Glycosylation of MBP with <i>E. coli</i> O157:H7 O-Ag and analyzed by (A), coomassie blue staining and western blot with (B), anti-6×-His antibody; (C), anti-MBP antibody; (D), O157:H7 antiserum.

<p>Glycosylation of MBP with <i>E. coli</i> O157:H7 O-Ag and analyzed by (A), coomassie blue staining and western blot with (B), anti-6×-His antibody; (C), anti-MBP antibody; (D), O157:H7 antiserum.</p

Spleen lymphocyte subsets assay.

<p>Spleen lymphocytes were isolated and stained for FITC-CD3, PE-CD4 or PE-CD8 antibodies and then analyzed by flow cytometry. The percentage of (<b>A</b>), CD3⁺ CD4⁺ T cells; (<b>B</b>), CD3⁺ CD8⁺ were expressed as the arithmetic mean ±SD indicated by error bars. Exprements were repeated three times. Differences of two groups are indicated with symbols (*: P<0.05; **: P<0.01).</p

Induction of Th1-biased responses by O-Ag-MBP.

<p>(<b>A</b>), Secretion of IFN-γ and IL-4 in serum samples of immunized mice. Differences of two groups are indicated with symbols (*: P<0.05; **: P<0.01). (<b>B</b>), <i>E. coli</i> O157:H7-specific IgG subtypes induced by O-Ag-MBP. BALB/c mice were immunized with O-Ag-MBP and serum samples were collected and <i>E. coli</i> O157:H7-specific IgG, IgG1, IgG2a and IgG2b titers were determined by ELISA. The cutoff value was OD_{negative control}×2.1. Results were expressed as the arithmetic mean ±SD indicated by error bars.</p

Antibody responses after third immunization.

<p>(<b>A</b>), <i>E. coli</i> O157:H7-specific IgG antibody titers of serum samples; (<b>B</b>), <i>E. coli</i> O157:H7-specific IgM antibody titers of serum samples; (<b>C</b>), <i>E. coli</i> O157:H7-specific IgA antibody titers of feces samples; (<b>D</b>), MBP-specific IgG antibody titers of serum samples. The cutoff value was OD_{negative control}×2.1. Results were expressed as the arithmetic mean ±SD indicated by error bars. Differences of two groups were indicated with symbols (*: P<0.05; **: P<0.01; ***: P<0.001).</p

Cytokines determined by ELISPOT.

<p>(<b>A</b>), IFN-γ; (<b>B</b>), IL-4. The spots for cytokine-producing lymphocytes after stimulation with MBP or O-Ag-MBP were counted and expressed based on 1×10⁵ cells. Results are expressed as the arithmetic mean ±SD indicated by error bars. Differences of three groups stimulated at the same dose of MBP are indicated with symbols (*: P<0.05; **: P<0.001).</p

Serum bactericidal activity against <i>E. coli</i> O157:H7.

<p>The % killing of <i>E. coli</i> O157:H7 are expressed as the arithmetic mean ±SD indicated by error bars.</p

MALDI-TOF MS analysis of (A) MBP and (B) O-Ag-MBP.

<p>(<b>A</b>), The peak with m/z 44017.39 corresponds to un-glycosylated MBP; (<b>B</b>), The peak with m/z 50530.62 and m/z 76617.71 correspond to glycosylated MBP (O-Ag-MBP).</p