Fumonisin, Aflatoxin, and DON Vomitoxin are mycotoxins produced by mold and fungus and found in seeds and grain products. Grain mycotoxins affect human health, animal health, and commodity value. Fluorescence Polarization (FPA) diagnostic technology provides an efficient way for grain producers and processors to check mycotoxin levels prior to shipment or final food processing. Diachemix uses FPA technology since it is quicker and easier to perform than other diagnostic technologies. FPA tests are performed in minutes, and require fewer steps, procedures, and materials than other methods. FPA uses a homogeneous format with none of the incubation or washing steps of heterogeneous diagnostic technologies. FPA is a quantitative assay performed in a field setting or in a central reference laboratory. Advanced lab technician skills are not necessary for FPA technology. FPA diagnostic technology has long been used in the human clinical market where it has proven to be both safe and effective. This project advances the development of FPA for mycotoxin detection (including Fumonisins, Aflatoxins, and Don vomitoxins) in grains and other grain products. FPA technology allows more efficient testing for mycotoxin levels by grain producers and processors. FPA detection tests are rapid (usually performed in minutes), easy to perform, and less expensive to perform than other detection technologies. FPA is a quanitative technology that can be performed either in the field, at the grain processing facility or central reference laboratory. OBJECTIVES: Diachemix LLC SBIR Phase II objectives are: (1) To develop, optimize and validate fluorescence polarization assays (FPAs) for the determination of Fumonisins, Aflatoxins, and DON Vomitoxins; (2) To modify FPA instrumentation software to obtain the concentration values (ppm or ppb) directly from a standard curve of polarization values without exporting the data to an external program; (3) To develop relatively large quanitites of antibodies and mycotoxin-fluorophores (tracers) for further analyisis with the same batch of antibodies and tracers; (4) To deliver a cost effective, simple, field portable and rapid quantititive test for these grain mycotoxins; and (5) To transfer the technology to industry for routine analysis of these mycotoxins. APPROACH: There are five main components to the Diachemix research and development plan leading to commercialization of Fluorescence Polariztion products for grain mycotoxin detection: (1) Antibody and Mycotoxin-fluorophore (tracer) Development for the Three Tests: Diachemix scientific staff will use existing antibody stocks to further test and validate each antibody with numerous toxin samples for each of the three mycotoxins studied. Selected antibodies will be developed in further bulk amounts from their cell lines as part of a CRADA with Dr. Chris Maragos of the USDA-ARS-NCAUR in Peoria, Illinois. Quality control methods will validate the scaling-up of manufactured antibodies. (2) New Antibody Development and Testing for Fumonisin: Diachemix scientific staff, along with Dr. Maragos (USDA-ARS-NCAUR)and scientific staff of the University of Illinois Immunology Resource Center will evaluate results of new monoclonal antibody candidates suggested by SBIR reviewers. These antibody candidates were developed using immunization, fusions, cloning and antibody production methods. (3) Synthesis and Study of Fumonisin-Conjugate (Tracer) Using Carboxylic Acid Route: New antibody-tracer combinations will be tested using the FP technology format. New fumonisin-fluorophore (tracer) will be synthesized, purified, and tested. Performance will be compared to other previously developed tracers for improved performance. (4) Instrument and Software Work will be performed to improve the portability of the instrument and to improve the software function so that it incorporates standard curves. Currently, an external computer and program is necessary to convert the raw FP score to a parts per million or parts per billion standard curve scores. By integrating this conversion software in the Diachemix FP instrument, the need for peripheral equipment and the added time for processing is eliminated. These developments increase the portability of the equipment while lessening equipment expense and operation time. (5) FPA Testing of Fumonsins, Aflatoxins and DON: A Comparison with HPLC: Through a collaboration among the staff of Diachemix and Trilogy Analytical Laoratories (Union, Missouri), side by side FPA and HPLC testing will be performed. GIPSA design criteria and test performance specifications will be used as the separate Fumonisin, Aflatoxin, and DON Vomitoxin candidate tracers are examined for their performance. As a result of these five stages, Diachemix expects to further optimize and finalize current development work leading to commercially available FP assays that can be used efficiently for grain mycotoxin detection by both producers and processors, either in the field at an elevator or at a central processing station. FPA grain mycotoxin testing is expected to improve the quality of human food and animal feed while improving the commodity value
