Recombinant Alpaca Anti-IAV NP Single Domain Antibody (PABC-575)

His-tagged NP was incubated briefly with His-tagged versions of the given VHHs. Biotinylated NP-VHH1 immobilized on streptavidin beads was then used to pull down NP-His. Precipitated protein was run on SDS-PAGE and detected via immunoblotting with His-HRP antibody.

Ashour, J., Schmidt, F. I., Hanke, L., Cragnolini, J., Cavallari, M., Altenburg, A., ... & Ploegh, H. L. (2015). Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization. Journal of virology, 89(5), 2792-2800.

MDCK cells stably expressing VHH-Ch were challenged with influenza virus (MOI, 1.0). Two hours postinfection, cells were metabolically labeled with [³⁵S]methionine for 20 min. Cells were lysed in NP-40-containing lysis buffer, and the lysate was subsequently probed with WSN serum and FluB1 antibody. Samples were analyzed by SDS-PAGE and autoradiography.

Ashour, J., Schmidt, F. I., Hanke, L., Cragnolini, J., Cavallari, M., Altenburg, A., ... & Ploegh, H. L. (2015). Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization. Journal of virology, 89(5), 2792-2800.

293T cellsweretransfectedwith plasmids expressing influenza virus PB1, PB2, PA, NP, and VHH-Ch and a plasmid transcribing a model genome segment encoding neuraminidase with a sortag motif inserted at the C terminus (NAsrt) (under the control of the pPol1 promoter). At 24 h posttransfection, cells were lysed in NP-40-containing lysis buffer. Samples were analyzed via Western blotting using antibody against the HA epitope, NP, mCherry, and GAPDH.

Ashour, J., Schmidt, F. I., Hanke, L., Cragnolini, J., Cavallari, M., Altenburg, A., ... & Ploegh, H. L. (2015). Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization. Journal of virology, 89(5), 2792-2800.

A549 cells expressing αNP-VHH1-HA in a doxycycline (Dox)-inducible manner were seeded 24 h before influenza A virus (IAV) infection. VHH expression was induced at the indicated time points relative to infection; cells were infected with IAV at an MOI of 1 at t=0 h. Cells were harvested 6 h postinfection (p.i.), stained for HA and NP, and analyzed by flow cytometry. Geometric mean of anti-HA-Alexa Fluor 488 (VHH expression level, red) and fraction of infected cells (NP positive, gray) are shown.

Hanke, L., Knockenhauer, K. E., Brewer, R. C., van Diest, E., Schmidt, F. I., Schwartz, T. U., & Ploegh, H. L. (2016). The antiviral mechanism of an influenza A virus nucleoprotein-specific single-domain antibody fragment. MBio, 7(6), e01569-16.

293T cells were transfected with expression vectors for influenza virus A/WSN/33 PA, PB1, PB2, and NP, as well as the indicated VHHs or Mx proteins. In addition, we cotransfected plasmid pPolI-RT, from which a synthetic genome segment was transcribed which encoded either EGFP (720 nt) or mCherry-T2A-EGFP (1,500 nt). Twenty-four hours posttransfection, EGFP-positive cells were quantified by flow cytometry. Since reduced EGFP levels were expressed from the mCherry-T2A-EGFP construct, values were normalized to EGFP-positive cells expressing VHH7 (control).

Hanke, L., Knockenhauer, K. E., Brewer, R. C., van Diest, E., Schmidt, F. I., Schwartz, T. U., & Ploegh, H. L. (2016). The antiviral mechanism of an influenza A virus nucleoprotein-specific single-domain antibody fragment. MBio, 7(6), e01569-16.