1a. Objectives (from AD-416):
Assays for detecting low molecular weight toxins rely upon components that can interact with the toxins and facilitate toxin isolation or detection. Because of this, the goal of developing assays with better performance characteristics (such as sensitivity, speed, and robustness) requires the development of improved materials that bind the toxins. To meet this goal, we propose four objectives: Objective 1: Develop novel biologically-based materials that bind foodborne toxicants; Objective 2: Design and develop synthetic-based materials for detection of agricultural and food-related toxins; Objective 3: Develop computational methods for foodborne toxins that enable new strategies to reduce exposure; Objective 4: Develop detection strategies for emerging toxins and toxins of commercial importance.
1b. Approach (from AD-416):
Toxins produced by fungi, mycotoxins, can cause devastating economic effects by affecting the safety and marketability of grain, and by causing disease in livestock. Diagnosis of health problems caused by mycotoxins is often difficult because while certain of the toxins can cause acute disease, most cause sub-acute or chronic effects that are more difficult to discern. For these reasons, attempts are made to detect mycotoxins at the many stages from crop production to finished product. As a result, a vital part of mycotoxin control is the availability of rapid, accurate, sensitive, and cost effective methods for toxin detection and quantitation. Fortunately, many such methods exist and are commercially available. With a few exceptions, most of these methods rely, in some fashion, on components that bind the toxins. Ideally, the binding components are highly specific for the toxins of interest and are capable of performing under their expected conditions of use. As analytical technologies progress, assays are required to perform under increasingly demanding conditions, requiring advances in their component materials. We propose to apply the expertise of the investigators in synthetic chemistry, antibody development, immunoassay and sensor development, and instrumental assay methodology, toward the development of the next generation of toxin binding materials, such materials being the fundamental basis of improved analytical technologies for these toxins. By improving technologies for detecting natural toxins, this project will have a direct impact on the ability of producers, processors, and regulatory agencies to improve monitoring programs for natural toxins, and thereby improve the safety of the U.S. food supply.
3. Progress Report:
This is the final report for Project 5010-42000-045-00D, which has been replaced by new Project 5010-42000-049-00D. For additional information, see the new project report. The overall goals of the project were the development of new materials and methods for mycotoxin detection. The first Objective was to develop novel biologically-based materials that bind foodborne toxicants. To this end we developed several biologically-based binding materials for those mycotoxins that have caused recurring problems for U.S. agriculture, specifically certain trichothecene toxins: deoxynivalenol (DON) and T-2 toxin. We developed a recombinant antibody fragment (single chain variable fragment: scFv) that bound DON and retained the excellent selectivity of a monoclonal antibody. The advantage of the scFv is that it can be produced in bacterial cell culture, which can reduce costs. Monoclonal antibodies were also developed that were used to detect the “masked” (glycosylated) form of T-2 toxin. These antibodies have an advantage in that they can simultaneously detect both masked and unmasked forms, reducing and simplifying the number of tests needed. Paxilline (PAX) is a tremorgenic neurotoxin that has been found in perennial ryegrass infected with certain fungi. We developed monoclonal antibodies directed against PAX and developed immunoassays based upon these antibodies. This resulted in a tool for the screening of PAX in corn silages. Finally, we developed materials, anti-idiotype antibodies that mimic the effects of DON and evaluated them in three immunoassay formats. These materials have the potential to replace the toxin in diagnostic test kits, making the kits safer to ship and use. The second Objective was to design and develop synthetic-based materials for detection of agricultural and food-related toxins. To this end we developed polymers for four groups of mycotoxins: patulin, ochratoxin A (OTA), citrinin, and fusaric acid. Patulin is a mycotoxin that can be found in beverages such as apple juice. With collaborators in Japan, a polyurethane cyclodextrin-based material (nanosponge) was used to develop a faster and easier way to isolate patulin from apple juice. OTA is a nephrotoxin found in many foods and beverages, and a nanosponge was developed that was suitable for removing OTA from red wine. The materials were fully characterized using spectroscopic techniques that confirmed the polymers possessed binding sites of a size appropriate for mycotoxins. This research provided a new type of material to selectively reduce exposure to OTA. Synthetic polymers based upon the principles of molecular imprinting (MIPs) were developed for two groups of mycotoxins: fusaric acid and citrinin. Fusaric acid can be found in maize, and a MIP was used to develop a more selective analytical method for its detection. The third Objective was to develop computational methods for foodborne toxins that enable new strategies to reduce exposure. To this end, along with collaborators at Bradley University, we completed computational studies of the three dimensional structures of citrinin, zearalenone (ZEA), and certain trichothecenes. Key features of the structures and properties of these mycotoxins were identified in order to aid in the design of improved materials for their detection. To develop more sensitive detection methods and to better understand the mechanisms by which fluorescence can be augmented, the binding interactions of cyclodextrins with T-2 toxin and other trichothecenes were investigated. A computational study predicted that a novel synthetic material could reduce levels of patulin in beverages. Density functional theory was used to study 35 trichothecenes and models were successfully developed to describe cytotoxicity, phytotoxicity, and detection cross-reactivity. These models possess suitable descriptive ability and serve as inexpensive tools to evaluate potential trichothecene toxicity and predict the potential for the occurrence of false-positives. Under this Objective the key factors of water’s influence on the fluorescence detection of ZEA were determined using computational and experimental methods. Results indicated the interaction of water with oxygen containing functional groups influences the molecule’s fluorescence. This phenomenon can be applied to develop more selective methods of detection. The fourth Objective was to develop detection strategies for emerging toxins and toxins of commercial importance. To this end, we developed detection systems based upon a novel biosensor platform (biolayer interferometry: BLI), fluorescence polarization immunoassay (FPIA), and ambient ionization mass spectrometry. We developed a rapid, reusable BLI-based biosensor for detecting DON. The technique was successfully used to measure DON in samples of naturally contaminated wheat, obtaining results much more quickly and that agreed well with a reference method. The result was a rapid and reusable sensor that can be used to more efficiently monitor contamination of wheat. We also assisted Italian scientists in the development of a FPIA method to detect DON in bran and whole-wheat flour. The method is being used there and has the potential to be widely used by industry as well. In collaboration with other scientists in our management unit, we contributed to the discovery that fungi growing on plants produce glucoside derivatives of T-2 toxin and HT-2 toxin. Also in collaboration with other scientists in our management unit we contributed to the development of ambient ionization mass spectrometric (MS) methods for determination of mycotoxins from a variety of surfaces. This included a Direct Analysis in Real Time (DART)-MS method for detection of aflatoxin B1 from corn extracts on paper surfaces. DART-MS was also used to detect aflatoxin M1 in milk, and T-2 and HT-2 toxins in corn. Most recently we evaluated detection of patulin on apples and apple leathers. The technique, an orthogonal method for ionization, presents opportunities for detecting toxins from sample extracts and to detect directly from food surfaces. The accomplishments across all four Objectives demonstrate significant impacts in the area of mycotoxin analysis. Direct impacts were demonstrated through the use and distribution of toxin-binding materials developed within the project. Such materials were widely distributed to university, government, and industrial laboratories. Furthermore, under this project state-of-the art detection methods were developed. These included biosensors and DART-MS. Indirect benefits from this project were also obtained. These included the advancement of knowledge in the area of mycotoxin analysis that was disseminated through publications and presentations. This project was highly successful both in terms of the number of publications and in terms of the number, and type, of presentations requested by outside organizations. Recent accomplishments that demonstrate significant indirect impact include the co-discovery of certain masked mycotoxins (T-2 glucosides), and the development of unique biosensors for toxin detection. Detection was also improved with synthetic receptors and, with the support of an industrial partner, computational methods were developed to explain aspects of toxin biosynthesis, toxicity, and detection. By improving technologies for detecting natural toxins, this project also facilitated the ability of producers, processors, and regulatory agencies to improve monitoring programs for natural toxins, and thereby improve the safety of the U.S. food supply.
1. Screening assay for monitoring of paxilline. Paxilline (PAX) is a toxin produced by certain fungi that grow on perennial ryegrass. It is a neurotoxin that causes tremors in animals and, along with a related toxin (Lolitrem B), has been associated with a disease in ruminants known as “ryegrass staggers.” In order to facilitate rapid screening for this toxin, ARS scientists in Peoria, Illinois, developed four monoclonal antibodies that bind PAX. One of the antibodies was used to develop a sensitive method for detecting PAX in maize silage. The assay was applied to 86 silage samples, with only very low levels of PAX detected. Results suggest the method can be applied as a sensitive technique for the rapid screening of PAX in silage. The antibody provides researchers with a new tool that will be used to develop better ways to detect this mycotoxin in plants, improving feed safety.
2. A new synthetic material for improved detection of citrinin in corn. In order to enable better detection of the mycotoxin citrinin in corn, ARS scientists in Peoria, Illinois, developed a new material to bind this toxin. Citrinin is a toxin produced by fungi that may contaminate agricultural commodities. Such contamination reduces the value of the commodity and poses health risks to humans and animals. The material was successfully applied to clean-up corn samples and enable accurate determination of citrinin using modern analytical instrumentation with fluorescence detection. The detection method provides the food industry with a new approach to monitor for citrinin in corn based upon a readily produced synthetic material that improves convenience and detection accuracy.
3. Improved reagent for use in detection of fumonisins. Fumonisins are a group of compounds produced by certain fungi that commonly infest cereal grains. To keep these natural toxins out of the food and feed supplies, commodities are routinely screened for their presence. This research, conducted by scientists at the Henan Academy of Agricultural Sciences, Henan, China, in cooperation with ARS scientists in Peoria, Illinois, was performed in order to improve a reagent used in a type of toxin screening assay: fluorescence polarization immunoassay. A less expensive alternative was developed to the existing reagent, reducing the costs for the detection kits and potentially reducing the costs for monitoring for this group of toxins.
5. Significant Activities that Support Special Target Populations:
Maragos, C.M. 2015. Immunologically-based methods for detecting masked mycotoxins. In: Dall'Asta, C., Berthiller, F., editors. Masked Mycotoxins in Food: Formation, Occurrence and Toxicological Relevance. Issues in Toxicology, Book 24. London, UK: Royal Society of Chemistry. p. 32-49.
Berthiller, F., Brera, C., Crews, C., Iha, M.H., Krska, R., Lattanzio, V.M.T., MacDonald, S., Malone, R.J., Maragos, C., Solfrizzo, M., Stroka, J., Whitaker, T.B. 2016. Developments in mycotoxin analysis: an update for 2014-2015. World Mycotoxin Journal. 9(1):5-29.
Liu, J-H., Maragos, C.M., Wang, M., Yin, H-Y., Zhang, L., Zhang, J-F., Wang, H-Q. 2015. Preparation and application of new fluorescein-labeled fumonisins B1 in fluorescence polarization analysis technique. Journal of Food Safety & Quality. 6(12):1-7.
Maragos, C.M. 2015. Development and evaluation of monoclonal antibodies for paxilline. Toxins. 7(10):3903-3915.
Sanders, M., McPartlin, D., Moran, K., Guo, Y., Eeckhout, M., O'Kennedy, R., De Saeger, S., Maragos, C. 2016. Comparison of enzyme-linked immunosorbent assay, surface plasmon resonance and biolayer interferometry for screening of deoxynivalenol in wheat and wheat dust. Toxins. 8(4):103.
Evans, K.O., Compton, D.L., Whitman, N.A., Laszlo, J.A., Appell, M., Vermillion, K.E., Kim, S. 2015. Octadecyl ferulate behavior in 1,2-dioleoylphosphocholine liposomes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 153:333-343. doi: 10.1016/j.saa.2015.08.009.
Appell, M.D., Jackson, M.A. 2014. Preface. In: Park, B; Appell, M., Editors. ACS Symposium #1143: Advances in Applied Nanotechnology for Agriculture. Washington, DC: American Chemical Society. p. ix.
Maragos, C.M. 2016. Multiplexed biosensors for mycotoxins. Journal of AOAC International. 99(4):1-12.
Appell, M., Jackson, M.A., Wang, L.C., Bosma, W.B. 2015. Determination of citrinin using molecularly imprinted solid phase extraction purification, HPLC separation, and fluorescence detection. Journal of Liquid Chromatography and Related Technologies. 38(20):1815-1819.
Appell, M., Mueller, A. 2016. Mycotoxin analysis using imprinted materials technology: Recent developments. Journal of AOAC International. 99(4):861-864.