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）：抗原夹心捕获分析是许多疾病诊断测试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）在产生针对有毒抗原的亲和试剂方面比传统杂杂瘤方法具有优势，因此开发这一丰富资源的兴趣越来越大。然而，筛选非竞争性的sdAb对并将其合并到最后的三明治试验中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，并从不纯抗体制剂中产生立即有用的测定。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，并直接从多样化中创建快速管道