1a. Objectives (from AD-416):
Provide toxicological data and analytical methodology for microbial toxins that will help ensure a safe food supply. (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. (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.
1b. Approach (from AD-416):
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.
3. Progress Report:
New high-affinity monoclonal antibodies (mAbs) were developed for sensitive detection of botulinum neurotoxins (BoNTs), including those with specificities for BoNT serotype E and non-toxic, neurotoxin-associated proteins (NAPs). Enzyme-linked immunosorbent assays (ELISAs) and immunosorbent assays using electrochemiluminescence (ECL) improved detection sensitivity in complex biological and food matrices for serotypes and complexes found in naturally contaminated samples. A new handheld immunochromatographic device was prepared using mAbs developed by the project and was effective in measuring BoNT/A and B spiked into simple matrices such as milk. Shiga toxin-producing Escherichia coli (STEC) cause disease by producing Shiga toxins 1 and 2 (Stx1 and 2) and other virulence factors. STEC are detected on the basis of their O-antigens as well as the toxins sercreted, generally necessitating multiple assays. We investigated a new, automated, bead-based chemiluminescence (CL) ELISA, in which up to 10 analytes can be measured within a single well. Data showed that this system can be used to determine the pathogenicity and serotype of E. coli isolates. Other studies led to the development of a highly selective culture method for STEC that exploits a medium that enhances production of Stx by STEC. The next step is co-culture with the protozoan Tetrahymena thermophila that is killed selectively when it ingests STEC. This system could potentially be combined into a single, biphasic medium for off-the-shelf use. New mAbs were developed for Shiga toxin 2 (Stx2), a toxin belonging to the type II group of ribosome-inactivating proteins (RIPs) that include biothreat toxins such as ricin. Assay systems for these RIPs were extended, with detection by CL and ECL that exploited mAbs developed by the project team. Mouse mAb and other antibodies were studied using a dip-and-read optical sensor system that measures the thickness of the bound molecular layer by the interference of reflected light beams. Other studies were directed toward understanding the etiology of intoxication. This knowledge will help in the design of more effective diagnostic and therapeutic methods for foodborne diseases. Studies were conducted to characterize the intragastric toxicity of BoNT/B and E complexes and the ability of antibodies to protect against botulism resulting from ingestion of BoNTs. In a system that mimics the interaction of ricin and its cellular receptor, it was determined that milk inhibits the binding of ricin to receptors, an observation that may lead to development of defenses to intentional food contamination with this toxin. Studies of Staphylococcal enterotoxin A (SEA), a frequent cause of food poisoning, demonstrated that primary T cells from the spleen are induced by SEA to secrete a regulatory molecule, tumor necrosis factor (TNF). Combined with an antibody-based method to isolate SEA from food samples, the quantitative determination of TNF provides a specific measure of active SEA, with improved selectivity compared to conventional ELISA.
Lin, A., Nguyen, L., Lee, T., Clotilde, L.M., Kase, J.A., Son, I., Carter, J.M., Lauzon, C.R. 2011. Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array. Journal of Microbiological Methods. 87(1):105-110. doi:10.1016/j.mimet.2011.07.019.