Hrp conjugated goat anti rabbit igg antibody

Western blotting and ELISA were used to determine the binding activity of rMSNOX to cPlg and hFn.
For western blot analysis, gradient diluted rMSNOX protein (2, 1, and 0.5 µg) and 2 µg BSA (Sigma) were subjected to 12.5% SDS-PAGE, then transferred to an NC membrane. After blocking with 5% skimmed milk, the NC membrane was incubated with 10 µg/mL of cPlg (Cell Sciences, USA) or hFn (Sigma) for 2 h at 37 °C. After excessive washing with PBST, the membrane was incubated with rabbit anti-cPlg/hFn polyclonal antibody (1:1000; Cell Sciences) for 1 h at 37 °C, then incubated with HRP conjugated goat anti-rabbit IgG antibody (1:5000; Thermo) for 1 h at 37 °C. Membranes were visualized with a Basic Luminol ECL kit (Yeasen).
For ELISA analysis, the 96-well plates (Corning) were coated with 1 µg/well of cPlg, hFn or BSA (negative control). After blocking with 5% skimmed milk, wells were incubated with various amounts of rMSNOX protein (1, 0.5, 0.15, 0.125, 0.0625, and 0.03125 µg/well) at 37 °C for 1.5 h. After being washed, the wells were treated with rabbit anti-rMSNOX serum (1:500) at 37 °C for 1 h, followed by HRP conjugated goat anti-rabbit IgG antibody (1:5000; Thermo) at 37 °C for 1 h. TMB substrate solution and 2 M H₂SO₄ were added successively and OD_{450 nm} values were measured as described above. The experiments were performed in triplicate.

Virus particles (1 × 10⁵ plaque-forming unit PFU/well) in PBS buffer were coated onto a Maxibinding Immunoplate (SPL Life Sciences, Republic of Korea) and incubated at 4°C overnight. The plate was washed five times with TBS-T and blocked with blocking buffer (5% skim milk in TBS-T buffer) for 2 h at RT. Then, 100 μL of the indicated proteins in a 2X serial dilution (starting from the 100 μg/well) containing blocking buffer was added and incubated at 37°C for 1 h. Polyclonal rabbit anti-HA antibodies (1:3000 dilution; Invitrogen, USA) were used as a control to ensure that the virions were present and intact for the assay. The proteins were detected using anti-6X His tag antibodies at a 1:3000 dilution (Abcam, UK) for 2 h at RT. After washing, 100 μL of a 1:5000 goat anti-mouse IgG H&L (HRP) (Abcam, UK) dilution for anti-6X His tag, and goat anti-rabbit IgG-HRP conjugated antibodies (Invitrogen, USA) for anti-HA antibodies were added and incubated for 2 h at RT. The TMB substrate and 1 M sulfuric acid were added last.

Influenza virions were diluted in PBS and coated onto an maxibinding immunoplate (SPL Life Sciences, Republic of Korea) at 4°C overnight (1 × 10⁵ plaque-forming unit PFU/well). The NVLH8 protein (100 μL/well) in a 2X serial dilution (starting from the 100 μg/well) was added to the plate after incubation with a blocking buffer (5% skim milk in TBS-T buffer) for 2 h at RT. Following an incubation at 37°C for 1 h, 100 μL anti-6X His tag antibodies at a 1:3,000 dilution (Abcam, United Kingdom) were added to the TBS-T-washed plate for 2 h at RT. The virions coated in the plate were validated using polyclonal rabbit anti-HA antibodies (1:3,000 dilution; Invitrogen, United States). Goat anti-mouse IgG H&L (HRP) (1:5,000 dilution, Abcam, United Kingdom) and goat anti-rabbit IgG-HRP-conjugated antibodies (1:5,000 dilution, Invitrogen, United States) were added and incubated for 2 h at RT. After adding TMB and 1 M sulfuric acid, the plate was ready at OD450. The experiment was reproduced three different times independently. Each batch was designed with five data points, and three data points were chosen to be analyzed.