Human Anti-B. anthracis PA Recombinant Antibody (W2) (CAT#: HPAB-0748-YJ)

Figure 1 ELISA titration of anti–protective antigen (PA)

Recombinant PA or LF or unrelated proteins, bovine serum albumin (BSA), thyroglobulin, lysozyme, and phosphorylase b were used to coat the wells of an ELISA plate. Wells then were incubated with various dilutions of scFvs. Bound scFv was detected by the addition of peroxidase-conjugated anti-His antibody, followed by tetramethylbenzidine substrate. The anti-PA and antiLF scFvs did not bind to the unrelated proteins; only BSA is shown as an example. OD, optical density measured at 450 nm.

Chen, Z., Moayeri, M., Zhou, Y. H., Leppla, S., Emerson, S., Sebrell, A., ... & Purcell, R. (2006). Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. The Journal of infectious diseases, 193(5), 625-633.

Figure 2 In vitro neutralization assay.

Anti–protective antigen (PA) IgG was mixed with anthrax toxin and was incubated for 1 h at 37°C. The mixture was added to RAW264.7 cells in a 96-well plate and was incubated for 4 h at 37°C. After washing, the cells were stained with MTT dye, and then lysis was performed in a solution containing 0.5% SDS in 90% isopropanol and 0.05 mol/L HCl. The plate was read at an optical density measured at 570 nm, and the cell survival was calculated relative to untreated cells. Results were plotted and analyzed using Prism software (version 4; Graphpad Software). EC50, effective concentration for 50% neutralization. ■, W1; ▼, W2; □, 14B7.

Chen, Z., Moayeri, M., Zhou, Y. H., Leppla, S., Emerson, S., Sebrell, A., ... & Purcell, R. (2006). Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. The Journal of infectious diseases, 193(5), 625-633.

Figure 3 Competitive ELISA.

Recombinant protective antigen (PA) was coated onto the wells of an ELISA plate. Wells then were incubated with anti-PA W2 Fab at the concentrations indicated. After incubation for 1 h at room temperature, anti-PA W2 Fab was removed from the wells, and mouse anti-PA monoclonal antibodies (MAbs) 14B7 and 2D3 were added to the wells. Bound MAbs were detected by the addition of peroxidase-conjugated anti–mouse antibody, followed by tetramethylbenzidine substrate. The binding to PA was calculated by dividing the optical density value in the absence of W2 by the optical density value in the presence of W2. ■, 14B7; ▲, 2D3.

Chen, Z., Moayeri, M., Zhou, Y. H., Leppla, S., Emerson, S., Sebrell, A., ... & Purcell, R. (2006). Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. The Journal of infectious diseases, 193(5), 625-633.

Figure 4 Inhibition of the binding of protective antigen (PA) to RAW264.7 cells by preincubation of toxin with monoclonal antibodies.

PA at a concentration of 6 nmol/L (500 ng/mL) was incubated with antiPA 14B7 or W2 antibodies at a molar ratio of 1:1 or 1:10 for 5 min. The mixture was added to RAW264.7 cells and was incubated for 20 min at 37°C. After the cells were washed and lysed, separation by SDS-PAGE was performed. The proteins were transferred to a membrane and were probed with anti-PA polyclonal antibody.

Chen, Z., Moayeri, M., Zhou, Y. H., Leppla, S., Emerson, S., Sebrell, A., ... & Purcell, R. (2006). Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. The Journal of infectious diseases, 193(5), 625-633.