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）：抗原夹心捕获测定构成了许多疾病诊断测试和研究疾病过程的实验测定的基础。由于重组抗体，尤其是单域抗体 (sdAb) 在生成亲和试剂（尤其是针对有毒抗原）方面比传统杂交瘤方法具有优势，因此人们对利用这一丰富资源越来越感兴趣。然而，非竞争性 sdAb 对的筛选以及随后将其纳入最终的夹心测定仍然是一个耗时的瓶颈，特别是当要筛选数百个克隆时。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方法。重要的是，该解决方案将通过“改造”适应任何现有的抗体或替代支架库，并直接从不同的来源创建快速管道 抗原捕获测定的曲目。我们假设，重组抗体的位点特异性体内半抗原化，以与现有展示技术兼容的方式，将能够快速筛选克隆，作为来自粗制大肠杆菌渗透休克物的捕获剂和示踪剂，以直接制定夹心测定法。我们有三个具体目标来证明这一点： 1. 我们将证明 sdAb 的单位点特异性生物素修饰能够通过中性亲和素使用预先存在的对多价马尔堡病毒核蛋白 (NP) 和非特异性的 sdAb 有条件封闭来区分捕获剂和示踪剂。抗肉毒神经毒素 (BoNT) sdAb 的竞争对。 2. 我们将设计一种大肠杆菌宿主菌株，既能够实现有效 sdAb 噬菌体展示的低水平表达，又能够实现有效可溶性 sdAb 生产的高水平表达，从而能够在噬菌体淘选过程的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 库，以将在 BoNT A 和埃博拉病毒扎伊尔两种模型抗原上选择的 sdAb 与之前使用常规方法分离的 sdAb 进行比较和对比。通过诊断免疫测定快速应对新出现的威胁部分取决于我们快速识别以所需测定形式发挥作用的抗体对的能力。无需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。尽管该策略旨在简化在遏制环境中独立操作的过程，但该方法将与高吞吐量自动化兼容。事实上，原理数据的证明将提高对任何感兴趣的抗原（包括癌症标志物和其他指示疾病的蛋白质）产生高质量诊断测定的可能性。