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 H1N1 neuraminidase showing the conversion of the 6′-sialyllactose to lactose in the presence of the transition state analog DANA at concentrations from top to bottom as 0.010, 0.050, 0.50, 1.5, 3.0, 6.0, 15, 41, 100 μM. The product peak area decreases with increasing inhibitor. The K i is approximated as the IC 50 . Traces are offset to aid in visualization. See Table S7A and Figure S2A in the Supporting Information for peak areas, offsets for the x - and y -axes, and data for replicate traces. The peak labeled with the asterisk is a contaminant present in the substrate standard or sialyllactose that has undergone spontaneously desialylation during the APTS labeling reaction. This contaminant peak is typically about 1–2% of the peak area of the sialyllactose. As required for evaluating activity, the sialyllactose peak area is significantly larger than the product peak area.

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, Activity Assay

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: Vaccine

Article Title: Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies.

doi: 10.1016/j.vaccine.2019.09.057

Figure Lengend Snippet: Fig. 2. Screening mammalian cell types for the influenza A/California/07/2009 (H1N1) VLP production (A) HA, NA, M1 protein detection by Western Blot with specific antibodies in the supernatant of the 293 T cells expressing VLPs (1) NIBSC influenza H1N1 antigen as positive control, (2) supernatant of 293 T cell as negative control, (3) supernatant of 293 T cell expressed influenza VLPs; (B) VLPs expressed in CHO-K1 cells (1), Vero cells (2), 293 T cells (3) and A/H3N2 influenza virus as positive control (4) were observed by Cryo-TEM performed by CBMN, UMR 5248, University of Bordeaux.

Article Snippet: After geltransfer and saturation, the blots were incubated with a rabbit polyclonal antibody against H1N1 influenza A virus NA (RD System), a polyclonal sheep antibody against H1N1 M1 (ThermoFisher), or a polyclonal rabbit antibody against the HA from A/California/07/2009 (H1N1) (produced in-house) for 1 h. Blots were then incubated with a secondary sheep anti-rabbit or antisheep antibody conjugated to IRDye 800 (Rockland) for 1 h. The membranes were visualized using Odyssey Infrared Imaging System (LICOR, Bioscience).

Techniques: Western Blot, Expressing, Positive Control, Negative Control, Virus

Journal: Vaccine

Article Title: Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies.

doi: 10.1016/j.vaccine.2019.09.057

Figure Lengend Snippet: Fig. 3. Production of influenza VLP from different strains in 293 T cells in disposable 3L-Bioreactor. The results of one representative experiment for each VLP batch are presented. (A) Time course of cell viability (1) and viable cell growth (2) during VLPs production (B) HAU and NA activities measured in the clarified cell supernatant 4 days after transfection. Data displayed as the mean ± standard deviation (C) TEM on the 293 T cells producing VLPs at D4 after transfection. Ultrathin sections of the cells showed the presence of numerous VLPs (green arrow) budding from the cell membrane (1) H3N2 VLP, (2) H1N1 VLP with granules visible around the aggregated VLPs (red arrow), (3) B/Phuket VLP, (4) B/Brisbane VLP. TEM analyses performed by IBiSA, University of Tours.

Article Snippet: After geltransfer and saturation, the blots were incubated with a rabbit polyclonal antibody against H1N1 influenza A virus NA (RD System), a polyclonal sheep antibody against H1N1 M1 (ThermoFisher), or a polyclonal rabbit antibody against the HA from A/California/07/2009 (H1N1) (produced in-house) for 1 h. Blots were then incubated with a secondary sheep anti-rabbit or antisheep antibody conjugated to IRDye 800 (Rockland) for 1 h. The membranes were visualized using Odyssey Infrared Imaging System (LICOR, Bioscience).

Techniques: Transfection, Standard Deviation, Membrane