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:
This is a new project, which was established in December 2015, and continues research from project 2030-42000-048-00D, "Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins." Progress was made on all four objectives. Under objective 1A, progress was made in the development of new monoclonal antibodies (mAbs) to botulinum neurotoxin (BoNT) serotype F. BoNT/F is one of the four BoNT subtypes known to cause human intoxication via food (albeit the least common). A type F assay is desirable to accompany detection assays already generated for subtypes A, B, and E. Non-toxic type-F fragments were cloned and expressed from genomic Clostridial DNA obtained from collaborators at the University of Wisconsin and were used to immunize mice and generate a monoclonal antibody based detection assay. Under objective 1B, progress was made to generate peptide antigens to all of the major types of botulinum neurotoxin accessory proteins. Monoclonal antibodies have now been generated and capture assays developed for select accessory proteins completed. Incorporation of these accessory protein antibodies into our toxin assays has not yet resulted in improved sensitivity. Cell fusion experiments to produce monoclonal antibodies to accessory proteins from serotype E have been completed. Under objective 1C, progress was made to develop in vitro assays for staphylococcal enterotoxin serotype E (SEE). Food poisoning caused by staphylococcal enterotoxins is among the leading causes of food-borne outbreaks. We developed an in vitro cell-based method for the detection of SEE toxin that has a sensitive detection limit of 1 fg/mL, which is 106 and 109 times more sensitive than a typical ELISA assay and the monkey and kitten bioassay, respectively. Under objective 1D, progress was made to develop antibodies to shiga toxin subtype variants from E. coli and non E. coli sources. Shiga toxin producing E. coli (STEC) are known to produce two subtypes of shiga toxins (Stxs), Stx 1 and Stx 2. Each type is further divided into subtypes, which have the potential to cause disease. New mAbs against Stx2b and polyclonal antibodies with broad reactivity to Stx1 were newly developed. Using these antibodies together with other Stx antibodies produced previously in our unit, a universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was developed. The ELISA assay was highly sensitive with a limit of detection for the different subtypes of Stx1 and Stx2 between 10 and 50 pg/mL in phosphate buffered saline (PBS). This new assay correctly indicated contamination with STEC by showing presence of Stxs in the culture fluids or even in single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterium. Shiga toxins produced from non-shiga toxin producing E. coli (STEC) type bacteria are also potential sources of foodborne contamination and subsequent intoxications. We developed new monoclonal antibodies against Stx1e, a new Stx1 toxin subtype produced by an Enterobacter cloacae strain isolated from a human clinical sample. Properties of Stx1e, such as the origin of the genes, conditions for toxin expression, receptor binding, and cytotoxicity, were also investigated with these new antibodies. Under objective 2, in collaboration with partners at the Department of Homeland Security (DHS), a collection of monoclonal antibodies to abrin has been obtained. These are being evaluated for their use in sensitive capture ELISA assays to detect abrin. Preliminary results indicate some antibody pairs will allow us to develop a detection method with a sensitivity of < 5 ng/mL. The detection capabilities of two commercially available abrin detection assays were also evaluated and compared to the in-house ELISA method. A commercial assay for alpha-amanitin toxins is also being evaluated. We also participated in inter-laboratory studies to evaluate the efficacy of lateral flow assays in the detection of abrin and Bacillus anthracis. Under objective 3A, an in vitro cell free translation assay was tested for use in the evaluation of abrin activity. The stability of abrin after treatment with different temperatures and pH were determined. Under objective 3B, the activity of BoNT, when in the presence or absence of neurotoxin associated proteins (NAPs), was evaluated using mouse bioassays. BoNT are secreted from bacteria together with a number of NAPs. Thus far, two different types of NAPs have been identified: hemaglutinin (HA) type proteins and non-HA complex proteins. We compared the toxicity of BoNT serotype E toxins in the presence or absence of NAPs in both in vitro cell-based assays and animal bioassays. Crude BoNT/E containing complex proteins were 20 times more toxic than BoNT/E holotoxin or purified BoNT/E complex (containing BoNT/E and a non-toxin non hemaglutinin protein) alone in the oral mouse bioassay suggesting that other protein component may play a role in BoNT/E toxicity. In collaboration with scientists at the University of California, Irvine, the role that individual or groups of BoNT/E NAPs play in toxicity was explored using mouse oral bioassays. Under objective 4, we developed a liquid chromatography/mass spectrometry (LC/MS) method to detect shiga toxins. 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 monoclonal antibodies (mAbs) to a new shiga toxin (Stx) subtype from a non-E. coli strain. Novel shiga toxins produced from non-E. coli bacteria are potential sources of foodborne illnesses but little is known about these toxins and no reagents are available for their detection. ARS researchers in Albany, California developed four new monoclonal antibodies against Stx1e, a new Stx1 subtype produced by an Enterobacter cloacae strain isolated from a clinical specimen. Enzyme-linked immunosorbent assay (ELISA) assays developed using these antibodies detected as little as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. Researchers also investigated the origin of the virulence genes; the conditions best suited for its production, its cytotoxicity, and identified the host receptor for this toxin. This sensitive detection assay for Stx1e-harboring bacteria will allow the identification of food contamination sources, can be used in disease diagnosis, and will help further understanding of the clinical significance and prevalence of new disease causing bacteria.
2. Development of a sensitive detection assay that can identify all known subtypes of shiga toxins. Shiga toxin producing E. coli (STEC) are known to produce two subtypes of shiga toxins (Stx 1 and 2), which are further classified into many subtypes. However, there is little information on these subtypes and few, if any reagents are available for use in their detection. ARS scientists in Albany, California developed a novel universal sandwich Enzyme-linked immunosorbent assay (ELISA) capable of detecting all known subtypes of Stx1 and Stx2 (including Stx1a, 1c, 1d, and Stx2a through 2g). The ELISA assay was highly sensitive, with a limit of detection of 10 to 50 pg/mL in phosphate buffered saline, was able to correctly indicate STEC contamination in culture fluids, or even from single bacterial colonies on agar plates without lengthy enrichment in liquid medium, and can identify ground beef contamination by a single bacterium. This research could have great impact on the fast and sensitive detection of all known shiga toxins in food processing or clinical settings.
3. Development of an in vitro method as an alternative to animal bioassays for the detection of staphylococcal enterotoxin type E (SEE) toxins. Food poisoning caused by staphylococcal enterotoxins is among the leading causes of food-borne outbreaks. The current method for detection of enterotoxins activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals and is impractical to test on a large number of samples. ARS researchers in Albany, California developed a robust cell based assay that combines the use of genetically engineered T cell-line expressing the luciferase reporter gene with a B-cell line that presents the toxin to the engineered T cell line. Exposure of the above mixed culture to active staphylococcal enterotoxin type E concentrations leads to measurable bioluminescence in a dose dependent manner. As little as 1 fg/mL of active SEE, which is 106 times more sensitive than a typical ELISA assay and 109 times more sensitive than the monkey and kitten bioassay can be detected making this new method an economical and effective alternative to current detection standards.
5. Significant Activities that Support Special Target Populations:
Rasooly, R., Do, P.M., Hernlem, B.J. 2016. Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1. Journal of Agricultural and Food Chemistry. 80:405-410.
Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X. 2015. Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum. Toxins. 7:5236-5253.
Babrak, L.M., Lin, A.V., Stanker, L.H., McGarvey, J.A., Hnasko, R.M. 2016. Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera. Toxins. 8(1):13.
Skinner, C.B., Patfield, S.A., Khalil, R., Kong, Q., He, X. 2016. New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum. mSphere. 1(1):e00099-15.
Khalil, R., Skinner, C.B., Patfield, S.A., He, X. 2016. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains. Pathogens and Disease. 74(5):1-11. doi: 10.1093/femspd/ftw037.
Carter, M.Q., Quinones, B., He, X., Louie, J.W., Zhong, W.W., Lee, B.G., Yambao, J.C., Mandrell, R.E., Cooley, M.B. 2015. An environmental shiga toxin-producing Escherichia coli O145 clonal population exhibits high-level phenotypic variation that includes virulence traits. Applied and Environmental Microbiology. doi: 10.1128/AEM.03172-15.