Anti np

To probe for NP content in the seasonal TIV (Fluzone), proteins from the vaccine were separated on a 4–12% gradient polyacrylamide gel (Bio Rad) and transferred to a PVDF membrane (Milipore). The membrane was then blocked in 5% milk in PBS for 1 h at room temperature. Immunodetection was performed using anti-NP (Millipore) antibody (1:1000 dilution) followed by secondary anti-mouse IgG antibody conjugated to horseradish peroxidase (GE Healthcare) (1:5000 dilution). NP specific band was detected by ECL western blotting detection system (ThermoFisher Scientific).

Samples were run on a 10% polyacrylamide SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked for 30 min with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (0.1% Tween 20 TBS-T). The membranes were probed with one of the following primary antibodies: anti-actin (Abcam) diluted 1:3,000, anti-NP (Millipore) diluted 1:1,000, anti-lamin B (Santa Cruz) diluted 1:1,000, or anti-MCOLN2 (Origene) diluted 1:200. For standard experiments, the membranes were washed with 0.1% Tween 20 TBS-T and probed with goat anti-rabbit or goat anti-mouse antibodies conjugated to horseradish peroxidase (HRP) (Pierce). The membranes were washed with 0.1% Tween 20 TBS-T, incubated with enhanced chemiluminescence (ECL) substrate (Pierce) according to the manufacturer’s instructions, and exposed to film. For quantitative experiments, membranes were probed with goat anti-mouse or donkey anti-goat antibodies conjugated to IRdye infrared dye (Li-Cor). The membranes were washed with Tris-buffered saline (TBS), and signal was detected using a Li-Cor Odyssey system.

One hundred TCID50 SN16/16 H3N2 virus was precultured with serially diluted sera for 1 h at 37°C and then transferred to the MDCK-SIAT1 monolayer cells in 96-well plates for 18 h at 37°C in a 5% CO₂ incubator. As reported previously, the cells were processed to be stained by anti-NP (Millipore, Burlington, MA, United States) (Rowe et al., 1999 (link)). The IC₅₀ of the tested sera was defined as the reciprocal of the last dilution that caused a 50% reduction of OD compared with the virus control.