*2.3. ELISA Detection of Anti-Influenza A IgG, IgG isotypes, and IgA*

IgG and ELISA measured IgA responses to influenza A in samples, as described [26]. The plates were coated with inactivated influenza A antigen (Swedish Institute for Communicable Disease Control, Solna, Sweden and Solvay Pharmaceuticals, BV, Weesp, Holland and recombinant HA/influenza A/H1N1/CA09pdm or NP Protein BioSciences, CT, USA) that was diluted to 2 μg/mL in sodium carbonate buffer pH 9.5–9.7 before 100 μL was added to each well. Influenza A positive mouse serum and naïve mouse serum were used as the controls for mouse anti-influenza A reactivity. The coated plates were washed with phosphate buffer saline (PBS)/0.05% Tween 20 (Sigma-Aldrich, S:t Louis, MO, USA) and then blocked with PBS/5% dry milk at 37 ◦C for 1 h followed by one wash. Mouse sera was diluted in PBS (pH 7.4)/0.5% bovine serum albumine (BSA, Boehring Mannheim, Mannheim, Germany)/0.05% Tween 20, and 100 μL of serial dilutions (1/50–1/5,000,000) were added to each well and then incubated at 37 ◦C for 90 min. After incubation, the plates were washed and 100 μL of HRP-conjugated goat-anti mouse IgG (BioRad, Richmond, VA, USA) or HRP-conjugated anti-mouse IgA (Southern Biotechnologies, Birmingham, AL, USA) (1:1000) diluted in 2.5% dry milk/0.05% Tween 20 (1:2000) was added to each well. The plate was incubated for 1 h at 37 ◦C and then washed. Ortho-phenylene diamine (OPD, Sigma) substrate was prepared by solving OPD-tablets 2 mg/mL in 0.1 M citrate buffer/0.003% H2O2. 100 μL was added to each well and the plate was then covered and incubated at room temperature for 30 min. The reaction was stopped by adding 100 μL 2.5M H2SO4 to each well and the absorbance was measured at OD 490 nm (24). The avidity index (AI) was determined by using the 8M urea wash procedure against the influenza antigens. IgG isotype reactivity to WIV was tested while using the ISO-2 ELISA reagent kit (Sigma), as recommended by the manufacturer. Isotype calculations of IgG1/IgG2a or 2c-ratios were calculated by dividing the OD 490 nm values for each subclass at dilution 1/100 or 1/1000. Inter-group ratio comparisons were made while using unpaired two-tailed, student t test. The ratio comparisons within each group were made using Pearsons correlation coefficient r.
