FIG 6.

Antibody neutralization of PTx in vitro. (A) Anti-PTx antibody binding to whole-cell B. pertussis monitored by flow cytometry. Antibodies were incubated with mid-log-phase bacteria and detected with anti-human IgG. Data are presented as means, and error bars represent standard deviations of the means. (B) In vitro inhibition of PTx-mediated clustering of CHO-K1 cells. Results are presented as the means for 6 replicates, and error bars represent the standard deviations from the means. Statistical significance was determined using Kruskal-Wallace analysis and Dunn's multiple-comparison test (α = 0.05; *, P < 0.05; **, P < 0.01, ***, P < 0.001). (C) Antibody-mediated blockade of PTx-receptor binding, with fetuin as a model receptor. Wells were coated with fetuin and blocked. Antibody and PTx were preincubated at the indicated molar ratios (molar excess of antibody ranging from 0.6× to 18.5×) and added to the blocked wells. PTx was detected with a murine anti-PTx antibody cocktail, followed by anti-mouse IgG–HRP and TMB substrate. The hu11E6 antibody, known to block PTx-receptor interactions, was used as a positive control, while wells lacking PTx were used as a negative control. The ELISA was performed twice, with similar results; error bars indicated are the standard errors of the means.