Figure 3.

Specificity of mAbs and associated antibody fragments to single-motif exopolysaccharide and self-antigens. Summary of indirect ELISA measurements of relative binding of each antibody fragment for 18B7, 2H1, and 3E5 antibody families to exopolysaccharide from four single motif strains and one mixed-motif strain (H99) are presented with respect to antibody fragment class. Each plate was coated with EPS at a concentration of 1 μg/ml. A, left panel: mAb binding to EPS from different strains at 5 μg/ml ([mAb] = 34 nM, [Fv] = 69 nM). Middle panel: Fab binding to EPS from different strains at 3.32 μg/ml ([Fab] = 69 nM, [Fv] = 69 nM). Right panel: scFv binding to EPS from different strains at 1.97 μg/ml ([scFv] = 69 nM, [Fv] = 69 nM). Absorbance values for 18B7, 2H1 and 3E5 antibody fragments are displayed as black, gray, and silver bars, respectively. For each fragment classification, binding to EPS isolated from each strain are displayed on the same graph. All A₄₀₅ values are normalized to the values obtained from a control reaction in each experiment on each plate: mAb 18B7 binding to 5 μg/ml H99 EPS. B, summary heat map of the data in A–C of each antibody fragment at 69 nM Fv is displayed to illustrate the differences in the magnitude of binding between each fragment class. C and D, reactivity of mAb, Fab, and scFv 18B7 toward a panel of self-antigens relative to binding to native GXM antigen. Binding profiles of each antibody fragment are displayed in black, red, green, blue, purple and pink for GXM, actin, tubulin, thyroglobulin, double-stranded DNA, and single-strained DNA antigens, respectively. Data represent the mean ± SD of two replicate measurements. Heatmap summaries of the polyspecificity binding data are displayed for the highest concentration of antibody or no dilution of Ag. C, antibody concentrations were held constant at 69.4 nM Fv (5 μg/ml mAb) and the antigen was serially diluted two-fold. D, antigen concentrations were held constant and antibody concentrations were serially diluted two-fold from an initial concentration of 34.7 nm Fv (2.5 μg/ml mAb).