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 United States food supply.
3. Progress Report
Ochratoxin A (OTA) is a toxin produced by certain fungi that contaminate agricultural commodities. In an effort to reduce exposure to OTA we investigated the ability of a carbohydrate-polymer material to assist in removal of OTA from water and wine. The interaction between OTA and the material was investigated using experimental and computational techniques. The results of this study will aid scientists looking for new kinds of materials to reduce exposure to OTA. Deoxynivalenol (DON) is a toxin produced by certain fungi that can infest wheat, barley, and corn. Because of the toxicity of DON, and the desire to protect human and animal health, extensive monitoring for this toxin is conducted in commodities and foods. In an effort to improve detection of DON, a new material for selectively binding the toxin was developed. A monoclonal antibody recognizing DON was used as the starting point in the development of a recombinant single chain variable fragment (scFv) antibody. The scFv can be produced in bacterial systems which may lower production costs. The scFv was characterized using two immunoassay formats. Results suggest the scFv retained the selectivity of the monoclonal antibody, in spite of its substantially smaller size. Citrinin is a toxin produced by several fungal species that frequently contaminate agricultural commodities. To gain insight into the properties of citrinin that may influence detection, a state-of-the-art computational study was carried out. Key chemical features of the different forms and properties of citrinin were investigated. Results provided information on the influence of the different forms on methods of detection. This study will aid researchers in the design of materials for citrinin detection and provides insight into the methods appropriate for studying related natural products. As part of an effort to reduce exposure to natural toxins, collaboration with an industrial partner to develop computational methods for Botulinum neurotoxin and mycotoxins was begun. Data from high through-put screening and from the scientific literature were evaluated using cheminformatics and molecular modeling. The cheminformatics studies consisted of analysis of the chemical structure and biological activity data using readily available programs. The analysis was conducted using software that included multivariate analysis. This information will be useful for researchers looking for drug candidates to reduce the effects of Botulinum neurotoxin. The techniques developed in this study will also be useful to researchers looking for new ways to analyze mycotoxin data.
1. Deoxynivalenol (DON) is a toxin produced by certain fungi that can infest wheat, barley, and corn. It results in substantial losses to the quality and value of grain worldwide. Because of the toxicity of DON, and the desire to protect human and animal health, extensive monitoring for this toxin is conducted in commodities and foods. As part of efforts to improve detection of DON in wheat, Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens & Mycology Research Unit of the USDA-ARS National Center for Agricultural Utilization Research, Peoria, IL examined the applicability of a new detection technology, biolayer interfereometry (BLI), to the measurement of DON. Among the advantages of BLI is that real time monitoring can be done without the need for reagents with enzymatic, fluorescent, or radioactive labels. The technique was used successfully to measure DON in whole wheat flour at levels relevant to food safety and animal health.