Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

Mishra, Manish; Tiwari, Shuchita; Gunaseelan, Anita; Li, Dongyang; Hammock, Bruce D.; Gomes, Aldrin V.

doi:10.1002/elps.201900059

Cited by 18 publications

(14 citation statements)

References 25 publications

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“…The limitation of sensitivity might be overcome by utilizing these sophisticated technologies combined with our monoclonal antibodies for HPeV3 VP0 antigen. Furthermore, we can potentially improve the detection sensitivity by altering the second antibody to recognize the poly-HRP complex [ 39 ] or by adding other antibodies, which can target VP1 or VP3 proteins.…”

Section: Discussionmentioning

confidence: 99%

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3

et al. 2020

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Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.

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Section: Discussionmentioning

confidence: 99%

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3

et al. 2020

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“…Western blotting assay was performed as described [ 11 ]. After the extraction of total protein from the tissues with a RIPA lysis buffer, the amounts of protein (40 µ g/line) were transferred to polyvinylidene difluoride membranes and immunoreacted with the following targeted primary antibodies: NF-κB p65, IκBα, C-casp-1, COX-2 (1:1,000), IL-1β, iNOS (1:500), and β-actin (1:2,000).…”

Section: Methodsmentioning

confidence: 99%

Granulocyte colony-stimulating factor protected against brain injury in a rat cerebral hemorrhage model by modulating inflammation

Piao

et al. 2022

Exp. Anim.

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Granulocyte colony-stimulating factor (G-CSF) has been reported to exert a protective effect against secondary brain damage, but the underlying mechanisms remain unknown.We explored the ability of G-CSF to protect the brain from injury in a rat autologous blood-induced model of intracerebral hemorrhage (ICH), with a special focus on the anti-inflammation effect. An ICH was induced in 8-week-old male rats by an infusion of autologous blood, and the rats were then randomly assigned to five treatment groups: sham, ICH, and ICH+ low-dose (25 μg/kg), middle-dose (50 μg/kg), and high-dose (75 μg/kg) G-CSF. We then evaluated the levels of brain inflammation-related genes and proteins. The levels of tumor-necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) mRNA increased between days 1 and 14 post-ICH, with the highest expression on day 3. These changes were rectified by G-CSF in a dose-dependent manner. At day 3 post-injury, an elevation of the nuclear factor-kappa B (NF-κB) p65 protein level and a reduction of the inhibitor of NF-κB alpha (IκBα) protein level were observed; G-CSF treatment exerted a beneficial effect on both protein expressions. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins were increased; these changes were rectified by the highest dose of G-CSF. The brain-protecting effects of G-CSF are likely to be attributable, at least in part, to attenuation of the TNF-α, IL-6, iNOS, and COX-2 expressions induced by NF-κB activation in the brain tissues of this autologous blood-induced ICH rat model.

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“…Anti-glyceraldehyde 3-phosphate dehydrogenase (1:1,000, 5174 T; Cell Signaling Technology) was used as the internal reference. 17 …”

Section: Methodsmentioning

confidence: 99%

Silencing of SPP1 Suppresses Progression of Tongue Cancer by Mediating the PI3K/Akt Signaling Pathway

Zhang

Lai

et al. 2020

Technol Cancer Res Treat

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Background: In the present study, we aimed to find an effective target for the treatment of tongue cancer using gene chip screening and signal pathway research. Methods: We used microarray screening and gene expression profile analyses to find important differentially expressed genes in tongue cancer. We constructed a protein-protein interaction network, and used enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes to screen for important genes. We then silenced the genes of interest in SCC154 cells to study the relationship with the Phosphatidylinositol 3-kinase/Akt signal pathway. Western blot analyses, the 3-(4,5Dimethylthiazol-yl)-2,5Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) test, and immunofluorescence assays were used to compare the expression levels of Phosphatidylinositol 3-kinase/Akt signal pathway-related proteins, cell viability, and cell proliferation ability in normal SCC154 cells, Si-RNA SCC154 cells, and gene-silenced SCC154 cells. The scratch test, Transwell test, and western blotting were used to determine migration, invasion, and carcinogenesis. Results: Using GSE9844, GSE13601, and GSE31056 gene chips, we identified 93 upregulated genes and 76 downregulated genes in tongue cancer. Using the protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we further identified 47 differentially expressed genes. Using Kaplan-Meier plotter online tools, we also identified 3 genes (SPP1, Recombinant Human Secreted Phosphoprotein 1; PLAU, plasminogen activator urinary; and APP, amyloid precursor protein). Compared with normal SCC154 cells and Si-RNA control SCC154 cells, the expressions of Phosphatidylinositol 3-kinase/Akt pathway proteins in si-SPP1 SCC154 cells were significantly decreased (*P < 0.05), and the protein activities and proliferation abilities were also significantly decreased (*P < 0.05), while the migration ability, invasion ability, and cancer forming ability were significantly increased (*P < 0.05). Conclusion: Inhibition of the SPP1 gene may have a therapeutic effect on tongue cancer, and could be an effective target for the treatment of this disorder.

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Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

Cited by 18 publications

References 25 publications

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3

Granulocyte colony-stimulating factor protected against brain injury in a rat cerebral hemorrhage model by modulating inflammation

Silencing of SPP1 Suppresses Progression of Tongue Cancer by Mediating the PI3K/Akt Signaling Pathway

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