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

快速高通量检测黄曲霉毒素C的新试剂和方法

• 批准号: 8396243
• 负责人: James N Herron
• 金额: $ 22.5万
• 依托单位: AGRI-ANALYSIS, LLC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-23 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396243
• 关键词: Affect; Affinity; Aflatoxins; Almond Nut; Animal Feed; Antibodies; Applications Grants; Aspergillus; Automation; Bacteriophages; Behavioral Research; Binding; Biological Assay; Biomedical Research; Businesses; California; Carcinogens; Categories; Cereals; Chemicals; Chronic; Complex; Country; DNA; Detection; Development; Enzyme-Linked Immunosorbent Assay; Epitopes; Exposure to; Farming environment; Flame Retardants; Fluorescence; Food; Goals; Government Agencies; Haptens; Harvest; Health; Human; Immunoassay; Institutes; Label; Laboratories; Legal patent; Letters; Literature; Livestock; Malignant neoplasm of liver; Mandatory Testing; Measurement; Methods; Milk; Mission; One-Step dentin bonding system; Peanuts - dietary; Peptide Library; Peptide antibodies; Peptides; Performance; Pesticides; Phage Display; Population; Prevention program; Primary carcinoma of the liver cells; Process; Product Approvals; Reagent; Reporting; Research; Research Infrastructure; Resources; Risk; Sampling; Signal Transduction; Small Business Innovation Research Grant; Solutions; Sorghum; Soybeans; Specificity; Speed; Structure; Techniques; Technology; Testing; Time; Tracer; Training; United States; United States National Institutes of Health; Universities; Utah; Vendor; Wheat; Work; base; combat; cost; cost effective; experience; fluorophore; fungus; high throughput screening; high throughput technology; improved; innovation; interest; meetings; mimetics; molecular dynamics; molecular recognition; nanobodies; new technology; next generation; novel; peptide A; professor; public health relevance; rapid technique; response; screening; skills; small molecule; success; tool

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.

描述（由申请人提供）：此 SBIR 拨款申请是对 PA-11-335 的回应，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“通过邀请小企业提交 SBIR 拨款申请，进一步开发与赞助 NIH 机构和中心的使命相关的此类技术，帮助将有用的技术从非商业实验室转移到商业市场。”在之前和当前的几份 RFP 中，NIH 和 NCI 都呼吁针对“致癌剂、接触致癌物”采用“稳健且可重复的技术/检测，并最终适应完全自动化”。 （例如，PA-09-187）。因此，为了应对这一国家挑战，我们建议开发针对一类致癌物黄曲霉毒素的快速、高通量技术。黄曲霉毒素由曲霉菌产生，可污染玉米、小麦、大豆、高粱、花生、杏仁和牛奶。它们影响着全世界 45 亿人，每年导致多达 15 万例肝细胞癌病例。在美国，1990 年《农业法案》要求进行强制性检测，其中规定“……对所有从美国出口的产品进行检测，以确定其黄曲霉毒素污染是否超过可接受的水平……”FDA 根据饲料和动物类型，规定食品中的黄曲霉毒素含量限值为 20 ng/g，动物饲料中的限值为 20 ng/g 至 300 ng/g。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，并且仅限于中心实验室。它们本质上运行缓慢且昂贵。我们建议开发能够将分子识别与信号转导相结合的技术，并且可以修改为稳健的、可重复的自动化和高通量测定。我们计划采用两种并行方法来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信标。 PI 和合作者（犹他大学 Herron 教授的小组）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 信标的美国关键专利。文献中已发现并报道了许多 AF 模拟肽，它们在抗体结合方面与 AF 特异性竞争。我们计划首先从商业供应商和文献中识别适合肽信标概念的抗体-肽对。一旦鉴定出配对，黄曲霉毒素的肽模拟物将用荧光团标记以形成肽信标（PB）。荧光团的选择将基于给定肽模拟物的特定结构和分子动力学。 PB 用作荧光示踪剂，与黄曲霉毒素竞争结合抗黄曲霉毒素抗体。 PB 与抗体结合时不会发出荧光，但在被 AF 取代时会发出高荧光，从而可以一步测量 AF。在一步检测中，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：开发黄曲霉毒素荧光夹心测定法。通常，由于小分子缺乏多个表位，因此无法进行夹心免疫测定。最近，我们在加州大学戴维斯分校的合作者 Hammock 教授的团队通过噬菌体展示筛选发现了几种肽，它们与农药 3-PBA 和阻燃化学品 BDE-47 的抗体半抗原复合物特异性结合。使用这些肽，他们成功地演示了几种小分子的夹心免疫测定。我们计划将这项新技术应用于 AF 检测。我们计划在噬菌体库中筛选与 AF-抗体复合物结合的肽，然后使用 ELISA 验证有前景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，以使用特定目标 1 中描述的同质荧光增强测定法来测试它们对 AF 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，获得 AOAC 和世界各地政府机构的批准，作为一种快速、高通量的工具部署，以应对 AF 的威胁。 公共卫生相关性：黄曲霉毒素是由真菌曲霉产生的，在收获前或储存期间定殖在谷物上。据估计，世界上有 45 亿人口接触黄曲霉毒素。长期接触会导致肝癌，并影响了全世界数百万人。在美国，1990 年农业法案要求进行强制性测试。及时、准确的结果是任何预防计划的一个组成部分。然而，当前的检测技术需要多个步骤和训练有素的操作员，因此成本高昂且耗时。对更高效、更具成本效益的测试方法的需求与当前技术之间存在着重大差距。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补这一差距。