Polyclonal anti influenza virus h3

A549 cells were seeded in microtiter wells using complete DMEM (1 × 10⁵ cells/well) and incubated overnight at 37°C. The wells were washed with PBS three times, and then rfhSP-D (10, 5, 2.5, and 1.25 µg/ml) was pre-incubated with pH1N1 or H3N2 virus (1.36 × 10⁶ pfu/ml) diluted in 200 µl of PBS + 5 mM CaCl₂; 10 µl of diluted virus was added to the corresponding wells, and incubated at RT for 2 h. Maltose-binding protein (MBP) was used as a negative control. The microtiter wells were then washed with PBS three times, and fixed with 4% paraformaldehyde (Fisher Scientific) for 10 min at RT. The wells were washed again with PBS three times, and blocked with 2% w/v BSA in PBS for 2 h at 37°C. Monoclonal anti-influenza virus H1 (BEI-Resources) and polyclonal anti-influenza virus H3 (BEI-Resources) in PBS (1:5,000) were added to each well and incubated for 1 h at 37°C. After washing with PBST three times, the corresponding wells were probed with goat anti-mouse IgG-HRP-conjugate (Thermo-Fisher), or Protein A-HRP conjugate (1:5,000) in PBS for 1 h at 37°C. The wells were washed again with PBST three times and the color was developed using TMB substrate. The reaction was stopped using 2 M H₂SO₄, followed by absorbance reading at 450 nm.

A549 cells (1 × 10⁵ cells/well) were seeded in 96 microtiter wells and incubated at 37°C overnight in 5% v/v CO₂. Once 80% confluency was reached, the cells were washed twice with sterile PBS. Factor H or VCP (10, 5, 2.5, 1.25 μg/ml), pre-incubated with H1N1 and H3N2 (1.36 × 10⁶ pfu/ml) IAV subtypes, were added to the wells (in 5 mM CaCl₂) and incubated at room temperature for 2 h. BSA was used as a negative control. Following washes with PBS three times, the corresponding wells were fixed with 4% v/v paraformaldehyde (Fisher Scientific) for 5 min at room temperature. The wells were then blocked with 2% w/v BSA for 2 h at 37°C. Polyclonal anti-influenza virus H3 (BEI-Resources) and monoclonal anti-influenza H1 (BEI-Resources) were added to the appropriate wells and incubated at 37°C for 1 h. After gentle washes with PBSST, the wells were probed with protein A-HRP conjugate or goat anti-mouse IgG-HRP-conjugate (Thermo-Fisher) diluted in PBS in 1:5,000 dilution and incubated at 37°C for 1 h. The wells were washed again with PBST; the color was developed by adding TMB substrate and the reaction was stopped by using 1M H₂SO₄. The absorbance was read at 450 nm using an ELISA plate reader.