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Western Blot - DOAJ

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Last Updated: 09 January 2023

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Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study.

BACKGROUND Trypanosoma cruzi is responsible for choking asthma. The presence of IgG anti-T. cruzi antibodies is characteristic of the chronic phase of CD; the diagnosis is performed by serological techniques. Because there is no reliable test that can be used as a reference measure, WHO recommends the simultaneous use of two different methods for CD serodiagnosis. In the chronic phase, PCR has low sensitivity in the initial phase, but only a few validated tests based on the WB method are commercially available worldwide. Methodology/principal results In this research, we investigated the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis using WB as the diagnostic platform. Conclusions/significance The present phase I research revealed the high diagnostic ability of these four IBMP antigens to distinguish between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with the WB.

Source link: https://doi.org/10.1371/journal.pntd.0010944


Use of the Western blot technique to identify the immunogenic proteins of Borrelia burgdorferi for developing a Lyme disease vaccine

Background: Lyme disease is a serious infectious disease with a restricted worldwide distribution for which there is no vaccine available for human use. This research was conducted to find common reactive antigens involved in Borrelia burgdorferi infections that are present in mammalian sera that may be useful for vaccine production. The electrophores of the electrophoresed proteins in the serum samples were then tested with anti-HRP IgG reagent. Several other Bb antigens with broad molecular weight ranges were also identified by rabbit and human sera, but less often with mouse sera. Conclusion: Following a bite of an infected tick, the strong immune response to the 41 kDa flagellin protein by the various mammalian species suggests the use of a potential vaccine targeting this protein, although other proteins may also be useful for preventing Lyme disease.

Source link: https://doi.org/10.1016/j.biopha.2022.114013


Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them.

Western blotting is a common laboratory technique used to detect proteins and determine their expression levels. Unfortunately, poor western blot image display techniques and a lack of detailed methods reporting can impede a reader's ability to analyze or replicate western blot findings. Although several organizations have examined the prevalence of common publication practices are uncertain, many researchers have investigated image manipulation or made suggestions for improving western blotting. Most published western blots are cropped and blot source data are not made available to readers in the supplement, according to our results. Publishing blots with visible molecular weight markers is rare, and many blots also lack molecular weight indicators. The amount of protein loaded on the gel, blocking steps, and antibody labeling procedures are all missing information about the antibody labeling protocol and antibody labeling protocols.

Source link: https://doi.org/10.1371/journal.pbio.3001783


Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls, as well as 50 sera samples from non-CL patients were collected. The Native strain of Leishmania major was cultured in Schnei-u00adder medium and soluble Leishmania antigens were made from amastigotes-like parasites. A 63 kDa subunit was identified by 59 cases, and 51 cases confirmed a 32-35 kDa component. Conclusion: Immunoblotting has a positive track record in diagnostics of CL, and this test can be used as an aid in proper diagnosis of CL.

Source link: https://doaj.org/article/0c15ec1e9add44d689785723d5f21f38


Diagnostic Accuracy of LDBIO-Toxo II IgG and IgM Western Blot in Suspected Seroconversion in Pregnancy: A Multicentre Study

Due to atypical reaction, the automated tests developed for Toxoplasma gondii serology's high sensitivity can result in false-positive IgM results. We conducted a multicentre research to determine the clinical validity of the Toxo II IgG and a new, not yet commercialized Toxo II IgM western blot on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity for IgM WB was 97. 8% and 99. 9% for IgG WB. The final diagnosis revealed that IgM and IgG Toxo WB were very good, with K = 0. 89 and K = 0. 99, respectively. IgM was detected in ten cases and in two cases for IgM. The association of IgG and IgM WB on the same sample not only confirmed all seroconversions, but also correctly identified the majority of the false-positive findings in the false-positive study.

Source link: https://doi.org/10.3390/pathogens11060665


Evaluation of the Chagas Western Blot IgG Assay for the Diagnosis of Chagas Disease

The World Health Organisation states that the diagnosis of T. cruzi infection is often based on antibodies against T. cruzi antigens and performed with two methodologically different assays. Native antigens derived from a T. cruzi strain of the TcVI genotype were used in a Chagas Western Blot IgG assay. This immunoblot is a fast and accurate test for the diagnosis of Chagas disease, with results in less than 2 h. This immunoblot is a convenient and fast test for determining antibodies against T. cruzi in serum samples.

Source link: https://doi.org/10.3390/pathogens10111455


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

Dystrophin is one of the largest cytoskeleton proteins coding by 79 exons. The absence of dystrophin causes muscular dystrophy in Duchenne muscular dystrophy. Hundreds of exon-specific human dystrophin monoclonal antibodies have been developed and used for DMD diagnosis over the past two decades. By immunofluorescence staining and western blot, we addressed the gap. To fill the void, we conducted a comprehensive review of 65 dystrophin monoclonal antibodies in normal and dystrophic dogs. We also included striated muscles from normal BL10 and dystrophin-null mdx mice for comparison. Both immunostaining and western blot, we found 15 antibodies that can reliably detect full-length canine dystrophin.

Source link: https://doi.org/10.1371/journal.pone.0088280


Western blot analysis of glucocorticoid receptor phosphoisoforms by one- and two-dimensional electrophoretic assays

Simple and easy-to-use non-radioactive protein mobility shift assays were developed using one- and/or two-dimensional gel electrophoresis based on variations in the pI and molecular mass of GR phosphoisoforms. Western blot screened GR cells, yeast BJ2168 cells that produce wild-type rat GR, rat hepatoma GRH2 cells grown in culture and brain tissue from Wistar rat experimental animals, and mouse tissue from Wistar rat experimental animals, respectively. The results obtained using the NMS assay were similar to previous findings obtained with the [uf067-32P] ATP standard assay assay.

Source link: https://doaj.org/article/c7705b83a60f45e39e730b2477210088


Identification of novel laminin- and fibronectin-binding proteins by Far-Western blot: capturing the adhesins of Streptococcus suis type 2

Bacterial cell wall and extracellular proteins are often involved in interactions with extracellular matrix proteins, such as laminin and fibronectin, which play key roles in adhesion and invasion. In this study, an effective way combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. From Streptococcus suis serotype 2 CW and EC proteins, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified with this strategy. Both human LN and human FN were shown with specific binding capability by Five proteins, as shown by their specific linking ability. In addition, seven key recombinant proteins were selected and found to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins and their corresponding polyclonal antibodies were found to reduce SS2 adhesion to Hep-2 cells, which indicates that these proteins play a role in SS2 adherence to the host cell surface.

Source link: https://doi.org/10.3389/fcimb.2015.00082


Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

Abstract Background Background: The use of purified protein standards containing the antigen is required for the replacement of radioligand binding assays with antibody-antigen interaction-based methods for quantitative examination of G protein-coupled receptor levels. In quantitative Western blot analysis of CB1 receptor expression in crude synaptosomes of the adult rat brain cortex, we produced highly soluble and stable recombinant protein constructs GST-CB141414-4142, including much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively. CB1 receptor density measured by quantitative Western blot was of the same order of magnitude but marginally larger than values obtained by the radioligand saturation binding assay, but just marginally higher than values obtained by quantitative Western blot. The differences between the results obtained by quantitative Western blot and radioligand saturation binding methods are discussed in the context of their particular theoretical bases and methodological constraints.

Source link: https://doi.org/10.1186/s12934-022-01914-1

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions