Fetuin from fetal bovine serum

Carbon nanoparticles (particle size less than 100 nm and specific surface area higher than 100 m²/g), ribonuclease B (RNase B) from bovine pancreas, fetuin from fetal bovine serum, human alpha-1 acid glycoprotein (AGP), human serum from human male AB plasma, 2,5dihydroxybenzoic acid (DHB), and iodomethane were purchased from Sigma-Aldrich (St. Louis, MO). Microspin columns were purchased from Harvard Apparatus (Holliston, MA) and PNGase F with 10×G7 reaction buffer (0.5 M sodium phosphate) was obtained from New England Biolabs (Ipswich, MA). HPLC grade ethanol, acetonitrile (ACN), and water were used for sample preparation and were purchased from Sigma-Aldrich (St. Louis, MO).

Before performing assays, A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A virus was titered to determine the optimum dilution. An NA inhibition assay with a small substrate was performed with the NA-Fluor Influenza Neuraminidase Assay kit (Thermo Fisher Scientific) according to protocol. For ELLA NI assays, 96-well ELISA plates were coated with 100 μL of 25 μg/mL of fetuin from fetal bovine serum (Sigma-Aldrich) diluted in 0.1 M PBS and incubated at overnight 4°C. 50 μL of three-fold serial dilutions of each antibody starting at a molar concentration of 66.7 nM (10 μg/mL) in PBS was added to 50 μL of pre-optimized virus dilution in PBS containing 0.9 mM CaCl₂, 0.5 mM MgCl₂, 1% BSA and 0.5% Tween. The fetuin-coated plates were washed, and the mAb-virus mixture was added to the plates and incubated for two h at 37°C. The plates were washed with PBST, and 100 μL of HRP-conjugated lectin from Arachis hypogaea (Sigma-Aldrich) at 5 μg/ mL was added to the plates and incubated for 1.5 h at RT. The plates were washed and 100 μL of TMB substrate was added to the plates, and the reaction was stopped with 1N HCl. The optical density values were measured at 450 nm wavelength on a BioTek plate reader. In both assays, each dilution was performed in triplicate, and the IC₅₀ values were calculated in Prism software (GraphPad) using non-linear regression analysis.