Detection of botulinum neurotoxin serotype B at sub mouse LD(50) levels by a sandwich immunoassay and its application to toxin detection in milk.

Detection of botulinum neurotoxin serotype B at sub mouse LD(50) levels by a sandwich immunoassay and its application to toxin detection in milk.

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Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H).We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified.

These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs).Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture Gym Mats 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 of BoNT/B heavy chain.

MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B BLOOD SUGAR SUPPORT in solution under physiological conditions.MAbs 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 that did not require SDS.We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay.

This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B.Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.