To assess anti-influenza immunoglobulin levels in serum, Immulon 2 HB plates (Thermo Fisher Scientific, Pittsburgh, PA) were coated with influenza A virus, A/Puerto Rico/8-9VMC3/1934 (H1N1) (1 μg per well) in bicarbonate buffer (pH 9.5) overnight at 4°C. Plates were washed three times with PBS containing 0.05% Tween 20 and blocked in PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 1 hour at room temperature. Heat-inactivated serum samples were diluted 1:5 in sterile PBS. Fifty microliters of each dilution was added to the antigen-coated plates for 4 hours at room temperature. Plates were washed three times with PBS containing 0.05% Tween 20 and incubated with horseradish peroxidase–conjugated goat anti-mouse IgG (1:2000; Southern Biotechnology Associates, Birmingham, AL) in separate wells for 2 hours at room temperature. Plates were washed, and 100 μl of substrate solution [o-phenylenediamine (0.4 mg/ml) (Sigma) in 50 mM phosphate-citrate buffer (pH 5.0) with 0.03% (v/v) hydrogen peroxide] was added to each well. The plate was incubated at room temperature for 10 min, and optical densities were read at 450 nm on a microplate reader (GloMax-Multi+ Detection System, Promega, Madison, WI).

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