Technical Abstract: Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of BoNT/B were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA, bindinbg to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain, the carboxyl end of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. Additional mAbs, b- F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA which did not require SDS. This assay has a detection limit of 100 fg BoNT/B (6.6 fMolar), fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this sandwich ELISA detected as little as 39 pg/ml BoNT/B in skim, 2% and whole milk.