The binding of SfbI to mouse IgG was further confirmed by Western blot analysis under denaturing conditions

The binding of SfbI to mouse IgG was further confirmed by Western blot analysis under denaturing conditions. for bacterial survival during the illness process. We have recently demonstrated that fibronectin-binding protein I (SfbI) of in vivo studies, we characterized the relationships between SfbI and mouse Igs. SfbI binds to mouse IgG.Microtiter plates coated with purified mouse IgA, IgG, or IgM (Dianova, Hamburg, Germany) were incubated with different concentrations of SfbI to test by enzyme-linked immunosorbent assay (ELISA) the ability of SfbI to bind mouse Ig. The SfbI-IgG complexes were recognized using rabbit polyclonal anti-SfbI antibodies and a peroxidase-conjugated goat anti-rabbit antibody as a secondary reagent. The results (Fig. ?(Fig.1A)1A) display that SfbI binds to immobilized IgG but not to IgA or IgM. The binding of SfbI to mouse IgG was further confirmed by Western blot analysis under denaturing conditions. Mouse IgA and IgG and human being IgG were immobilized onto nitrocellulose and incubated with the SfbI protein. Blots were then exposed to an SfbI-specific rabbit antiserum, which was recognized using a peroxidase-conjugated goat anti-rabbit antibody. Appropriate settings were used to exclude possible cross-reactions with secondary reagents. The results that we JMS obtained confirmed that SfbI bound to mouse IgG (Fig. ?(Fig.1B).1B). Open in a separate windowpane FIG. 1 Binding of SfbI protein to mouse Ig. (A) Binding of SfbI to immobilized mouse IgA (), IgG (), and IgM (?) mainly because determined by ELISA. The reported data are representative of three self-employed experiments. Results are the averages of triplicate samples. Standard deviations were lower than 10%. (B) GIBH-130 Western blot analysis of SfbI binding to mouse (m) and human being (h) Igs. SfbI interacts with mouse IgG through the F(abdominal)2 component of the Ig molecule.To identify the binding site within the mouse IgG molecule, purified IgG, IgG F(ab)2, and IgG Fc fragments (Dianova) were tested for his or her binding to SfbI. The results demonstrate that SfbI interacts with mouse IgG through the F(ab)2 portion (Fig. ?(Fig.2A).2A). These results were further confirmed by Western blotting (Fig. ?(Fig.2B).2B). The biological significance of a pathogen expressing a single protein with different mammalian Ig-binding patterns is not clear. However, this type of multipattern binding is not unprecedented but rather is definitely common among bacterial Ig-binding proteins (2C5, 11), suggesting the expression of these proteins may play a role in the adaptive response of the pathogen to an unfavorable sponsor environment. Open in GIBH-130 a separate window FIG. 2 SfbI binds specifically to the F(abdominal)2 fragment of mouse IgG. (A) ELISA of SfbI binding to immobilized mouse IgG, IgG F(abdominal)2, or IgG Fc fragments. Results are the averages of triplicate samples. Standard deviations are indicated by vertical lines. (B) Western blot analysis of SfbI binding to mouse (m) and human being (h) IgG, IgG F(abdominal)2, and IgG Fc fragments. Mouse IgG F(ab)2 inhibits the binding of SfbI to human being IgG Fc. Inhibition experiments were performed to determine whether the binding of SfbI to human being IgG Fc and mouse IgG F(ab)2 was mediated by either a solitary site or two independent sites. The binding of SfbI to human being IgG Fc was tested in the presence of increasing concentrations of mouse IgG F(ab)2. Number ?Figure3A3A demonstrates mouse IgG F(ab)2 competitively inhibited GIBH-130 the binding of SfbI to human being IgG Fc inside a dose-dependent manner. No effect was observed when human being IgG F(ab)2 fragments were used in the competition test. These results suggest either the same website of the SfbI protein is responsible for binding to both human being IgG Fc and mouse IgG F(abdominal)2 or the binding sites for both molecules are near each other. On the other hand, the binding of the SfbI website to one of GIBH-130 the moieties may either impact the overall conformation of SfbI or sterically hinder the binding capacities of a putative second website. Open in a separate windowpane FIG. 3 (A) Mouse IgG F(abdominal)2 fragments inhibit the binding of SfbI to human being IgG Fc. The binding of SfbI to human being IgG Fc was performed in the presence of increasing concentrations of either mouse or human being IgG F(ab)2 fragments. GIBH-130 The ideals are means of three determinations; one representative out of three self-employed experiments is demonstrated. Standard deviations were lower than.