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<p>Provide toxicological data and analytical methodology for microbial toxins that will help ensure a safe food supply.</p>
<p>(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. Develop new monoclonal antibody (mAb)-based assays for botulinum neurotoxins (BoNTs), non-toxic neurotoxin-associated proteins, and Shiga toxins (Stx), and optimize antibodies for biosensor applications. Develop methodology for detection of Shiga toxin-producing E. coli (STEC) and a multiplex bead-array assay for detecting Stx and STEC pathogenicity/virulence factors. Develop improved activity assay for staphylococcal enterotoxins.</p>
<p>(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. Determine the bioavailability of different botulinum neurotoxin serotypes. Validate new toxin assays using activity assays.</p>
Approach:
For the first objective, the general approaches are to exploit immunoassays, especially enzyme-linked immunosorbent assay (ELISA), immuno-polymerase chain reaction (iPCR), and bead array assays because of their versatility, robustness, and sensitivity; and to develop activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation and potential for diagnostic/therapeutic applications. Development of new toxin-specific mAbs will exploit a variety of immunogens, including toxoids and recombinant polypeptide chains corresponding to different domains of the toxin chains. Methodologies to optimize antibodies include the use of flow cytometry to test and select hybridoma cell lines. Structurally different antibodies such as IgY and single-chain antibodies will also be developed and compared to mAbs. Optimal capture/detection antibody pairs will be identified using ELISA and assay performance will be investigated with respect to robustness. Selected capture antibodies will be coupled to immunomagnetic beads for use in sample preparation. Assays will be evaluated in the food matrices of principal interest: milk, juices, liquid eggs, and ground meat and poultry products. For samples that produce a high background signal, matrix interference, or poor recovery, simple preparative methods will be tested, such as differential centrifugation, filtration, or immunomagnetic bead separations. Similar methodology will be used to develop antibodies and assays for accessory proteins found in toxin preparations as secreted by bacteria. Activity assays report active toxin. They will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are found in processed food samples. Assays that measure the activity of selected toxins (such as Stx and staphyloccocal enterotoxins) will utilize existing and new cell lines that are sensitive to active toxin. Suitable readout systems include cell lines that produce reporter molecules in response to toxin. For the second objective, the general approach is to relate variations in toxin structure to toxicity, bioavailability, and responses in detection systems. Bioassays and cell-based assays will be used to assess the impact of food processing on toxin activity and bioavailability. Dose-response and bioavailability will be determined for BoNT holotoxins and toxin complexes. We will determine the effect of accessory proteins on toxicity. The transit time for passage through the intestinal epithelium will be determined for holotoxin and toxin complexes in a model system, polarized colonic epithelial translocation assay. The protective effect of newly developed antibodies will be determined for various BoNTs. Assay validation is based on side-by-side comparison of samples in different assay systems. Active toxin concentration will be estimated by biochemical and cellular assays. Bioassays will be used for assessment of toxicity of unknown concentrations of toxins for comparison with in vitro assays, especially for toxin in raw and processed food matrices.