Novel Reagents and Methods for Rapid and High Throughput Detection of Aflatoxin C

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DESCRIPTION (provided by applicant): This SBIR grant application is in response to PA-11-335, from Lab to Marketplace: Tools for Biomedical and Behavioral Research. The intent of PA-11-335 is "to help move useful technologies from non-commercial laboratories into the commercial marketplace by inviting SBIR grant applications from small businesses for further development of such technologies that are relevant to the missions of the sponsoring NIH institutes and centers." In several previous and current RFPs, both NIH and NCI have called for "technologies/assays that are robust and reproducible and, eventually, adaptable to full automation" for "carcinogenic agents, exposure to carcinogens". (e.g., PA-09-187). Therefore, to meet this national challenge, we propose to develop rapid and high throughput technologies for aflatoxins - a Category I carcinogen. Aflatoxins are produced by the fungi Aspergillus, which can contaminate corn, wheat, soy beans, sorghum, peanuts, almonds, and milk. They affect 4.5 billion people worldwide and causes up to 150,000 cases of hepatocellular carcinoma each year. In the U.S., mandatory testing is required by the 1990 Farm Bill that stipulates "... all cor exported from the United States be tested to ascertain whether it exceeds acceptable level of aflatoxin contamination ..." The FDA sets the limit of 20ng/g in food products and between 20 ng/g to 300 ng/g in animal feeds depending upon the feed and animal type. Current methods of Aflatoxin detection require multiple steps and highly trained operators and are limited to central laboratories. They are inherently slow and expensive to operate. We propose to develop technologies that can combine molecular recognition with signal transduction, and that are amendable to robust, reproducible automated and high throughput assays. We plan to work on two parallel approaches in order to mitigate risks and maximize success rate. Both approaches are innovative applications of proven technologies as called for by PA-11- 335: from Lab to Marketplace. Specific Aim # 1: Developing Peptide Beacons for Aflatoxin Detection. The PI and Collaborator (Professor Herron's group at University of Utah) were the first to develop the peptide beacon concept. Based on their work, the University was issued three key U.S. patents on peptide and DNA beacons. Many AF-mimetic peptides have been discovered and reported in the literature that specifically competes with AF in antibody binding. We plan to first identify antibody-peptide pairs from commercial vendors and the literature that are suitable for the peptide beacon concept. Once pairs are identified, peptide mimetics of aflatoxin will be labeled with fluorophores to make peptide beacons (PB). Fluorophore selection will be based on the particular structure and molecular dynamics of a given peptide mimetic. A PB is used as a fluorogenic tracer in competition with aflatoxin for binding to anti-aflatoxin antibodies. The PB will be non-fluorescent when bound to the antibody but becomes highly fluorescent when displaced by AF, thereby allowing for one-step measurement of AF. The level of aflatoxin is directly related to the fluorescence level of the PB tracer in a one-step assay. Specific Aim # 2: Developing a Fluorescent Sandwich Assay for Aflatoxins. Usually, sandwich immunoassays of small molecules are not possible due to their lack of multiple epitopes. Recently, our collaborator Professor Hammock's group at UC Davis discovered several peptides from phage display screening that bind specifically to the antibody-hapten complex for the pesticide 3-PBA and the fire retardant chemical BDE-47. Using these peptides, they successfully demonstrated sandwich immunoassays for several small molecules. We plan to apply this novel technology for AF detection. We plan to screen a phage library for peptides that bind to the AF-antibody complex, then validate promising sequences with ELISA for specificity and affinity. We would then make fluorescent beacons from these peptides to test for their usefulness for one-step sandwich immunoassays of AF using the homogenous fluorescence enhancement assay described in Specific Aim 1. Our long term goal is to develop these technologies into robust products for approvals by AOAC and government agencies worldwide for deployment as a rapid and high throughput tool to combat the threat of AF.