DEVELOPMENT OF DETECTION TECHNOLOGIES FOR TOXINS AND THEIR VALIDATION IN FOOD MATRICES
Location: Foodborne Contaminants Research
Project Number: 5325-42000-043-00
Start Date: Dec 28, 2005
End Date: Dec 27, 2010
Determine enteric dose-response relationships for crude preparations of botulinum neurotoxin (BoNT) and ricin in rodent models and document the pathophysiological, immunological, and histological responses, as well as potential toxin synergies. Optimize sample preparation for recovery of toxins from food. Develop rapid immunological, biochemical, and/or molecular biological tests for botulinum toxin and ricin or appropriate surrogates. The results should provide both basic and applied knowledge useful in countering intentional biothreats, including an understanding of the effects of complex food matrixes on the toxicity of BoNT and ricin following exposure by ingestion and new analytical technology to detect these biothreat toxins in foods.
Toxicity studies will be performed to better define the safety and security problems and help to define the analytical needs. The stability of pure and crude toxins in food matrices will be determined, and dose-response relationships will then be established for enteric exposure to pure and crude toxins in three food matrices (raw milk, liquid eggs, and ground beef). Oral administration of toxins will include feeding and gavage. The acute toxicity as well as the histopathology of intoxication will be studied. Milk, liquid eggs, and ground beef are the three foods of primary interest in this project, primarily because they comprise major commodities processed in large batches via a highly decentralized system. Immune responses will be characterized with regard to specificity for A chain, B chain, hemagglutinins, and other components of crude toxins. We will determine whether these antibodies are protective by challenging immunized mice and naïve controls with toxin. Sample preparation technology for toxins of interest that is compatible with real-time and multi-analyte testing of large numbers of samples will be developed. Extraction and capture procedures that extract toxin(s)or marker(s)from the matrices, concentrate the analytes, and remove most of the impurities that would otherwise interfere with the assay will be developed. These will include techniques that could be used in a field setting, e.g., a mobile laboratory or "black box" assay machine. These sample preparation methods must be simple and extremely robust; immunoaffinity capture is an example of a possible methodology. We will determine matrix effects for existing assays and use this information as a starting point for developing preparative techniques applicable to these assays as well as new techniques developed in this project. Assay protocols will be validated using food spiked with active toxin. The best methods identified for each toxin-food combination will be compared to the mouse bioassay. All sample preparation procedures will be characterized for throughput and robustness, as well as their impact on the accuracy, precision, sensitivity, and dynamic range of assays for which they are used. New, rapid tests for BoNT and ricin that can be used to test a variety of food samples will be developed utilizing multiple methodologies. Monoclonal antibody ligands, nanoparticle labels, and immunosensor techniques offer the possibility of ultrasensitive assays. An additional analytical approach will be to develop biochemical and/or cellular assays that could possibly provide even greater biological relevance.
FY01 program increase $269,370. 1 SY added. Bridging project replacing 5325-42000-027-00D (Feb. 05) FY05 Prog. Inc. $400,000. Add 1 SY. Formerly 5325-42000-042-00D (11/05). Combining 5325-32000-006-00D (1/09)