Virus-specific IgG ELISA and cytokine analysis in BNV immunized mice. (a,b) Swine influenza H1N1 and H3N2-specific IgG titers in serum two weeks after the final immunization. The minimum detectable dose of mouse IgG is less than 1.0 µg/mL. (c,d) Levels of IgG1 and IgG2a in serum of immunized mice were determined using ELISA two weeks after boost immunization. The minimum detectable dose of mouse IgG1 and IgG2a is less than 1.0 µg/mL and 0.1 µg/mL, respectively. (e,f) IFN-γ and IL-4 levels in serum from immunized mice were analyzed by mouse IFN-γ and IL-4 enzyme immunoassay kit. The minimum detectable dose of mouse IFN-γ and IL-4 is typically less than 1.0 pg/mL. Values are presented as mean ± standard deviation (S.D). Statistical significance analysis was performed using one-way ANOVA and then multiple comparison tests (** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, p > 0.05), and n.s. indicates no significance between the two compared groups (n = 8). Three parallel experiments in each group were conducted.