Concentration dependence curves for the neutralization of protective antigen by MAbs AVR1046 and F20G75 in the J774A.1 and CHO cell-based assays. The indicated concentrations of MAb AVR1046 and MAb F20G75 were used to neutralize a constant concentration of either LT in the J774A.1 cell-based assay (A and C) or ET in the CHO cell-based assay (B). In panel C, FcγRIIB/III receptors were blocked by the addition of MAb 2.4G2 as described in Materials and Methods. Each point corresponds to the mean of the values obtained for three independent sample preparations, with the standard deviation (SD) indicated by the error bar. The samples were run on duplicate plates for the J774.1A cell-based assay and on a single plate for the CHO cell-based assay. Each curve is representative of three independent assays run on different days.

Concentration dependence curves for the neutralization of protective antigen by MAbs 2F9 and C3 in the J774A.1 and CHO cell-based assays. The indicated concentrations of MAb 2F9 and MAb C3 were used to neutralize a constant concentration of either LT in the J774A.1 cell-based assay (A and C) or ET in the CHO cell-based assay (B). In panel C, FcγRIIB/III receptors were blocked by the addition of MAb 2.4G2 as described in Materials and Methods. Each point corresponds to the mean of the values obtained for three independent sample preparations, with the SD indicated by the error bar. The samples were run on duplicate plates for the J774.1A cell-based assay and on a single plate for the CHO cell-based assay. Each curve is representative of three independent assays each run on different days.

Concentration dependence curves for toxin neutralization by the combination of MAbs AVR1046 and F20G75. The concentration of one antibody was varied in the manner indicated on the x axis, while that of the other was held constant. Cell viability is indicated for concentrations of the serially diluted antibody assayed either individually or combined with the other MAb held constant at the concentration indicated. Neutralization obtained with the antibody that was held constant in the absence of the serially diluted antibody is indicated on the y axis. In panel B, assays were conducted in the presence of the Fcγ receptor-blocking antibody MAb 2.4G2 as described in Materials and Methods. Each point corresponds to the mean of the values obtained for three independent sample preparations run on the same plate, with the SD indicated by the error bar. For each independent assay, the samples were run on duplicate plates. Each curve is representative of independent assays each run on at least three different days.

Concentration dependence curves for toxin neutralization of protective antigen by the combination of MAbs AVR1046 and 2F9. The concentration of one antibody was varied in the manner indicated on the x axis, while that of the other was held constant. Cell viability is indicated for concentrations of the serially diluted antibody assayed either individually or combined with the other MAb held constant at the concentration indicated. Neutralization obtained with the antibody that was held constant, in the absence of the serially diluted antibody, is indicated on the y axis. Each point corresponds to the mean of the values obtained for three independent sample preparations, with the SD indicated by the error bar. For each independent assay, samples were run on duplicate plates. Each curve is representative of three independent assays each run on different days.

Concentration dependence curves for toxin neutralization by the combination of MAbs C3 and 2F9. The concentration of one antibody was varied in the manner indicated on the x axis, while that of the other was held constant. Cell viability in the J774A.1 cell-based assay (A and B) or % RLU in the CHO cell-based assay (C and D) is indicated for concentrations of the serially diluted antibody assayed either individually or combined with the other MAb held constant at the concentration indicated. Neutralization obtained with the antibody that was held constant, in the absence of the serially diluted antibody, is indicated on the y axis. Each point corresponds to the mean of the values obtained for three independent sample preparations, with the SD indicated by the error bar. For each independent assay, samples were run on duplicate plates. Each curve is representative of three independent assays each run on different days.

Analysis of MAb AVR1046 binding in the presence or absence of MAb 2F9 using a competitive PA ELISA with soluble PA as the competitor. The indicated concentrations of soluble PA mixed with either biotinylated AVR1046 IgG (B-AVR1046) in the absence and presence of 2F9 (A) or B-AVR1046 Fab fragments in the absence and in the presence of 2F9 (B) were assayed for binding using the competitive ELISA format described in Materials and Methods. The ability of soluble PA to inhibit binding of B-AVR1046 or B-AVR1046 Fab fragments to the PA coating the plate was determined. The OD₄₀₅ readings for each competition curve were normalized to the OD₄₀₅ of its upper asymptote, set as 100%, and the data points were then fitted using a 4-PL regression model. Each point corresponds to the mean of the values obtained for three independent sample preparations run on the same plate, with the SD indicated by the error bar. Each figure is representative of the three independent assays, each run on different days.