGG tools work in serial and parallel and have been used to model complex reactor core models consisting of conical pins, load pads, several thousands of axially varying material properties of instrumentation pins and other interstices meshes.

  6. Bears. Interactive Animal Kit. Grades 1-3.

    Science.gov (United States)

    Bernard, Robin

    This kit was created to make learning about bears a fun and meaningful experience for teachers and students. It offers students opportunities to learn about favorite animals through an assortment of fun activities filled with information. The activities interact with science, language arts, critical thinking, music, social studies, math, art, and…

  7. Influence of nest box environment on kit survival

    DEFF Research Database (Denmark)

    Lund, V.H.; Malmkvist, Jens

    2012-01-01

    . In conclusion, access to different nesting materials reduced the risk of dying in farm mink in relation with factors as e.g. nest quality and maternal behaviour. A decrease in kit mortality from 20% to 5% would give the Danish mink production a profound number of Danish kroner extra per year....

  8. A new IgG immunoblot kit for diagnosis of toxoplasmosis in pregnant women.

    Science.gov (United States)

    Khammari, Imen; Saghrouni, Fatma; Lakhal, Sami; Bouratbine, Aida; Ben Said, Moncef; Boukadida, Jalel

    2014-10-01

    The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG® kit recently recommended as a confirmatory test (96.7% of concordance).

  9. C-kit gene mutation in human gastrointestinal stromal tumors

    Institute of Scientific and Technical Information of China (English)

    Ying-Yong Hou; Ai-Hua Zheng; Tai-Ming Zhang; Wen-Zhong Hou; Jian Wang; Xiang Du; Xiong-Zeng Zhu; Yun-Shan Tan; Meng-Hong Sun; Yong-Kun Wei; Jian-Fang Xu; Shao-Hua Lu; Su-Jie A-Ke-Su; Yan-Nan Zhou; Feng Gao

    2004-01-01

    AIM: To investigate the significance of c-kit gene mutation in gastrointestinal stromal tumors (GIST).METHODS: Fifty two cases of GIST and 28 cases of other tumors were examined. DNA samples were extracted from paraffin sections and fresh blocks. Exons 11, 9 and 13 of the c-kit gene were amplified by PCR and sequenced.RESULTS: Mutations of exon 11 were found in 14 of 25 malignant GISTs (56%), mutations of exon 11 of the c-kit gene were revealed in 2 of 19 borderline GISTs (10.5%),and no mutation was found in benign tumors. The mutation rate showed significant difference (X2=14.39, P<0.01)between malignant and benign GISTs. Most of mutations consisted of the in-frame deletion or replication from 3 to 48 bp in heterozygous and homozygous fashions, None of the mutations disrupted the downstream reading frame of the gene. Point mutations and frame deletions were most frequently observed at codons 550-560, but duplications were most concentrated at codons 570-585. No mutations of exons 9 and 13 were revealed in GISTs, Neither c-kit gene expression nor gene mutations were found in 3 leiomyomas, 8 leiomyosarcomas, 2 schwannomas, 2malignant peripheral nerve sheath tumors, 2 intraabdominal fibromatoses, 2 malignant fibrous histiocytomas and 9 adenocarcinomas.CONCLUSION: C-kit gene mutations occur preferentially in malignant GISTs and might be a clinically useful adjunct marker in the evaluation of GISTs and can help to differentiate GISTs from other mesenchymal tumors of gastrointestinal tract, such as smooth muscle tumors,schwannomas, etc.

  10. Pharmacological inhibitors of c-KIT block mutant c-KIT mediated migration of melanocytes and melanoma cells in vitro and in vivo

    Science.gov (United States)

    Posch, Christian; Moslehi, Homayoun; Sanlorenzo, Martina; Green, Gary; Vujic, Igor; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-01-01

    Mutations in the receptor tyrosine kinase c-KIT (KIT) are frequent oncogenic alterations in melanoma and are predominantly detected in tumors of acral, mucosal, and chronically sun-damaged skin. Research indicates that melanocytes with aberrant KIT signaling can be found in the distant periphery of the primary tumor; However, it is hitherto unknown whether KIT might confer a migratory advantage, thereby enabling genetically abnormal cells to populate a distal area. In this study, we investigated the role of mutant KIT in melanocyte- and melanoma cell migration using KIT mutant lines as well as genetically manipulated murine and primary human melanocytes. Our results revealed that melanocytes, stably transduced with mutant KIT closed a gap inflicted on cell monolayers faster than wild-type controls. Similarly, KIT mutant human melanoma lines were able to populate a larger area in a 3D in vitro skin model compared to KIT wild type and BRAF mutant lines. Genomic profiling revealed that genes associated with increased cell-dispersal of KIT mutant variants were linked to a statistically significant up-regulation of 60 migratory genes (z-score 1.334; p=0.0001). In addition, in vivo experiments harnessing a mouse xenograft model of early melanoma development demonstrated rapid lateral migration of KIT mutant cells compared to respective controls. The specific kinase inhibitors imatinib and nilotinib, could abrogate this migratory advantage in vitro and in vivo. Our work suggests that KIT inhibition might help to target migratory active, KIT mutant melanoma cells, thus representing a potential strategy to reduce spread and local recurrence. PMID:27322141

  11. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries

    DEFF Research Database (Denmark)

    Høyer, Poul Erik; Byskov, Anne Grete; Møllgård, Kjeld

    2005-01-01

    Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry......Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry...

  12. The Use of Kits in the Analysis of Tissue Lipids Requires Validation.

    Science.gov (United States)

    Rider, T; LeBoeuf, R C; Tso, Patrick; Jandacek, R J

    2016-04-01

    The ready availability and ease of use of kits for the measurement of serum lipids has greatly facilitated these measurements. In many cases it would be convenient to use these kits in the determination of lipid concentrations in tissues. The successful application of serum kits in tissue analysis requires that two important issues be considered. First, the solvent system for the extraction of the lipids and the solvent used for analysis by the kit must be compatible with the reactions in the kit. Second, the concentration range in the analyzed solution must be within the range for which the kit is used. We report here that lipids in liver and adipose tissues may be significantly underestimated by the use of some kits. We recommend that the use of kits for tissue analysis of lipids be validated for the specific analysis.

  13. Evaluation of the Beckman Antistreptolysin O and Antideoxyribonuclease B (ASO/ADB) Test Kit

    Science.gov (United States)

    Klein, George C.

    1976-01-01

    The Beckman antistreptolysin O kit agreed within one-dilution increment for 70% of the specimens tested. The antideoxyribonuclease B kit agreed within onedilution increment for 96% of the specimens tested. PMID:1262456

  14. Evaluation of beckman antistreptolysin O and antideoxyribonuclease B (ASO/ADB) test kit.

    Science.gov (United States)

    Klein, G C

    1976-04-01

    The Beckman antistreptolysin O kit agreed within one-dilution increment for 70% of the specimens tested. The antideoxyribonuclease B kit agreed within onedilution increment for 96% of the specimens tested.

  15. A sensitive non-radioactive northern blot method to detect small RNAs.

    Science.gov (United States)

    Kim, Sang Woo; Li, Zhihua; Moore, Patrick S; Monaghan, A Paula; Chang, Yuan; Nichols, Mark; John, Bino

    2010-04-01

    The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

  16. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.

    Science.gov (United States)

    Albers, Eliene; Sbroggiò, Mauro; Martin-Gonzalez, Javier; Avram, Alexandra; Munk, Stephanie; Lopez-Contreras, Andres J

    2017-01-19

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

  17. Simultaneous preparation of RNA and nuclei for Northern blot and flow cytometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Tesfaigzi, J.; Jaramillo, R. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States)

    1996-11-01

    Several methods have been developed to quantify RNA synthesis during the progression of the cell cycle. The rate of RNA synthesis can be detected during different stages of the cell cycle by staining cells with agents that intercalate with nucleic acids. For example, following staining of mammalian cells with acridin orange, the green and red fluorescence that correlates with DNA and RNA content, respectively, can be analyzed by flow cytometry. Increase in RNA content during the progression of cells through the cell cycle can be measured after staining with acridin orange. RNA synthesis resulting from the stimulation of quiescent cells with various growth factors has also been demonstrated by labeling cells with bromo-uridine and using the anti-bromo-deoxyuridine antibody. These methods allow measurement of the overall RNA content in cells; however, they do not allow the measurement of the levels of specific mRNAs throughout the cell cycle. Current methods to quantify specific mRNAs generally require the preparation of a large number of cells (5--10 {times} 10{sup 6} cells) to carry out flow cytometric analyses and to isolate RNA for Northern blot analysis or solution hybridization. In this report, the authors describe a method of simultaneously preparing RNA and nuclei for Northern blot and flow cytometric analyses, respectively. The minimum number of nuclei required to obtain flow cytometric data and the effect of conserving nuclei in methanol for several days are also presented.

  18. Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2011-06-01

    Full Text Available To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs were prepared. Ginsenosides were developed on thin layer chromatography (TLC plates which were covered by a polyvinylidene difluoride (PVDF membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

  19. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  20. BIOTECON diagnostics foodproof Listeria monocytogenes Detection Kit, 5' nuclease in combination with the foodproof ShortPrep II Kit.

    Science.gov (United States)

    Junge, Benjamin; Grönewald, Cordt; Berghof-Jäger, Kornelia

    2012-01-01

    A method was developed for the detection of Listeria monocytogenes in food. The method is based on real-time PCR using hydrolysis probes (5' Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the BIOTECON foodproof ShortPrep II Kit designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is carried out using the foodproof Listeria monocytogenes Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For the internal comparison study, three different foods (soft cheese, coalfish, and smoked ham) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for detection of L. monocytogenes. From each food, 20 samples were inoculated with a low level (1-10 CFU/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.

  1. Extending Mechanical Construction Kits to Incorporate Passive and Compliant Elements for Educational Robotics

    DEFF Research Database (Denmark)

    Assaf, Dorit; Larsen, Jørgen Christian; Reichardt, Markus

    2012-01-01

    artificial intelligence and biomechanics to students with different backgrounds. The robots we use both for research and education are usually built incorporating compliant materials as well as passive dynamics. These kind of properties are often not available in classical robot kits or mechanical...... and mechanical construction kits we describe interface solutions between several kits. Further we show some solutions to incorporate compliant materials and passive dynamics to traditional mechanical construction kits by using cheap and widely available materials....

  2. Gene Duplication of the zebrafish kit ligand and partitioning of melanocyte development functions to kit ligand a.

    Directory of Open Access Journals (Sweden)

    Keith A Hultman

    2007-01-01

    Full Text Available The retention of particular genes after the whole genome duplication in zebrafish has given insights into how genes may evolve through partitioning of ancestral functions. We examine the partitioning of expression patterns and functions of two zebrafish kit ligands, kit ligand a (kitla and kit ligand b (kitlb, and discuss their possible coevolution with the duplicated zebrafish kit receptors (kita and kitb. In situ hybridizations show that kitla mRNA is expressed in the trunk adjacent to the notochord in the middle of each somite during stages of melanocyte migration and later expressed in the skin, when the receptor is required for melanocyte survival. kitla is also expressed in other regions complementary to kita receptor expression, including the pineal gland, tail bud, and ear. In contrast, kitlb mRNA is expressed in brain ventricles, ear, and cardinal vein plexus, in regions generally not complementary to either zebrafish kit receptor ortholog. However, like kitla, kitlb is expressed in the skin during stages consistent with melanocyte survival. Thus, it appears that kita and kitla have maintained congruent expression patterns, while kitb and kitlb have evolved divergent expression patterns. We demonstrate the interaction of kita and kitla by morpholino knockdown analysis. kitla morphants, but not kitlb morphants, phenocopy the null allele of kita, with defects for both melanocyte migration and survival. Furthermore, kitla morpholino, but not kitlb morpholino, interacts genetically with a sensitized allele of kita, confirming that kitla is the functional ligand to kita. Last, we examine kitla overexpression in embryos, which results in hyperpigmentation caused by an increase in the number and size of melanocytes. This hyperpigmentation is dependent on kita function. We conclude that following genome duplication, kita and kitla have maintained their receptor-ligand relationship, coevolved complementary expression patterns, and that

  3. 21 CFR 864.9650 - Quality control kit for blood banking reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control kit for blood banking reagents... Manufacture Blood and Blood Products § 864.9650 Quality control kit for blood banking reagents. (a) Identification. A quality control kit for blood banking reagents is a device that consists of sera,...

  4. Take-Home Art Appreciation Kits for Kindergartners and Their Families.

    Science.gov (United States)

    Mulcahey, Christine

    2002-01-01

    Describes collaborative project between an art specialist and kindergarten teacher to develop take-home art appreciation kits for kindergartners. Discusses benefits of the collaboration for teachers, children, and families. Focuses on aesthetic inquiry approach to the art kits to develop inquiry and critical thinking skills. Describes the kits'…

  5. DEMONSTRATION BULLETIN: HNU-HANBY PCP IMMUNOASSAY TEST KIT - HNU - SYSTEMS, INC.

    Science.gov (United States)

    The HNU-Hanby test kit rapidly analyzes for petroleum hydrocarbons in soil and water samples. The test kit can be used to estimate pentachlorophenol (PCP) concentrations in samples when the carrier solvent is a petroleum hydrocarbon. The test kit estimates PCP concentrations in ...

  6. HNU-HANBY PCP IMMUNOASSAY TEST KIT - INNOVATIVE TECHNOLOGY EVALUATION REPORT

    Science.gov (United States)

    The HNU-Hanby pentachlorophenol (PCP) test kit rapidly analyzes for PCP in soil samples. The test kit can only detect those PCP carriers that contain aromatic compounds. The test kit estimates PCP concentrations in soil samples indirectly by measuring petroleum hydrocarbon carrie...

  7. 21 CFR 866.2480 - Quality control kit for culture media.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control kit for culture media. 866.2480... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2480 Quality control kit for culture media. (a) Identification. A quality control kit for culture media is a device...

  8. 33 CFR 149.323 - What are the requirements for first aid kits?

    Science.gov (United States)

    2010-07-01

    ... first aid kits? 149.323 Section 149.323 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF... Lifesaving Equipment Manned Deepwater Port Requirements § 149.323 What are the requirements for first aid kits? (a) Each manned deepwater port must have an industrial first aid kit, approved by an...

  9. Prolonged expression of the c-kit receptor in germ cells of intersex fetal testes

    DEFF Research Database (Denmark)

    Rajpert-De Meyts, Ewa; Jørgensen, N; Müller, Jørn

    1996-01-01

    Stem cell factor (SCF) and its receptor Kit encoded by the c-kit proto-oncogene are crucial for the development and migration of primordial germ cells in rodents. The expression of Kit has been examined immunohistochemically in gonads obtained from five specimens of fetal tissues with intersex...

  10. Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Akin, C; Gilfillan, A M

    2008-01-01

    KIT is a member of the tyrosine kinase family of growth factor receptors which is expressed on a variety of haematopoietic cells including mast cells. Stem cell factor (SCF)-dependent activation of KIT is critical for mast cell homeostasis and function. However, when KIT is inappropriately activa...

  11. Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds

    Science.gov (United States)

    Albuquerque, Pedro; Ribeiro, Niza; Almeida, Alexandre; Panschin, Irena; Porfirio, Afonso; Vales, Marta; Diniz, Francisca; Madeira, Helena; Tavares, Fernando

    2017-01-01

    Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious

  12. Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

    Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

  13. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10(5) cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  14. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

    Science.gov (United States)

    Fontanot, Marco; Iacumin, Lucilla; Cecchini, Francesca; Comi, Giuseppe; Manzano, Marisa

    2014-10-01

    The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively.

  15. The detection of KIT mutations in acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Luis Eduardo Silva Machado

    2012-09-01

    Full Text Available Objective: This study describes a new method used in the clinicallaboratory at Hospital Israelita Albert Einstein to detect mutationsin exons 8 and 17 of the KIT gene in patients with acute myeloidleukemia. Methods: Genomic DNA extraction was performed on 54samples of peripheral blood or bone marrow from patients with acutemyeloid leukemia. The extracted DNA was amplified by polymerasechain reaction and sequenced, and the fragments were analyzed.Results: Within the analyzed samples, we detected four mutations inexon 8, two mutations in exon 17, and mutations or a double mutationin one sample. Conclusion: The tests detecting mutations in exon 8and 17 on the KIT gene were successfully standardized. The test isnow included among the routine diagnostics employed for patients atHospital Israelita Albert Einstein clinical laboratory.

  16. 3D vision upgrade kit for TALON robot

    Science.gov (United States)

    Edmondson, Richard; Vaden, Justin; Hyatt, Brian; Morris, James; Pezzaniti, J. Larry; Chenault, David B.; Tchon, Joe; Barnidge, Tracy; Kaufman, Seth; Pettijohn, Brad

    2010-04-01

    In this paper, we report on the development of a 3D vision field upgrade kit for TALON robot consisting of a replacement flat panel stereoscopic display, and multiple stereo camera systems. An assessment of the system's use for robotic driving, manipulation, and surveillance operations was conducted. The 3D vision system was integrated onto a TALON IV Robot and Operator Control Unit (OCU) such that stock components could be electrically disconnected and removed, and upgrade components coupled directly to the mounting and electrical connections. A replacement display, replacement mast camera with zoom, auto-focus, and variable convergence, and a replacement gripper camera with fixed focus and zoom comprise the upgrade kit. The stereo mast camera allows for improved driving and situational awareness as well as scene survey. The stereo gripper camera allows for improved manipulation in typical TALON missions.

  17. Assessment of RFID Read Accuracy for ISS Water Kit

    Science.gov (United States)

    Chu, Andrew

    2011-01-01

    The Space Life Sciences Directorate/Medical Informatics and Health Care Systems Branch (SD4) is assessing the benefits Radio Frequency Identification (RFID) technology for tracking items flown onboard the International Space Station (ISS). As an initial study, the Avionic Systems Division Electromagnetic Systems Branch (EV4) is collaborating with SD4 to affix RFID tags to a water kit supplied by SD4 and studying the read success rate of the tagged items. The tagged water kit inside a Cargo Transfer Bag (CTB) was inventoried using three different RFID technologies, including the Johnson Space Center Building 14 Wireless Habitat Test Bed RFID portal, an RFID hand-held reader being targeted for use on board the ISS, and an RFID enclosure designed and prototyped by EV4.

  18. Shankar: reproductive health teaching aids kit for adolescent boys.

    Science.gov (United States)

    1999-12-01

    Currently being developed by the Thoughtshop Foundation in collaboration with the Child In Need Institute in West Bengal, this kit is intended to help peer educators in generating awareness about reproductive health issues among rural adolescent boys and men through discussions and other activities. The central theme is men's responsibility for their sexual behavior and the reproductive and sexual health of their partners. In five modules, the kit tells the story of Shankar, a boy 13 years old. Each module consists of a flip chart and accompanying visual aids/activities. The modules are as follows: 1) Puberty, self-esteem, responsibility; 2) Knowledge of changes which girls experience during puberty; 3) Childbirth; 4) Contraception--why/how; 5) Safer sex, hygiene, STD/HIV/AIDS.

  19. Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning; Zhang, Dawei; Tasker, Andrew S.; Bürli, Roland; Hungate, Randall; Yu, Violeta; Nguyen, Yen; Whittington, Douglas A.; Meagher, Kristin L.; Plant, Matthew; Tudor, Yanyan; Schrag, Michael; Xu, Yang; Ng, Gordon Y.; Hu, Essa (Amgen)

    2010-01-12

    Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

  20. Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transp...