Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 18, 2010
Publication Date: June 10, 2010
Citation: Scotcher, M.C., Cheng, L.W., Stanker, L.H. 2010. Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and its Application to Toxin Detection in Milk. PLos One. 5(6):e11047.doi:10.1371/journal.pone.0011047. Interpretive Summary: Botulism is a serious, often fatal neuroparalytic foodborne disease in humans and animals caused by a toxin produced by the bacterium Clostridium botulinum. Botulinum neurotoxin is considered the most toxic biological agent known and it is classified as a class ‘A’ bioterrorism agent. Seven different serotypes (A-G) of toxin are known. The majority of human cases of botulisum are due to serotypes A and B. The "gold standar" for detection of botulinum toxin in food is a time consuming rodent bioassay. A faster, non animal-based test is needed. In a previous report we described the development of such a test, that is even more sensitive than the mouse bioassay. This faster sandwich immunoassay, developed in the Foodborne Contaminants Research Unit (FCRU), Albany CA, uses monoclonal antibodies and is specific to toxin serotype A. Here we report the development of a comparable assay, also based on monoclonal antibodies but specific for the second most common serotype, botulinum neurotoxin serotype B, and its application for toxin detection in milk. Our ability to rapidly screen milk and other foods for the two most common forms of botulinum neurotoxin is enhanced by the development of these immunoassays and contributes to the safety of the food supply.
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.