2.10. ELISA assay
The ORF8-specific antibodies (IgG, IgG1, IgG2a and IgM) titers were measured by ELISA, essentially as described previously.[20] The 96-well plates were pre-coated with ORF8 (0.75 μg/well) in 50 mM NaHCO3 (pH 9.6) at 4 °C overnight. The plates were blocked with 200 μL of 4% skimmed milk in PBS buffer (PBS-milk, pH 7.4) at 37 °C for 1 h, followed by washing 3 times with PBS buffer (pH 7.4). Diluted sera (100 μL) were added the plate, followed by incubation in the wells for 1 h at 37 °C and washing with PBS buffer containing 0.1% Tween 20 (PBST, pH 7.4). The plates were added with 100 μL of the diluted HRP-IgG, HRP-IgG1, HRP-IgG2a or HRP-IgM at 37 °C for 1 h, respectively. After washing 3 times with PBST, 100 μL of 0.015% (w/v) TMB was added and incubated at 37 °C for 30 min, followed by adding 25 μL of 2 M H2SO4. The resultant solution was determined at 450 nm. The antibody titers were presented as the highest sample dilution, which led to in an OD value greater than 2.1 times the OD mean of the sera of the PBS group.