ELISA antibody titer

Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA). In brief, 96-well ELISA plates were coated with 10 μg/mL OVA or 1 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), HRP-conjugated sheep anti-mouse IgG secondary antibodies (1:5,000, 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 stopped by addition of 3 M H₂SO₄. Optical absorbance (OD_450nm) was read in a microplate reader (Molecular Device). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD_450nm that was ∼3 times higher than the background value.