Quantitative Assay

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Keith P. Klugman - One of the best experts on this subject based on the ideXlab platform.

  • The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens
    PLoS ONE, 2016
    Co-Authors: Melina Messaoudi, Milen Milenkov, Werner C. Albrich, Mark P. G. Linden, Thomas Bénet, Monidarin Chou, Mariam Sylla, Patricia Barreto Costa, Nathalie Richard, Keith P. Klugman
    Abstract:

    For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or Quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR Assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the Assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the Assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, Quantitative Assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

Richard H Ebright - One of the best experts on this subject based on the ideXlab platform.

  • fluorescence anisotropy rapid Quantitative Assay for protein dna and protein protein interaction
    Methods in Enzymology, 1996
    Co-Authors: Tomasz Heyduk, Hong Tang, Richard H Ebright
    Abstract:

    Publisher Summary This chapter discusses fluorescence anisotropy. The Quantitative Assay for protein-DNA and protein-protein interaction is discussed. Fluorescence spectroscopy involves excitation of a fluorochrome with light of a wavelength at which the fluorochrome exhibits significant absorption, creating fluorochrome excited state that can be followed by the detection of emitted light. Measurement of fluorescence polarization involves excitation of a sample with polarized light and determination of the extent of polarization of the emitted light. Fluorescence anisotropy Assays can be performed using any standard spectrofluorometer equipped with polarizers or using a dedicated fluorescence polarization instrument. Standard spectrofluorometers permit use of a range of excitation and emission wavelengths, and thus a range of different fluorochromes, and are compatible with sample volumes from -0.05 to 4.0 ml. Fluorescence anisotropy analysis of protein-protein interaction requires careful experimental design.

Michel J Tremblay - One of the best experts on this subject based on the ideXlab platform.

  • Modulation of Human Immunodeficiency Virus Type 1-Induced Syncytium Formation by the Conformational State of LFA-1 Determined by a New Luciferase-Based Syncytium Quantitative Assay
    Journal of Virology, 1998
    Co-Authors: Benoit Barbeau, Jean-françois Fortin, Nicolas Genois, Michel J Tremblay
    Abstract:

    The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a Quantitative Assay, a new luciferase-based Assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this Assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this Assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa–CD4–LTR–β-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.

Yasutami Mitoma - One of the best experts on this subject based on the ideXlab platform.

Melina Messaoudi - One of the best experts on this subject based on the ideXlab platform.

  • The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens
    PLoS ONE, 2016
    Co-Authors: Melina Messaoudi, Milen Milenkov, Werner C. Albrich, Mark P. G. Linden, Thomas Bénet, Monidarin Chou, Mariam Sylla, Patricia Barreto Costa, Nathalie Richard, Keith P. Klugman
    Abstract:

    For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or Quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR Assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the Assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the Assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, Quantitative Assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.