3 mL of 1 × 1010/mL EHEC O157:H7. The mice were provided with food and water from 12 hr after being infected, and their deaths were recorded. All the data was expressed as . F testing was used to analyze the antibody OD values of each group and χ2 testing to analyze the

differences in survival rates between the immunized and control groups CHIR-99021 manufacturer after infection with EHEC O157:H7. We analyzed β-turn, flexibility, hydrophilicity, accessibility, and antigenicity of IntC300 using the methods of Hopp-Woods (14), Chou-Fasman (15), Karplus-Schulz (16), Emini (17), Jameson-Wolf (18) and Kolaskar-Tongaonakar (19). The results are shown in Figure 1. We performed a comprehensive analysis of the outcome predicted by different approaches and the possible locations of B-cell epitopes are shown in Table 1. Table 1 shows that the peptide segments of 658–669, 711–723, 824–833, 897–914, 919–931 are consistent with the prediction using β-turn, flexibility, hydrophilicity, accessibility, and antigenicity as indices. This indicates

that the B-cell epitopes are located within or near the above peptide segments. The amino acid sequences of five peptides are given in Table 2. We chose one of the predicted EHEC O157:H7 IntC300 B-cell epitopes, KT-12. We synthesized it and coupled it with KLH, then immunized mice by subcutaneous injection and intranasal delivery on days 1, 14 and 28. Orbital blood was taken on days 0, 21 and 35 and indirect ELISA used for detection of OD values for IgG (Fig. Quisqualic acid 2) and IgA antibodies (Fig. 3). As seen in Figure 2, after subcutaneous and intranasal immunization Nutlin-3a manufacturer serum IgG antibody concentrations gradually increased from day 0 through days 21 and 35, indicating that both kinds of immunization were able to induce high concentrations of IgG antibodies compared to the control groups. The differences in serum IgG concentrations were statistically significant (P < 0.05). Further, a higher concentration of IgG antibody was produced in the group that received

subcutaneous immunization than in the intranasal immunization group (P < 0.05). Figure 3 shows that after intranasal immunization serum IgA antibody concentrations increased gradually from day 0, through days 21 and 35 compared with the control group. The difference in serum IgA concentrations was statistically significant (P < 0.05). In contrast, the difference between the test and the control group in IgA antibody concentrations was not statistically significant for subcutaneous immunization. Intranasal mucosal immunization induced high concentrations of IgA antibodies, whereas subcutaneous immunization did not. A higher concentration of IgA antibody was produced by mice that received intranasal immunization than by those that received subcutaneous immunization. Enterohemorrhagic Escherichia coli O157:H7 strain 882364 (1 × 1010 CFU/mL) was used to infect mice by the oral route.