Fig. 6

Immunopotentiation following immunization with an H7:ND (1/12, w/w) conjugate. a Immunization scheme in mice. b Measurement of H7 specific IgG amount in mouse sera via ELISA. In total, 50 ng of purified H7 [Influenza antigen A/Anhui/1/2013 (H7N9), NIBSC] per well were coated at the plate. The sera were diluted 1:5000 and a monoclonal mouse anti- H7N9 haemagglutinin/HA antibody (SinoBiological InC.) at concentrations of 0.5, 0.75, 1, 1.25, 2.5, 5, 12.5, 25, 50, 100, 150 μg/mL was applied as a standard, then analysed via ELISA. Specific immune responses were measured at 450 nm after 1 and 2 booster immunizations with H7 (group 1), H7:ND (group 2) and PBS:ND (group 3). The responses were recalculated according the standard values as µg/mL anti H7N9 antibody in the sera. The BSA background was subtracted. A standard curve was built by the help of OD450 values corresponding to known amounts of H7N9 haemagglutinin/HA antibody. The amount of H7 specific IgG antibody in mouse sera was measured via the standard curve. Statistical analyses were performed using the t-test (SigmaPlot) and are presented. A single dot indicates the value of a single mouse serum. SD was included on a single dot that corresponds to an ELISA data variation of a single mouse serum with three replications. The bars indicate the average value of the test groups. P < 0.05 was defined as a statistically significant difference. c Detection of H7-specific IgG antibodies via a Western blot. Sera from five mice from each group (against H7, H7:ND and PBS:ND as a negative control) were mixed, diluted 500 times and used as a primary antibody for detecting 100 ng of purified H7 [Influenza Antigen A/Anhui/1/2013 (H7N9), NIBSC]. Anti-mouse H7N9 haemagglutinin/HA antibody (SinoBiological InC.) was used as a primary antibody (positive control: P). HRP-linked goat anti-mouse IgG was used as a secondary antibody