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:
Aflatoxins are toxins produced by a variety of fungi during their infestation of growing grain kernels. Animals consuming infested grain can pass aflatoxin M1 (AFM1) into their milk. ARS scientists at the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, developed a technique to measure AFM1 in milk based on the coupling of ambient ionization with mass spectrometry. The procedure allows the rapid and sensitive detection of AFM1 from milk samples without extensive sample preparation or the normally required chromatographic separation. This work provides a rapid, sensitive and convenient analytical tool to food processors seeking to assure the safety of milk based products. Deoxynivalenol (DON) is a toxin which contaminates certain agricultural commodities, and is frequently monitored by antibody-based detection methods. However, co-occurring metabolites can cause false-positive results, and complicate accurate analysis. To gain insight into the cross-reactivity of DON antibodies with structurally related trichothecene metabolites, including T-2 toxin and nivalenol, ARS developed computational methods for modeling the interactions. The binding of the trichothecenes to the antibody was related to the polarity, and the antibody selectivity was associated with the change in the magnitude of the polarity. This information will be helpful in the generation of better detection methods for trichothecenes. A new class of polymers were developed that could bind and facilitate detection of multiple toxins in food and beverages. In our studies nanosponge materials were synthesized and fully characterized. The efficacy of these materials for use with multiple mycotoxins (ochratoxin A, zearalenone, beta-zearalenol, and patulin) was evaluated using sorption studies and solid phase extraction protocols. The nano-sized channels and composition of the materials were investigated using Fourier Transformation Infrared Spectroscopy, Atomic Force Microscopy, and computational techniques. This research is important to scientists looking for robust materials to improve detection of several small molecule toxins in agricultural commodities.
1. Development of novel antibodies for detection of masked mycotoxins. Certain fungi that commonly infest grains are known to produce a potent toxin known as T-2 toxin, which can be found in crops such as wheat, corn, barley, oats, and rye and in human food and animal feeds. A variety of methods for detecting T-2 toxin have been developed. However masked forms of T-2 toxin, including T-2 glucosides, have been found in grain and are of interest as potential reservoirs of T-2 toxin that are not detected by many analytical methods. In this research, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, generated an antibody for the sensitive detection of both T-2 toxin and its glucoside. The antibody provides scientists with a new tool to detect this heretofore masked mycotoxin and to assure the safety of food and feed.
2. A rapid method to detect deoxynivalenol in wheat bran and whole-wheat flour. Deoxynivalenol (DON) is among the most important mycotoxins commonly found in wheat, barley, and corn. When grains are processed, DON does not distribute equally among the fractions. The increasing inclusion of bran into food formulations, as a source of dietary fiber, necessitates increased monitoring for DON in such fractions. As part of efforts to improve monitoring of the toxin, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, collaborated with scientists at the Institute of Sciences of Food Production (ISPA) in Bari, Italy, to develop a method for detecting DON in wheat bran and whole-wheat flour using fluorescence polarization immunoassay. The technique was successfully used to measure DON in samples of naturally contaminated bran and whole-wheat flours, with results that agreed well with a reference method. The result is a rapid method that can be used to monitor DON in wheat bran and whole-wheat flour providing a more safe food supply.
3. A new reagent to reduce the need for toxin in toxin-detection kits. As part of efforts to improve toxin monitoring, kits for detecting deoxynivalenol (DON) based upon specific antibodies (immunoassays) have been widely developed. In such kits, the toxin is generally included as a standard against which test samples are compared. This means that the toxin is shipped with the test kits, and that analysts have the potential to be exposed to the toxin when the kits are used. However, materials other than the toxin can potentially be used, provided they mimic the toxin in the assay. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, developed a material (an anti-idiotype antibody) that mimics the effects of the toxin in assays and evaluated the material in three immunoassay formats. This material has the potential to replace the toxin in diagnostic test kits, which would make the kits safer to use and to ship.
4. New materials and methods to detect fusaric acid. Fusaric acid (FA) is a toxin produced by a variety of fungi, and is of concern because of its potential to contaminate agricultural commodities. However, it is currently difficult to determine how much FA is present in corn. To aid in the isolation and detection of FA, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with researchers at Central Michigan University, developed a synthetic material with binding sites capable of selectively isolating FA. This durable material was applied in a new method to detect FA in corn and improved detection accuracy by removing interferences from samples. The development of this novel binding material is important to food safety scientists and regulators looking for accurate methods to monitor FA and other toxins in the food supply.
5. Rapid detection of aflatoxin B1 in corn. Aflatoxin is a highly carcinogenic toxin produced by a variety of fungi during their infestation of growing corn kernels. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, developed a technique to determine levels of aflatoxin from corn using ambient ionization from a paper surface coupled to mass spectrometry. The procedure allows the rapid and sensitive detection of aflatoxin from corn samples without extensive sample preparation or the normally required chromatographic separation. This work provides a rapid, sensitive, and convenient analytical tool to grain processors seeking to assure the safety of corn based products.
Appell, M., Jackson, M.A., Wang, L.C., Ho, C., Mueller, A. 2014. Determination of fusaric acid in maize using molecularly imprinted SPE clean-up. Journal of Separation Science. 37(3):281-286.
Maragos, C.M. 2014. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms. Mycotoxin Research. 30(2):103-111.
Peterson, S.C., Jackson, M.A., Appell, M. 2013. Biochar: sustainable and versatile. In: Park, B., Appell, M., editors. Advances in Applied Nanotechnology for Agriculture. New York, NY: American Chemical Society Symposium Series. p. 193-205.
Maragos, C.M., Kurtzman, C.P., Busman, M., Price, N.P., McCormick, S.P. 2013. Development and evaluation of monoclonal antibodies for the glucoside of T-2 toxin (T2-Glc). Toxins. 5(7):1299-1313.
Appell, M.D., Jackson, M.A. 2014. Applications of nanoporous materials in agriculture. In: Park, B., Appell, M., editors. ACS Symposium Series: Advances in Applied Nanotechnology for Agriculture. Washington, DC: American Chemical Society. p. 167-178.
Compton, D.L., Laszlo, J.A., Appell, M., Vermillion, K., Evans, K.O. 2014. Synthesis, purification, and acyl migration kinetics of 2-Monoricinoleoylglycerol. Journal of the American Oil Chemists' Society. 91(2):271-279.
Lippolis, V., Maragos, C.M. 2014. Fluorescence polarization immunoassays for rapid, accurate and sensitive determination of mycotoxins. World Mycotoxin Journal. DOI: 10.3920/WMJ2013.1681.
Valenzano, S., Lippolis, V., Pascale, M., De Marco, C., Maragos, C.M., Suman, M., Visconti, A. 2014. Determination of deoxynivalenol in wheat bran and whole-wheat flour by fluorescence polarization immunoassay. Journal of Food Analytical Methods. 7(4):806-813.
Busman, M., Liu, J., Zhong, H., Bobell, J.R., Maragos, C.M. 2014. Determination of the aflatoxin AFB1 from corn by direct analysis in real time-mass spectrometry (DART-MS). Food Additives & Contaminants. 31(5):932-939.
Berthiller, F., Burdaspal, P.A., Crews, C., Iha, M.H., Krska, R., Lattanzio, V.M., MacDonald, S., Malone, R.J., Maragos, C.M., Stroka, J., Whitaker, T.B. 2014. Developments in mycotoxin analysis: an update for 2012 – 2013. World Mycotoxin Journal. 7(1):3-33.