Rapid Ligand Pairing Strategy to Simplify Diagnostic Immunoassay Assembly

描述（由申请方提供）：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性试验的基础。由于重组抗体，特别是单结构域抗体（sdAb）可以提供优于常规杂交瘤方法的优点，用于产生特别是针对毒性抗原的亲和试剂，因此人们对利用这种丰富的资源越来越感兴趣。然而，sdAb的非竞争性对的筛选及其随后并入最终夹心测定中仍然是耗时的瓶颈，特别是当要筛选数百个克隆时。我们建议加速配对过程，并从不纯的抗体制备物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，并直接从不同的抗体或替代支架库创建快速管道。 抗原捕获测定的所有组成部分。我们假设，重组抗体的位点特异性体内半抗原化，以与现有展示技术相容的方式，将能够从粗制E.大肠杆菌渗透休克，以直接配制夹心测定。我们有三个具体目标来证明这一点：1。我们将显示sdAb的单位点特异性生物素修饰使得能够通过中性抗生物素蛋白的条件性封闭来区分捕获物与示踪物，所述条件性封闭使用对多价马尔堡病毒核蛋白（NP）特异的预先存在的sdAb和抗肉毒杆菌神经毒素（BoNT）sdAb的非竞争性对。2.我们将设计一个宿主菌株。大肠杆菌，其能够实现用于有效sdAb噬菌体展示的低水平表达和用于有效可溶性sdAb产生的高水平表达，以使得能够在噬菌体淘选过程中的任何阶段立即配对克隆。3.我们将联合收割机结合这些方法，并将其应用于现有的免疫和非免疫sdAb谱系，将在两种模型抗原BoNT A和扎伊尔埃博拉病毒上选择的sdAb与之前使用常规方法分离的sdAb进行比较和对比。快速响应新出现的威胁与诊断免疫测定在一定程度上取决于我们的能力，以快速识别抗体对的功能，在所需的测定格式。在没有蛋白纯化的情况下筛选功能加速了这一发现过程，并扩大了可分析的克隆数量。虽然该策略旨在简化在密闭环境中的独立操作，但该方法将与高通量自动化兼容。事实上，原理数据的证明将提高产生针对任何感兴趣的抗原（包括癌症标志物和指示疾病的其他蛋白质）的高质量诊断测定的可能性。

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.