## 3 Results
### Functional and Structural Characterization of the Purified Haemagglutinin (HA) Proteins
The Artificial H5.c1 Protein Strongly Elicited Haemagglutinin (HA)-Specific IgG and Neutralizing Antibodies  
Immunogenicity of the artificial H5.c1 protein and native HSTG was tested by immunization of mice. Five BALB/c mice from each group were immunized with IMAC-purified artificial H5.c1 protein (group 1), IMAC-purified native HSTG protein (group 2), or PBS, as a negative control (group 3). The resulting antibody-dependent humoral immune responses against the SEC-purified HA proteins were first evaluated by SDS-PAGE and Western blot (Figure 6a). Five mouse sera collected after the second and third immunizations of each group (artificial H5.c1 protein, native HSTG and PBS) were mixed and used as a primary antibody. Specific IgG antibodies against SEC-purified HA proteins were detected following the second and the third immunizations. After the third immunization, a much stronger band signal was clearly visible compared to that following the second immunization in both the artificial H5.c1 and native HSTG groups. The band signal of the artificial H5.c1 group was significantly stronger than that of the native HSTG group following the second immunization. In the negative control mice group vaccinated with PBS, there was no HA-specific IgG antibody response induced. ELISA is the most common immunoassay used for detecting and quantifying a target substance (typically an antigen or antibody) in which an enzymatic reaction is used to amplify the signal if the target substance is present [35]. In this study, the immune responses in the sera of five mice of groups 1 and 2 were measured against the SEC-purified HA proteins by indirect ELISA. Five individual sera in each group were characterized and compared by t-test analysis in Sigma Plot software. Figure 6b shows the mean OD450 values of each mouse, with the standard deviations represented as bars, individual values as dots, and geometric mean titres as horizontal bars for each test group. The mean OD450 value of group 1 (artificial H5.c1) mouse sera at the \(16\times 10^{-3}\) serum dilution was not significantly different from that of group 2 (the native HSTG) following the second immunization (P value = 0.130) and the third immunization (P value = 0.070). No immune response against HA proteins was detected in mouse sera from mice vaccinated with PBS. Figure 6: Western blot to detect haemagglutinin (HA)-specific IgG antibodies in mice sera (**a**) and an indirect ELISA to detect HA-specific IgG antibodies in mice sera at the \(16\times 10^{-3}\) serum dilution (**b**). Hemagglutination inhibition (HI) assays are commonly used to measure neutralizing antibodies induced in serum after immunization with HA protein. In this study, four HI assays, including one for homologous artificial HA proteins and three for heterologous strains, were used for testing induced neutralizing antibodies in mouse sera following the second and third immunizations. The results are shown in Figure 7, in which a single dot indicates the HA titre of a single serum sample, and the horizontal bars represent the geometric mean titre of each test group. In our first HI assay, the SEC-purified artificial H5.c1 and H5 TG proteins were used as the homologues for the tested mouse sera. In the mouse group that was vaccinated with the artificial H5.c1 protein, the HI titre ranged from 16-32, and the HI titre mean was 22.4 after the second immunization. Following the third immunization, all of the sera had HI titres from 32-128, and the HI titre mean was 70.4. In the mouse group that was vaccinated with the native H5.c1 protein, the HI titre ranged from 8-32, and the HI titre mean was 16 after the second immunization. Following the third immunization, all of the sera had HI titres from 16-128, and the HI titre mean was 70. However, the difference between these two groups was not statistically significant, with a P value \(>\) 0.05 (as shown in Figure 7a). Second, a heterologous inactivated virus strain whose HA protein derives from A/Vietnam/1194/2004(H5N1) NIBRG-14, clade 1, was used.