ELISA-based Serum Antibody Titer Measurement

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) as in our previous report^{25 (link)}. In brief, 96-well ELISA plates were coated with 2 µg/ml rHA at 4 °C overnight. After blocking with 5% non-fat milk, two-fold serial dilutions of immune sera were added and incubated at room temperature for 90 min. After washing in PBS supplemented with 0.05% Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibodies (1:2500, NA931, GE Healthcare Life Sciences) were added and incubated at room temperature for 1 h. After washing in PBST, TMB substrates were added and reactions were then stopped by addition of 3 N H₂SO₄. Optical absorbance (OD_450nm) was determined using a microplate reader (Molecular Device). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD_450nm ~ 3 times higher than the background values.