Mass Spectrometry Assays for Biodefense Agents

生物防御剂的质谱分析

• 批准号: 6880460
• 负责人: RANDALL W NELSON
• 金额: $ 29.5万
• 依托单位: INTRINSIC BIOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6880460
• 关键词: affinity labeling; antibacterial antibody; antigen antibody reaction; biohazard detection; biomarker; bioterrorism /chemical warfare; buffers; diagnosis design /evaluation; diagnosis quality /standard; food contamination; immunologic assay /test; mass spectrometry; matrix assisted laser desorption ionization; milk; molecular weight; staphylococcal enterotoxin; water sampling /testing; water supply

DESCRIPTION (provided by applicant): The objective of this Phase I Research Proposal is to develop affinity-based mass spectrometry assay for detection of the NIAID High Priority Biodefense Agent Staphylococcal Enterotoxin B (SEB). The assay will utilize affinity capture in pipette-fitted microcolumns modified with anti-SEB antibody to selectively retrieve SEB from buffer, tap water, and milk samples. Detection will be achieved by measuring the molecular weight of the eluted SEB via MALDI-TOF mass spectrometry. The assay is unique as it offers dual specificity: the first comes from the use of a capturing antibody, and the second comes from the observation of a specific m/z value, characteristic of the analyzed toxin, in the resulting mass spectra. The use of polyclonal antibodies in combination with MS enables capture and detection of protein toxins and related variants in a single assay, and in the process significantly reduces the cost and time of analysis. In such, the proposed assay offers significant advantages over existing and commercially available sandwich-based SEB assays. Under the three specific aims under this proposal, we will demonstrate the reproducibility and sensitivity of the SEB assay, and develop and apply quantitative approaches for detection of SEB via the proposed affinity-based mass spectrometric assay. The successful completion of these Phase I Specific Aims will result in a fully-functional SEB-MSIA assay, and will lay the foundation for an experimental/developmental Phase II program that will focus on increasing the number of pathogen assays, developing multiplexed assays capable of detecting families and cohorts of toxins, integrating data evaluation methods into the assays, and describe the use of the assays and the accompanying instrumental platform in the laboratories that are part of the CDC's Laboratory Reference Network.

描述（由申请人提供）：本第一阶段研究提案的目的是开发基于亲和力的质谱分析法，用于检测 NIAID 高优先级生物防御剂葡萄球菌肠毒素 B (SEB)。该检测将利用抗 SEB 抗体修饰的移液管微柱中的亲和捕获，选择性地从缓冲液、自来水和牛奶样品中回收 SEB。通过 MALDI-TOF 质谱测量洗脱的 SEB 的分子量来实现检测。该测定的独特之处在于它提供了双重特异性：第一个来自捕获抗体的使用，第二个来自在所得质谱中对特定 m/z 值（所分析毒素的特征）的观察。将多克隆抗体与 MS 结合使用，可以在一次测定中捕获和检测蛋白质毒素和相关变体，并在此过程中显着降低分析成本和时间。因此，所提出的检测方法比现有的和市售的基于三明治的 SEB 检测方法具有显着的优势。根据该提案的三个具体目标，我们将证明 SEB 测定的重现性和灵敏度，并开发和应用通过所提出的基于亲和力的质谱测定检测 SEB 的定量方法。这些第一阶段具体目标的成功完成将产生功能齐全的 SEB-MSIA 检测，并将为实验/开发第二阶段计划奠定基础，该计划将侧重于增加病原体检测的数量，开发能够进行多种检测的多重检测。检测毒素家族和群体，将数据评估方法整合到检测中，并描述检测和随附仪器平台在属于 CDC 实验室参考网络一部分的实验室中的使用。