The binding activity of mAb was determined by ELISA (A) and Western blot analysis (B). Neutralizing mAbs were selected by in vivo neutralization assay(C). For the initial screening of the positive hybridoma that produced the neutralizing antibody, 200 µl of culture supernatant from hybridomas grown in a 24-well tissue culture flask for 3–5 days, was pre-incubated with 4 LD50 of BoNT/B for 1 hour at 37°C, and the reaction mixtures intraperitoneally injected to each of six mice. More than 4 survivals in four days were considered as positive. Serum of the immunized mice in the dilution of 1∶100 was set as control. Left: PBS, 1D12, 8D1, 8E10, 4E5, Serum. Middle: PBS, 2F4, 2H12, 2G11, 3F11, 2H7, Serum. Right: PBS, 5G10, 1F4, 2B2, 3D8, 3C1, 8H5, Serum. The error bars represented standard deviations.