Journal: The Journal of Biological Chemistry

Article Title: Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

doi: 10.1074/jbc.M114.560748

Figure Lengend Snippet: Inhibitory effect of bezlotoxumab on activity and binding of TcdB in Vero cells. A, neutralization of the cytotoxic effects of purified TcdB by bezlotoxumab. The concentration of TcdB used (10 pg/ml) was previously determined to cause a ∼95% decrease in cell viability in this assay. B, Western blots of Vero cell membranes following incubation of cells with 50 ng/ml (lanes 3–5) or 100 ng/ml (lanes 6–8) TcdB, in the absence and presence of bezlotoxumab or actoxumab (200 μg/ml). Binding experiments were carried out at 37 °C in the presence of the endocytosis inhibitor chlorpromazine fixed at 14 μm. Lane 1, 1 ng of purified TcdB (no membranes); lane 2, membranes from cells incubated without TcdB; lanes 3 and 6, membranes from cells incubated with TcdB in the absence of bezlotoxumab; lanes 4 and 7, membranes from cells incubated with TcdB in the presence of bezlotoxumab; lanes 5 and 8, membranes from cells incubated with TcdB in the presence of actoxumab. Levels of TcdB are shown in the top panel, and levels of cadherin (loading control) are shown in the bottom panel. C, quantification of membrane-bound TcdB (normalized to the 1 ng of TcdB control lane and to cadherin band densities) following incubation of Vero cells in the presence of 50 or 100 ng/ml TcdB alone (black bars) or in the presence of bezlotoxumab (white bars) or actoxumab (hatched bars). Values are averages ± S.D. of two independent determinations.

Article Snippet: Nitrocellulose membranes were also probed with rabbit anti-cadherin polyclonal antiserum (Cell Signaling, Beverly, MA) to ensure that equivalent amounts of protein were loaded in each lane.

Techniques: Activity Assay, Binding Assay, Neutralization, Purification, Concentration Assay, Western Blot, Incubation