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:
Progress reported relates to objectives 1 and 2. For the study and diagnosis of bacterial foodborne disease, it is essential to know which bacterial strains and toxins are present. New reagents were developed that permit detection of attomolar concentrations of botulinum neurotoxins (BoNTs) in animal blood after oral intoxication. Monoclonal antibodies (mAbs) to neurotoxin-associated proteins (NAPs) such as hemagglutinin-70 were produced and incorporated into rapid, sandwich enzyme-linked immunosorbent assays (ELISAs) for these components that are thought to affect the toxicity and bioavailability of BoNTs. Studies in mouse models of botulism demonstrated that components found in the crude bacterial lysates contributed to BoNT/E toxicity. The structure of the 14-subunit botulinum “progenitor toxin complex” was studied by x-ray crystallography and electron microscopy, revealing features that protect toxin from degradation in the intestine and facilitate its absorption. A new electrochemiluminescence assay outperformed ELISA in several food matrices and proved more sensitive than the mouse bioassay. With collaborators from University of Massachusetts and Sandia National Laboratory, a rapid, ultra-sensitive assay for BoNT was developed using a novel biosensor that purifies and measures the toxin in a single device. Shiga toxin (Stx)-producing E. coli (STEC) are responsible for serious foodborne disease. Analyses and diagnostic procedures are complex because there are multiple strains of STEC and several variants of Stx2. A simple sandwich ELISA using a conventional, polyclonal antibody was developed for sensitive detection of all subtypes of Stx2 thus far isolated from human and environmental samples. Monoclonal antibodies are generally advantageous for specific assays, and new mAbs that specifically bind Stx2 were used to develop an immunoassay for Stx2. The assay was effective in milk, and testing in other matrices is in progress. Some of the new mAbs blocked the toxic effects of Stx2 in mice. The study of protective antibodies may provide new information with clinical application. One of the unusual variants of Stx is serotype 2f (Stx2f), known to be associated with avian disease and suspected of causing some human disease. A purification scheme was devised for this unusual Stx variant. New mAbs to Stx2f were made and used to develop a highly sensitive immunoassay that will help elucidate the importance of this Shiga toxin variant in human infections. A bead-based antibody system developed last year correctly identified 78 of the 79 STEC strains tested. It was validated against 160 field isolates of STEC and compared to a molecular polymerase chain reaction assay developed by cooperators at Food and Drug Administration. In studies of staphylococcal enterotoxin A, monitoring cell surface markers in mouse CD4+ T-cells provided a convenient and rapid method to detect and quantify active toxin. The response can be used to detect toxin in contaminated samples of milk and other food matrices. The availability of these new antibodies and assays will enhance our ability to monitor for toxins in food, providing enhanced food safety and biosecurity.
1. Test for 7 Shiga toxin-producing E. coli (STEC) strains. Some types of E. coli bacteria produce disease in humans by releasing Shiga toxin. The seven most common STEC are known as O26, O45, O103, O111, O121, O145, and O157. Control of these bacteria is dependent on detecting them, and in the case of outbreaks it is very helpful to know which type is present. A test that determines which of the 7 most common types of E. coli are present developed by ARS scientists in Albany, California, was compared to a polymerase chain reaction-based assay developed by Food and Drug Administration. Both tests use a microbead format and were used to characterize a panel of 160 field isolates of STEC. This test facilitates and automates testing of foods possibly contaminated with STEC and extends our capability to improve food safety.
2. Rapid biochemical assays for 3 serotypes of botulinum neurotoxin. Although it is very sensitive, the mouse bioassay for botulinum neurotoxins (BoNTs) takes 4-7 days to complete, uses death as an end point, and is only is available in a few laboratories. A test is needed that specifically measures the activity of individual toxin serotypes in food and serum samples within a few hours. ARS scientists in Albany, California, cooperated with BioSentinel Pharmaceuticals Inc. (Madison, Wisconsin) to develop a set of rapid biochemical assays that measures activity of BoNT serotypes A, B, and F, 3 of the 4 serotypes associated with human disease. The test uses specific monoclonal antibodies and magnetic beads to purify the toxin complexes from food samples such as whole milk. These tests will further our ability to monitor for botulinum neurotoxins and improve the safety and biosecurity of the food supply.
3. Purification, characterization, and immunoassay of Shiga toxin 2f. Shiga toxin 2 (Stx2) subtype f is commonly found in avian species. Its role in human disease may be underestimated because it has been difficult to detect. ARS scientists in Albany, California, developed a purification scheme for this toxin and characterized its binding to cell surface glycolipids that serve as receptors. Stx2f-specific monoclonal antibodies (mAbs) were then generated that were specific for Stx2f. An antibody-based test was developed with a detection limit of 1 ng/mL Stx2f. This highly sensitive immunoassay will contribute to understanding the role of Stx2f in Shiga toxin-producing E. coli (STEC) disease in humans and animals.
4. New means of detecting staphylococcal enterotoxins. Staphylococcal enterotoxins (SEs) are produced by the bacterium S. aureus, and generate a superantigen immune response and gastrointestinal disease at low concentrations. ARS scientists in Albany, California, measured the responses of mouse immune cells exposed to S. Aureus and Staphylococcal Enterotoxins A (SEA) and S. Aureus and Staphylococcal Enterotoxins B (SEB) using a flow cytometer. This instrument detects the binding if specially labeled antibodies to specific cell surface molecules by means of a laser-excited fluorescent signal. Cell surface changes were observed within 6 hours of exposure to Staphylococcal Enterotoxins (SEs). This response provided the basis for a test for SEs that proved effective in spiked samples of milk and other food matrices. This rapid new method detects only active toxin and does so with exquisite sensitivity.
5. Reagents for biothreat detection. As part of an intense research effort to develop detection reagents, vaccines and therapeutics for biothreat agents, ARS scientists in Albany, California, developed high-affinity monoclonal antibodies (mAbs) for the sensitive detection of botulinum neurotoxins (BoNTs) and other toxins. New methods using electrochemiluminescence were developed. Some of the mAbs were also tested for their ability to provide therapeutic protection. The timing of antibody neutralization in animal models revealed windows of opportunity for antibody therapeutic treatment as well as information on the movement of BoNTs between blood and other tissues. Technology has been transferred to federal and state laboratories via cooperative work with the Department of Homeland Security, participation in the Pacific Southwest Regional Center of Excellence, and other agreements. These reagents better equip the nation for assuring food safety and biosecurity.
Singh, A.K., Stanker, L.H., Sharma, S.K. 2013. Botulinum neurotoxin: Where are we with detection technologies. Critical Reviews in Microbiology. 39(1):43-56. doi:10.3109/1040841X.2012.691457.