Project Number: 2030-42000-053-00-D
Project Type: In-House Appropriated
Start Date: Dec 28, 2020
End Date: Sep 30, 2021
The overall objective of this project is to advance the development of technologies for detecting toxins that impact food safety and food defense and to determine their stability and bioavailability. Specifically, the project will focus on the following four objectives: Objective 1: Advance the development of structure- and activity-based detection methods for protein toxins. Subobjective 1A: Develop new antibodies (Abs) to botulinum neurotoxin (BoNT) serotype F, with serotypes C, D, and G as secondary priorities. Subobjective 1B: Determine the impact of different types of accessory proteins on the detection of BoNTs. Subobjective 1C: Develop activity-based detection methods for staphylococcal enterotoxin (SE) serotype E. Subobjective 1D: Develop monoclonal antibodies (mAbs) to Shiga toxin (Stx) subtypes and variants, including those from non-E. coli. Objective 2: Advance the development of detection methods for non-bacterial toxins. Subobjective 2A: Develop new detection methods for plant-derived protein toxins such as abrin. Subobjective 2B: Develop new detection methods for mushroom toxins such as amatoxins. Objective 3: Assess foodborne risks through examination of toxin stability and bioavailability in relation to intrinsic and extrinsic stresses. Subobjective 3A: Use activity-based assays to assess impact of food processing, matrices and accessory proteins on toxin activity. Subobjective 3B: Determine the factors that affect the bioavailability of toxins using rodent bioassay. Objective 4: Advance the development of instrumental, portable, and field-deployable testing methods. Subobjective 4A: Develop platforms such as optical array technologies to detect toxins. Subobjective 4B: Utilize instrumental methods to detect toxins based on mass spectra and/or other physicochemical characteristics.
Objective 1 has 2 general approaches: (1) Exploit ELISA and related technologies because of their versatility, robustness, and sensitivity. The mAbs developed for immunoassay will also be useful for sample preparation and for establishing design criteria for protective antibodies with clinical utility. (2) Develop activity assays for Stx2 variants and SEs. Activity assays will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are expected in processed food samples. Both approaches address practical analytical problems. The following hypotheses will be tested:(a) High affinity mAbs and recombinant Abs for BoNT serotypes and NAPs as analytical targets will provide useful reagents for ELISA and sensor methodology. (b) Neurotoxin-associated proteins (NAPs) influence the physicochemical properties of BoNT complexes, and their ease of detection in food matrices. These effects depend on the types of accessory proteins present. (c) A cell line can be engineered to provide a cell-based activity assay for SEE to measure active toxin in food matrices to replace bioassay and improve upon structure-based immunoassay. (d) New mAbs will be able to distinguish new subtypes of Stx1 or Stx2 produced by non-E. coli bacteria such as Enterobacter cloacae. Objective 2 will exploit immunoassays, especially ELISA and related technologies (as in Objective 1) and also develop and utilize activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation. The following hypotheses will be tested: (a) High affinity variant-specific mAbs will provide useful reagents for ELISA and sensor methodology for detecting nonbacterial protein toxins like abrin. (b) High affinity toxin-specific and group-specific mAbs will provide useful reagents for ELISA and sensor methodology for mushroom toxins. Objective 3 will exploit activity assays and rodent bioassay to better define the vulnerabilities of our food supply and the analytical needs. The following hypotheses will be tested: (a) Food processing conditions, food matrices, and accessory proteins impact toxin activity. (b) The oral bioavailabilities of BoNT and abrin vary among subtypes of toxin and state of the toxin (pure, complexed, crude) and depend on the food matrix. Objective 4 will exploit instrumental and portable technologies for toxin detection. Some of these technologies will will utilize binding molecules and activity assays developed under Objectives 1 and 2. To advance the development of instrumental and field-deployable testing, the following hypotheses will be tested: (a) A robust cell-based activity assay for SEE in a food matrix can be developed using a small fluorescence-detecting charge-coupled device to read data. (b) Mass spectral data and other physicochemical properties are useful for detection of toxin proteins and peptides. Contingency plans are built into the Approach for each objective and sub-objective. For example, contingencies for Objective 1 include use of alternative immunogens, sample preparation strategies, and assay formats.