Monoclonal anti influenza virus h1

Factor H or VCP (5, 2.5, 1.25, and 0.625 μg/well in 100 μl volume) were coated onto 96-well microtiter plates using carbonate-bicarbonate buffer (CBC), pH 9.6, and incubated at 4°C overnight. After washing the microtiter wells with PBS, the wells were blocked with 2% w/v BSA in PBS and incubated at 37°C for 2 h, followed by three PBST (PBS + 0.05% v/v Tween 20) washes. Twenty microliters of H1N1 or H3N2 virus (1.36 × 10⁶ pfu/ml) in PBS were added to each well and incubated at 37°C for 2 h in the presence of 5 mM CaCl₂. VSV-G pseudotyped lentivirus was used as a negative control. Following PBST washes, the corresponding wells were incubated with primary antibodies (100 μl/well): polyclonal anti-influenza virus H3 and monoclonal anti-influenza virus H1 (1:5,000) (BEI-Resources). The wells were again washed with PBST three times and probed with Protein A-HRP-conjugate, or goat anti-mouse IgG-horseradish peroxidase (HRP)-conjugate (1:5,000) (Fisher Scientific), followed by incubation at 37°C for 1 h. Color was developed using 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich), and reaction was stopped using 1M H₂SO₄, followed by measuring absorbance at 450 nm, using iMark™ microplate absorbance reader (Bio-Rad).

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.