Journal: Emerging Microbes & Infections

Article Title: Cross-reactive antibodies binding to H4 hemagglutinin protect against a lethal H4N6 influenza virus challenge in the mouse model

doi: 10.1080/22221751.2018.1564369

Figure Lengend Snippet: Antibody binding to H4 on infected cells and in Western blot. (A–F) Immunofluorescence analysis to assess binding of antibodies to HA on the surface of infected cells. MDCK cells were infected with (A) A/duck/Czechoslovakia/1956 (H4N6-PR8), (B) A/shorebird/Delaware Bay/718/1988 (H4N6), (C) A/blue-winged teal/Illinois/10OS1563/2010 H4N6, (D) A/duck/Zhejiang/D9/2013 (H4N6-PR8), (E) A/Caspian seal/Russia/T1/2012 (H4N6-PR8), and (F) A/swine/Missouri/A01727926/2015 (H4N6-PR8) followed by staining with 30 μg/ml of each antibody and secondary antibody treatment (anti-mouse Alexa Fluor 488). A mAb binding to the Lassa virus glycoprotein served as negative control while a pan HA mAb, CR9114, served as positive control. (G) Western blot analysis. Recombinant H4 (A/duck/Czechoslovakia/1956) and recombinant H11 HA were denatured and reduced, run on an SDS-PAGE and then blotted onto a nitrocellulose membrane. Membranes were probed with 30 μg/ml of each antibody and then treated with anti-mouse IgG alkaline phosphatase secondary antibody. Both recombinant proteins feature a hexahistidine tag, and an anti-hexahistidine antibody was used as a positive control.

Article Snippet: Hundred micro litre of secondary antibody, goat anti-mouse IgG heavy plus light chain (H + L)–Alexa Fluor 488 (Abcam), which was also prepared in 1% non-fat milk in PBS, was added to the cells afterwards at a dilution of 1:1000 for an hour.

Techniques: Binding Assay, Infection, Western Blot, Immunofluorescence, Staining, Negative Control, Positive Control, Recombinant, SDS Page