*2.4. Total IgA Quantification and Detection of Lung Anti-Influenza A IgA Responses*

Lung-washes were harvested by flushing the lungs with PBS that was supplemented with protease inhibitors (Complete Mini, Roche, Mannheim, Germany) and then subjected to total IgA isolation while using the Kaptive IgA/IgE reagents (Biotech IgG, Copenhagen, Denmark) as recommended by the manufacturer. Total isolated IgA quantities were determined using an in-house murine IgA capture ELISA. Briefly, purified lung-wash IgA and standard mouse IgA (1 mg/mL, Sigma) was diluted ten-fold (PBS/5% dry-milk/0.05% Tween 20). 100 μL/per dilution was added to a 96-microwell plate that was precoated with rabbit anti-murine IgA (Dakopatts AB, Copenhagen, Denmark) and then incubated at 37 ◦C for 1 h. The plates were washed four times with PBS/0.05% Tween 20 before 100 μL HRP-conjugated goat anti-murine IgA was added to each well (Southern Biotechnologies) (1:1000). After 1 h incubation at 37 ◦C plates were washed and bound conjugate was detected by using OPD, as described above. The reactions were terminated using 100 μL/well 2.5M H2SO4 and the absorbance was measured at OD 490 nm. Total IgA was determined by comparing the OD-values of the test samples with the IgA standard. Detection of anti-influenza A IgA in total lung-wash IgA was done as with IgA from serum (above).
