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

描述(申请人提供)：此SBIR拨款申请是对PA-11-335的响应，从实验室到市场：生物医学和行为研究的工具。PA-11-335的目的是“通过邀请小企业的SBIR拨款申请，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此类技术。”在以前和现在的几个RFP中，NIH和NCI都呼吁对“致癌剂和致癌物暴露”进行“可靠和可重复的，并最终适用于完全自动化的技术/分析”。(例如，PA-09-187)。因此，为了迎接这一国家挑战，我们建议开发黄曲霉毒素--一种I类致癌物质--的快速高通量技术。黄曲霉毒素是由真菌曲霉产生的，它可以污染玉米、小麦、大豆、高粱、花生、杏仁和牛奶。它们影响着全球45亿人，每年导致多达15万例肝细胞癌。在美国，1990年的《农场法案》要求进行强制性检测，该法案规定“……所有从美国出口的COR都要进行检测，以确定其是否超过可接受的黄曲霉毒素污染水平……”FDA规定，根据饲料和动物类型的不同，食品中的限量为20 ng/g，动物饲料中的限量在20 ng/g到300 ng/g之间。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员，而且仅限于中央实验室。它们天生就很慢，运营成本也很高。我们建议开发可以将分子识别与信号转导相结合的技术，并且可以修改为健壮、可重复的自动化和高通量分析。我们计划采取两种并行的方法，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多肽信标。PI和合作者(犹他大学Herron教授的团队)是第一个发展多肽信标概念的人。根据他们的工作，该大学获得了多肽和DNA信标的三项关键美国专利。在文献中已经发现和报道了许多与AF特异性竞争抗体结合的AF模拟多肽。我们计划首先确定来自商业供应商的抗体-多肽对以及适合多肽信标概念的文献。一旦确定了多肽对，黄曲霉毒素的多肽模拟物将被荧光团标记以制造多肽信标(PB)。荧光团的选择将基于给定肽模拟物的特殊结构和分子动力学。PB被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。当PB与抗体结合时是非荧光的，但当被AF置换时变得高度荧光，从而允许一步测量AF。黄曲霉毒素的水平与一步法中PB示踪剂的荧光水平直接相关。具体目标2：建立黄曲霉毒素的荧光夹心法。通常，小分子的夹心免疫分析是不可能的，因为它们缺乏多个表位。最近，我们的合作者Hammock教授在加州大学戴维斯分校的团队在噬菌体展示筛选中发现了几个肽，它们专门与杀虫剂3-PBA和阻燃剂BDE-47的抗体-半抗原复合体结合。使用这些多肽，他们成功地展示了对几个小分子的夹心免疫分析。我们计划将这项新技术应用于房颤检测。我们计划筛选一个噬菌体文库，寻找与AF抗体复合体结合的多肽，然后用ELISA法验证有希望的序列的特异性和亲和力。然后，我们将用这些多肽制作荧光信标，以测试它们对使用特定目标1中描述的均相荧光增强分析进行房颤一步夹心免疫分析的有效性。我们的长期目标是将这些技术开发成强大的产品，供AOAC和世界各地的政府机构批准，作为一种快速和高通量的工具来对抗房颤的威胁。 与公众健康相关：黄曲霉毒素是由真菌曲霉产生的，在收获前或储存期间定植在谷物上。据估计，世界上有45亿人口接触到黄曲霉毒素。慢性接触会导致肝癌，并已影响到全球数百万人。在美国，1990年《农场法案》要求进行强制性检测。及时和准确的结果是任何预防计划不可或缺的一部分。然而，目前的检测技术需要多个步骤和训练有素的操作员，因此成本高且耗时。在对更有效和更具成本效益的测试方法的需求与当前技术之间存在着严重的差距。我们的研究旨在通过开发快速、高通量和自动化的黄曲霉毒素检测技术来弥合这一差距。

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. PUBLIC HEALTH RELEVANCE: Aflatoxins are produced by the fungus Aspergillus, colonized on grains before harvest or during storage. It is estimated that 4.5 billion of the world's population are exposed to aflatoxins. Chronic exposure leads to liver cancer, and have affected millions of people worldwide. In the U.S., mandatory testing is required by the 1990 Farm Bill. Timely and accurate result is an integral part of any prevention program. However, current detection technologies require multiple steps and highly trained operators, and are therefore costly and time consuming. There is a critical gap between the need for more efficient and cost effective testing methods and the current technology. Our research aims to bridge this gap by developing rapid, high throughput and automated technologies for aflatoxin detection.