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Development of Detection Technologies for Toxins and Their Validation in Food Matrices


<oL> <LI> 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. <LI>
Optimize sample preparation for recovery of toxins from food. <LI>Develop rapid
immunological, biochemical, and/or molecular biological tests for botulinum toxin and
ricin or appropriate surrogates. </ol> 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.

More information

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 naive 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. <p>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. <p>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-<P>
027-00D (Feb. 05) FY05 Prog. Inc. %400,000. Add 1 SY. Formerly 5325-42000-042-00D

Wong, Rosalind; Stanker, Larry; Rasooly, Reuven; Hernlem, Bradley; Cheng, Luisa Wai Wai; Carter, John Mark ; Brandon, David
USDA - Agricultural Research Service
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