Abstract

BackgroundBotulinum 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.Methods and FindingsRecombinant 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 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 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.ConclusionsWe 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.

Highlights

  • Foodborne botulism is a serious condition in which the patient experiences a gradual flaccid paralysis, 18 to 36 hours following consumption of contaminated food

  • We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay

  • We report the development of a new sandwich ELISA, capable of detecting Botulinum neurotoxin (BoNT)/B in buffer at concentrations undetectable by the mouse bioassay

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Summary

Introduction

Foodborne botulism is a serious condition in which the patient experiences a gradual flaccid paralysis, 18 to 36 hours following consumption of contaminated food. Botulinum neurotoxins (BoNTs) are the causative agents of botulism, and are the most potent naturally-occurring toxins known [3]. There are seven serotypes of BoNTs, designated A through G, with serotypes A, B, E and F most frequently associated with human cases of botulism [4]. BoNT/A is the most widely studied and best characterized of the BoNT serotypes - a cursory survey of the scientific literature indicates that there are approximately three times as many publications about BoNT/A than the most frequent serotype, BoNT/B. 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

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