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

描述（由申请人提供）：此SBIR资助申请是为了响应PA-11-335，从实验室到市场：生物医学和行为研究工具。PA-11-335的目的是“通过邀请小企业的SBIR资助申请，帮助将有用的技术从非商业实验室转移到商业市场，以进一步开发与赞助NIH研究所和中心的使命相关的技术。在之前和当前的几个RFP中，NIH和NCI都呼吁“技术/检测是强大的和可重复的，最终，适应于完全自动化”的“致癌物质，暴露于致癌物质”。(e.g., PA-09-187）。因此，为了应对这一国家挑战，我们建议开发快速和高通量的黄曲霉毒素技术-一类致癌物质。黄曲霉毒素是由曲霉菌产生的，它可以污染玉米、小麦、大豆、高粱、花生、杏仁和牛奶。它们影响全球45亿人，每年导致多达15万例肝细胞癌。在美国，1990年农业法案要求进行强制性测试，该法案规定“......所有从美国出口的玉米都要进行检测，以确定其黄曲霉毒素污染是否超过可接受的水平。“FDA规定食品中的限量为20 ng/g，动物饲料中的限量为20 ng/g至300 ng/g，具体取决于饲料和动物类型。目前的黄曲霉毒素检测方法需要多个步骤和训练有素的操作人员，并且仅限于中心实验室。它们的运行速度本来就很慢，而且成本很高。我们建议开发技术，可以结合联合收割机的分子识别与信号转导，并可用于强大的，可重复的自动化和高通量的测定。我们计划采用两种并行的方法，以降低风险并最大限度地提高成功率。这两种方法都是PA-11- 335所要求的成熟技术的创新应用：从实验室到市场。具体目标#1：开发用于黄曲霉毒素检测的肽类抗生素。PI和合作者（犹他州大学的Herron教授小组）是第一个开发肽信标概念的人。基于他们的工作，该大学获得了三项关于肽和DNA信标的关键美国专利。在文献中已经发现并报道了许多AF模拟肽，其特异性地与AF竞争抗体结合。我们计划首先从商业供应商和文献中确定适合肽信标概念的抗体-肽对。一旦识别出对，黄曲霉毒素的肽模拟物将用荧光团标记以制备肽信标（PB）。荧光团选择将基于给定肽模拟物的特定结构和分子动力学。PB用作与黄曲霉毒素竞争结合抗黄曲霉毒素抗体的荧光示踪剂。PB在与抗体结合时将是非荧光的，但在被AF置换时变为高荧光的，从而允许AF的一步测量。在一步测定中，黄曲霉毒素的水平与PB示踪剂的荧光水平直接相关。具体目标#2：开发黄曲霉毒素的荧光夹心测定法。通常，小分子的夹心免疫测定由于其缺乏多个表位而不可能。最近，我们的合作者加州大学戴维斯分校的Hammock教授的小组从噬菌体展示筛选中发现了几种肽，它们特异性地结合农药3-PBA和阻燃化学品BDE-47的抗体-半抗原复合物。使用这些肽，他们成功地证明了几种小分子的夹心免疫测定。我们计划将这种新技术应用于AF检测。我们计划筛选噬菌体文库中与AF-抗体复合物结合的肽，然后用ELISA验证有希望的序列的特异性和亲和力。然后，我们将从这些肽中制备荧光信标，以测试它们对于使用特异性目的1中描述的均相荧光增强测定的AF的一步夹心免疫测定的有用性。我们的长期目标是将这些技术开发成强大的产品，供AOAC和全球政府机构批准，作为快速和高通量的工具部署，以应对AF的威胁。

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.