Development of Detection Technologies for Bacterial Neurotoxins and Their Validation in Food Matrices
Foodborne Toxin Detection and Prevention
2013 Annual Report
1a.Objectives (from AD-416):
The objectives of this cooperative research project are to determine the dosage
response of mice to pure and crude toxin preparations and to develop monoclonal
antibodies against botulinum neurotoxins and associated proteins for use in
detection or diagnostic technologies.
1b.Approach (from AD-416):
There are seven serotypes of BoNT (BoNT/A to G) and, among them, 32 known subtypes of BoNT. Amino acids of subtypes from each serotype can differ greatly. It is therefore important and a challenge to develop monoclonal antibodies (mAbs) that recognize a wide range of serotypes and their subtypes for use in detection or diagnostics assays. BoNTs are also secreted from the bacterium in association with other proteins, forming large complexes that have been shown to be important for oral intoxication. Different serotypes and even subtypes of toxins can form different types and sizes of toxin complexed. Little information is available on the toxicities of different serotypes and subtypes important in oral intoxications. We plan to determine and compare the toxicities and bioavailability of different BoNT serotypes and their subtypes in mouse systemic and oral models of intoxication in both buffer and in the presence of complex food matrices. Furthermore, we will develop mAbs against BoNTs and their associated proteins and use them to develop highly sensitive detection assays. We will further characterize mAbs for epitope binding sites, binding affinity, specificity and cross reactivity, and in in vivo toxin neutralization assays.
Cooperator collaborates on botulinum toxin assay development and provides critical crude and purified botulinum neurotoxin reagents to ARS scientists at Albany, California. These services are valuable and cost-effective. All material shipments were documented and contents confirmed upon arrival. The Cooperator and ARS scientists in Albany, California, have co-authored several presentations and a paper on characterization of high-affinity monoclonal antibodies to botulinum neurotoxins. Collaborator provided crude BoNT/E toxins for oral bioavailability studies that yielded important clues that non-traditional toxin-associated proteins (hemagglutinin proteins) were also important in oral toxicity. Collaborator also provided genomic DNA for the production of recombinant proteins used to develop monoclonal antibodies against BoNT/E and BoNT/F. Progress reported addresses objectives 1: "Develop new assays for bacterial toxins and their variants, using immunological and other methods, with emphasis on applicability to practical problems facing the food industry and regulatory agencies" and 2: "Calibrate in vitro methodology against established animal bioassays, and develop new data on the bioavailability of toxins, the impact of food processing on toxin activities, and the significance of antibody-mediated clearance on toxicity, especially via the oral route of intoxication" of the parent project.