Journal: Analytical Chemistry

Article Title: Native Capillary Nanogel Electrophoresis Assay of Inhibitors of Neuraminidases Derived from H1N1 and H5N1 Influenza A Pandemics

doi: 10.1021/acs.analchem.4c06127

Figure Lengend Snippet: Depicts the traces obtained with H5N1 neuraminidase showing the conversion of the 6′-sialyllactose substrate to the lactose product in the presence of peramivir at concentrations listed from top to bottom as 0.010, 1.0, 3.0, 5.0, 10., 14, 75, 750 nM. The area of the product, which decreases with increasing inhibitor, is used to create the dose response curve. The K i is approximated as the IC 50 . Traces are offset to aid in visualization (see Table S13, and Figure S7 in the Supporting Information for peak areas and offsets for the x - and y -axes). The peak labeled with the asterisk is a contaminant present in the substrate preparation prior to enzyme treatment.

Article Snippet: Recombinant influenza A virus neuraminidase proteins H1N1 (4858-NM-005 lot RJJ0822031) and H5N1 (7597-NM-010 lot DCLS0121052) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Labeling

Journal: PLoS ONE

Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells

doi: 10.1371/journal.pone.0097574

Figure Lengend Snippet: (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of PNGase F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.

Article Snippet: The identity of the purified protein was determined by immunoblotting with mouse AIV H5N1 HA polyclonal antibodies (ProSci, Poway, CA) and anti-mouse IgG-horseradish peroxidase conjugate as secondary antibodies (Santa Cruz Biotechnology, Dallas, TX).

Techniques: SDS Page, Recombinant, Purification, Affinity Chromatography, Filtration, Affinity Column, Chromatography, Incubation, Migration

Journal: PLoS ONE

Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells

doi: 10.1371/journal.pone.0097574

Figure Lengend Snippet: (A) The antiviral effect of selected aptamers on the viability of MDCK cells infected by the influenza virus was evaluated using the MTT method. MDCK cells were infected with influenza virus H3N2 at an MOI of 0.1 TCID 50 and then treated with the selected RNA aptamers (5 pmol). The highest protective activity was observed by HA12-16, as compared with other selected RNA aptamers. (B) Microscopic analysis of the effect of HA12-16 on viral infection of cells. Immunofluorescence images of MDCK cells after 24 h of incubation under various conditions were obtained using fluorescence microscopy; (a) MDCK cells only, (b) MDCK cells infected with influenza virus (MOI of 0.1 TCID 50 ), (c) MDCK cells treated with the influenza virus and the HA12-16 aptamer (30 pmol), (d) MDCK cells only treated with the HA12-16 aptamer. The cells were fixed with 3% paraformaldehyde followed by permeabilization with 0.5% Triton X-100. The cells were then immunostained to test for the presence of the influenza virus by using mouse anti-HA antibodies and goat anti-mouse polyclonal antibodies conjugated to FITC as secondary antibodies. Nuclei were visualized by staining with DAPI; HA and nuclei are shown in green and blue fluorescence, respectively.

Article Snippet: The identity of the purified protein was determined by immunoblotting with mouse AIV H5N1 HA polyclonal antibodies (ProSci, Poway, CA) and anti-mouse IgG-horseradish peroxidase conjugate as secondary antibodies (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Infection, Virus, Activity Assay, Immunofluorescence, Incubation, Fluorescence, Microscopy, Staining

Journal: Plotkin's Vaccines

Article Title: Regulation and Testing of Vaccines

doi: 10.1016/B978-0-323-35761-6.00079-1

Figure Lengend Snippet: Viral Vaccines Currently Licensed in the United States

Article Snippet: Influenza A (H5N1) virus monovalent vaccine, adjuvanted , ID Biomedical Corporation of Quebec.

Techniques: Vaccines, Recombinant, Virus