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)：抗原夹心捕获试验构成了许多疾病诊断试验和研究疾病过程的实验分析的基础。由于重组抗体，特别是单域抗体(SdAb)在制备亲和试剂，尤其是针对有毒抗原的杂交瘤方法方面具有传统杂交瘤方法无法比拟的优势，人们对开发这一丰富的资源越来越感兴趣。然而，筛选非竞争性的sdAb对并随后将它们合并到最终的三明治试验中仍然是一个耗时的瓶颈，特别是在要筛选数百个克隆的情况下。我们建议加速配对过程，并从不纯的抗体制剂中产生立即有用的检测。重要的是，该解决方案将适合于任何现有的抗体或替代支架保存库，并创建一个快速管道直接从不同的 抗原捕获试验曲目。我们假设，以与现有展示技术兼容的方式，重组抗体在体内的位点特异性半抗原基化将使从粗制大肠杆菌渗透休克物中快速筛选克隆作为捕获器和示踪剂，以直接形成三明治分析。我们有三个具体的目的来证明这一点：1.我们将证明sdAb的单一位点特异性生物素修饰能够通过使用预先存在的针对多价MarburgVirus核蛋白(NP)的sdAb和非竞争性的抗肉毒杆菌神经毒素(BONT)sdAb对，通过中性亲和素的条件阻断来区分捕获者和示踪物。2.我们将设计一种大肠杆菌宿主菌株，使其既能低水平表达有效的sdAb噬菌体展示，又能高水平表达有效的可溶性sdAb生产，以便在噬菌体淘洗过程的任何阶段立即进行克隆配对。3.我们将把这些方法结合起来，应用于现有的免疫和非免疫sdAb库中，将针对两种模式抗原BONT A和埃博拉病毒扎伊尔的sdAb与以前用常规方法分离的sdAb进行比较和对比。用诊断免疫分析方法快速应对新出现的威胁在一定程度上取决于我们是否有能力快速识别以所需的分析形式发挥作用的抗体对。没有蛋白质纯化的功能筛选加速了这一发现过程，并扩大了可分析的克隆数量。虽然该战略旨在简化在安全壳环境中独立操作，但该方法将与高吞吐量自动化兼容。事实上，原则数据的证明将提高对任何感兴趣的抗原产生高质量诊断分析的可能性，包括癌症标记物和其他指示疾病的蛋白质。