detection of botulinum neurotoxin serotype b at sub mouse ld50 levels by a sandwich immunoassay and its application to toxin detection in milk检测肉毒神经毒素b在子鼠标ld50水平的血清型夹心免疫测定牛奶中及其毒素检测中的应用.pdfVIP

Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk Miles C. Scotcher, Luisa W. Cheng, Larry H. Stanker* Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California, United States of America Abstract Background: 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. Methods and Findings: 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 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. Conclusio