Rapid Ligand Pairing Strategy to Simplify Diagnostic Immunoassay Assembly

简化诊断免疫测定组装的快速配体配对策略

• 批准号: 8492614
• 负责人: ANDREW HAYHURST
• 金额: $ 27.45万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8492614
• 关键词: Affinity; Antibodies; Antibody Formation; Antibody Repertoire; Antigens; Automation; Bacteriophages; Biological Assay; Biological Markers; Biotin; Bontoxilysin; Botulinum Toxin Type A; Communicable Diseases; Containment; Crude Extracts; Data; Democratic Republic of the Congo; Development; Diagnostic; Diagnostic tests; Disease; Economics; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Escherichia coli; Gold; Human; Hybridomas; Immune; Immunoassay; In Vitro; Laboratories; Lateral; Libraries; Ligands; Liquid substance; Malignant Neoplasms; Methodology; Methods; Mining; Modeling; Modification; Noise; Nucleoproteins; Phage Display; Post-Translational Protein Processing; Preparation; Process; Proteins; Reagent; Recombinant Antibody; Reporter; Resources; Signal Transduction; Site; Social Welfare; Solubility; Solutions; Speed; Staging; Surface; Suspension substance; Suspensions; Taxes; Techniques; Technology; Time; Toxin; Tracer; Transfer RNA; Zaire Ebola virus; antibody engineering; antigen antibody binding; base; cost; improved; in vivo; interest; meetings; neutravidin; novel; pathogen; protein purification; public health relevance; scaffold; screening; success

DESCRIPTION (provided by applicant): Antigen sandwich capture assays form the basis of many diagnostic tests for disease and experimental assays for studying disease processes. Since recombinant antibodies, especially single domain antibodies (sdAb) can offer advantages over conventional hybridoma methods for generating affinity reagents especially towards toxic antigens, there is increasing interest in tapping this rich resource. However, the screening of non-competitive pairs of sdAb and their subsequent incorporation into a final sandwich assay is still a time- consuming bottleneck, especially when hundreds of clones are to be screened. We propose to accelerate the pairing process and produce an immediately useful assay from impure antibody preparations. Importantly, the solution will fit in to any existing antibody or alternative scaffold repertoire by "retrofitting" and create a rapid pipeline directly from diverse repertoire to antigen capture assay. We hypothesize that site specific in vivo haptenylation of recombinant antibodies, in a manner that is compatible with existing display technologies will enable rapid screening of clones as both captor and tracer from crude E. coli osmotic shockates to directly formulate sandwich assays. We have three specific aims to demonstrate this: 1. We will show that a single site specific biotin modification of sdAb enables distinction of captor fro tracer via conditional occlusion by neutravidin using pre-existing sdAb specific for polyvalent Marburgvirus nucleoprotein (NP) and non- competitive pairs of anti-botulinum neurotoxin (BoNT) sdAb. 2. We will engineer a host strain of E. coli that enables both low level expression for effective sdAb phage display, and high level expression for effective soluble sdAb production to enable immediate pairing of clones at any stage in a phage panning process. 3. We will combine the approaches and apply them to existing immune and non-immune sdAb repertoires, to compare and contrast sdAb selected on two model antigens BoNT A and Ebola virus Zaire with those isolated previously using conventional methods. Rapidly responding to emerging threats with diagnostic immunoassays in part depends on our ability to quickly identify pairs of antibodies that function in the desired assay format. Screening for function without protein purification accelerates this discovery process and expands the number of clones that can be analyzed. Though the strategy is geared towards simplicity to operate stand-alone in containment environments, the methodology will be compatible with high throughput automation. Indeed, proof of principle data would improve the likelihood of generating high quality diagnostic assays for any antigen of interest including cancer markers and other proteins indicative of disease.

描述（由申请人提供）：抗原三明治捕获测定法构成了许多疾病诊断测试和研究疾病过程的实验测定法的基础。由于重组抗体，特别是单个结构域抗体（SDAB）可以提供比常规杂交瘤方法具有产生亲和力试剂的优势，尤其是对有毒抗原的亲和力试剂，因此对利用这种丰富资源的兴趣越来越高。但是，筛选非竞争力对SDAB及其随后的掺入最终的三明治测定法仍然是一种耗时的瓶颈，尤其是在要筛选数百个克隆时。我们建议加速配对过程，并通过不纯净的抗体制剂产生有用的测定法。重要的是，该解决方案将通过“改造”直接从不同的不同的抗体或替代脚手架曲目中进行。 抗原捕获测定法的曲目。我们假设以与现有的显示技术兼容的方式，该位点特异性的体内体内haptenylation将其作为绑架者和示踪剂既可以从粗糙的大肠杆菌渗透性冲击物中快速筛选，从而可以快速筛选克隆，从而直接制定三明治分析。我们有三个具体的目的来证明这一点：1。我们将证明，SDAB的单一特定于SDAB的特定于生物素修饰可以通过中性素通过条件闭塞来区分绑架者，并使用特定于多价Marburgvirus核蛋白（NP）和非竞争力的抗生素的SDAB进行的SDAB进行隔离。 2。我们将设计一个大肠杆菌的宿主菌株，该宿主既可以实现有效的SDAB噬菌体显示，又可以使有效的可溶性SDAB产生的高水平表达表达，以便在噬菌体平移过程中在任何阶段立即配对克隆。 3。我们将结合这些方法，并将其应用于现有的免疫和非免疫SDAB曲目，以比较和对比两种模型抗原Bont A和埃博拉病毒Zaire与先前使用常规方法隔离的SDAB。通过诊断免疫测定的迅速响应新兴威胁部分取决于我们快速识别以所需测定格式起作用的抗体对的能力。在没有蛋白质纯化的情况下筛选功能会加速这一发现过程，并扩大可以分析的克隆数量。尽管该策略旨在简单地在控制环境中操作独立，但该方法将与高吞吐量自动化兼容。确实，原理数据证明将改善为任何感兴趣的抗原产生高质量诊断测定的可能性，包括癌症标志物和其他指示疾病的蛋白质。