FIG 4.
Characterization of the A8 epitope on PTx S1-220. (A) Epitope binning of isolated antibodies by competition ELISA. PTx was used to coat ELISA wells, followed by the addition of a biotinylated human antibody and an unlabeled competitor antibody at various concentrations. Bound biotinylated antibody was detected by use of streptavidin-HRP. Results for each antibody pair are from at least one ELISA performed with duplicate antibody samples. (B) Subset of the fitness metric heat map for A8 IgG binding to yeast-displayed PTx S1-220 variants. Deep sequencing and entropy analysis of the variants selected from A8 IgG binding to a PTx S1-220 mutagenesis library implicated candidate residues (circled sections) predicted to be involved in binding. (C) Validation of A8 candidate epitope residues identified from deep sequencing of yeast-displayed PTx S1-220 variants. Binding of A8 IgG to the yeast-displayed PTx S1-220 variants was assessed by ELISA. Data are represented as normalized binding relative to the wild-type level (A450, variant/A450, wild type) at an antibody concentration of 2 μg/ml, so lower values indicate reduced binding. The means were compared to that for wild-type PTx S1-220 by using ANOVA and Tukey's test (α = 0.05; *, P ≤ 0.05; ****, P < 0.0001). (D) Representative flow cytometry histogram of A8 antibody binding to yeast display variants. The mean fluorescence intensities are shown next to the peaks.