1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Under Sub-objective 1A, following immunization and cell fusion experiments with recombinant derived peptide fragments of botulinum neurotoxin serotype F (BoNT/F), nine monoclonal antibodies (mAbs) were isolated. The binding domain of each mAb is localized to the carboxyl terminal domain of the toxin (N1087—N1278). Only a single mAb of this group was observed to capture BoNT/F in solution. Mice were also immunized with an inactivated BoNT/FA (new serotype). However, several screens for antibodies did not yield usable monoclonal antibodies specific for BoNT/FA. Under Sub-objective 1B, peptide antigens for nontoxic associated proteins of BoNT/A, /B, and /E were generated and used to generate monoclonal antibodies. Studies using a newly developed monoclonal antibody to BoNT/A NAP HA 70 did not demonstrate improved detection of BoNTs. Monoclonal antibodies to all of the neurotoxin associated proteins (NAPs) of BoNT serotype A have been developed and will aid in new detection schemes. Under Sub-objective 1C, progress was made to develop activity-based detection methods for staphylococcal enterotoxin serotype E (SEE). SEE is one of the leading causes of foodborne illness outbreaks. Researchers developed and evaluated a luminometry assay for SEE using a bioluminescence imaging system developed in-house and a genetically engineered T lymphocyte (T-cell) line that produces light when exposed to active SEE. The imaging system and assay were capable of measuring active SEE over an 8-log range of SEE concentrations from 100 femtogram/milliliter to 1000 nanogram/milliliter. Shiga toxin-producing E. coli (STEC) are known to produce two types of Shiga toxins (Stxs), Stx 1 and Stx 2. Each type is further divided into subtypes, which differ in receptor preference and toxin potency. Under Sub-objective 1D, progress was made to identify antibodies and immunoassays that detect new subtypes of Stx2h and Stx2k produced by E. coli strains STEC299 and STEC388 isolated from wild marmots and pigs in the Qinghai-Tibetan plateau by collaborators in the Chinese Centers for Disease Control and Prevention (CDC). These antibodies and immunoassays are useful for epidemiological surveillance of STEC infections. Under Sub-objective 2A, progress was made to develop toxoids for abrin isoforms a through d. Abrin toxin is a select agent, known to have the potential to pose a severe threat to both human and animal health. Four isoforms of abrin, abrin-a, -b, -c, and –d have been identified from the seeds of the plant, Abrus precatorius. These isotoxins have similar amino acid composition, but very different cytotoxicity. Currently, there are no standards available for each abrin isotoxin. These toxoids are useful resources for abrin studies and for the future development of specific antibodies. Under Sub-objective 2B, both alpha and beta amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. The presence of toxin was confirmed using spectral scans, mass spectrometry, and commercial antisera. Polyclonal antibodies useful for development of a competitive enzyme-linked immunoassay (cELISA) were observed using two of the four linkage strategies. These efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays. ELISA detected the alpha, beta, and gamma forms of amanitins. The newly developed ELISA were more sensitive than current methods such as using high performance liquid chromatography (HPLC), and were able to detect the presence of amanitins in mushrooms previously thought to be toxin free. Production of new monoclonal antibodies to amanitin, the mushroom toxin found in death cap mushrooms, is in progress. Rabbit polyclonal antibodies to amanitins were also incorporated into rapid lateral flow (LFA) or dipstick assays but were not sensitive enough to be usable. Under Objective 3, the bioavailability of abrin toxin treated with a range of pH, in different foods and food processing temperatures was assessed using in vitro assays for activity (Vero cell and the cell free translation assays) and the mouse bioassay. In food matrices, common times and temperatures used for food processing are not sufficient to inactivate abrin. Under Sub-objective 4A, progress was made on the development of flow cytometric methods for the detection of active staphylococcal enterotoxin serotype E (SEE). Assays were developed using human B- and T-cell lines, the latter cells responding to active SEE by secretion of the cytokine Interleukin-2 (IL-2) and by reduced surface expression of the T-cell receptor (TCR) protein Vß8. IL-2 secretion by T-cells is a specific response to SEE as compared with subtypes SEA and SEB. Further, IL-2 secretion by Jurkat T-cell line exhibits a dose-response relationship to SEE concentrations as low as one picogram/milliliter. The assay based on reduced Vß8 expression exhibited SEE dose dependence and had a detection limit below one femtogram/milliliter, a billion times more sensitive than live animal testing for active SEE. A new PathSensor Canary Zephyr portable biosensor detection system was also evaluated for its effectiveness in the detection of BoNT/A. The assay is rapid (40 minutes), requires minimal processing, and can detect as little as nanogram (ng) or lower levels of BoNT/A in different complex food matrices. Under Sub-objective 4B, a liquid chromatography/mass spectrometry (LC/MS) method to detect shiga toxins was developed. This method detected as low as 10 attomoles of toxin in a human serum matrix, illustrating its potential use in clinical settings.
1. Development of new sensitive detection assays for shiga toxins in human serum. Shiga toxins (Stxs) are the major virulence factors associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by Escherichia coli. Presence of Stx in human sera is a risk indicator for HUS development. However, detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. ARS researchers from Albany, California, improved Stx detection in serum by treating samples with guanidinium chloride. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using samples from STEC-infected patients. This new method should be useful for the early detection of Stx2 in human serum, thereby preventing severe complications associated with HUS.
2. Development of a sensitive detection assay for staphylococcus enterotoxin serotype E. Staphylococcal enterotoxin serotype E (SEE) is one of the leading causes of clinical infections and foodborne illnesses. Traditional methods for detection use live animals such as kittens, and other antibody-based methods do not distinguish active or inactive toxins. Researchers in Albany, California, identified interleukin 2 production by T lymphocyte (T-cells) as a specific biological marker for the quantitative detection of SEE. The new assay can detect SEE concentrations in very low amounts. Rapid and sensitive detection of SEE would lead to improved food safety.
3. Development of sensitive detection assays for abrin toxin. Abrin is a Select Agent toxin and a potential bioterror weapon. Researchers in Albany, California, developed new monoclonal antibodies against abrin and assembled sandwich enzyme-linked immunosorbent assays (ELISA) capable of detecting a mixture of abrin isoforms. The ELISA can detect as little as 1 nanogram/milliliter of the abrin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. Some of these antibodies can also neutralize abrin toxicity in cell-based assays. Improved methods of detection for abrin toxins would be useful during incidences of deliberate or suspected food contamination.
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