A scalable platform for highly-multiplexed analysis of antibody reactivity from <1uL of blood

项目摘要 抗体是高度特异性、多样性和广泛测定的生物标志物，用于确定最近或历史的 病原体暴露，测量疫苗赋予的保护，了解自身免疫的基础 疾病或评估宿主的免疫功能。传统的抗体检测集中在一个或一小部分 一次反应的数量，因此与个体反应的规模和多样性不相称。 抗体反应使用小样本量更全面地询问反应性多样性的工具 将使系统免疫学、疾病关联和流行病学的新一代研究成为可能。 监视在这里，我们建议优化和显着扩展的方法，我们已经开发了高度- 多路复用、可重复和廉价的检测，能够对抗体进行灵敏和高分辨率的分析 <1微升血液中100，000个抗原的反应性。我们的方法利用了快速、全面的 产生100，000个DNA条形码化肽（“PepSeq”）作为高度多路复用的 使用DNA测序来询问血清抗体。作为概念验证，我们将在此重点介绍 靶向已知感染人类的所有病毒的测定（即，人类病毒组）。病毒组是一个理想的用例， 这项技术，作为病毒代表了一个令人难以置信的多样化和无处不在的挑战，免疫系统， 由于它们的基因组尺寸小，完整的病毒组可以覆盖在单个文库中， 多样性的丧失。我们用这种病毒组测定法的初步数据确立了这种方法的可行性。在这里， 我们将优化多种样品类型的测定程序以提高灵敏度和特异性， 同时降低成本。我们还将建立同位素特异性分析的标准化方案， 技术，使抗原特异性，单细胞表征，并建立一套开放获取的数据分析 和可视化工具，以促进更广泛的研究社区使用这项技术。在整个 在这个过程中，我们将产生一组抗病毒抗体谱，包括纵向分布的队列- 这些资料会透过入境处的入门网站向市民公布。如果成功，该项目将 提供：（i）优化的测定SOP和库，用于使用小型 样本量，（ii）大量公开的抗病毒免疫数据集，以及（iii）用于 可直接扩展到其他靶标的多重血清学检测开发。

PROJECT SUMMARY Antibodies are highly-specific, diverse and widely-assayed biomarkers used to determine recent or historical pathogen exposures, measure the protection conferred by a vaccine, understand the basis of autoimmune diseases or evaluate a host's immunological function. Traditional assays for antibodies focus on one or a small number of reactivities at a time, and so are incommensurate with the scale and diversity of an individual's antibody response. A tool to more holistically interrogate this diversity of reactivities using a small sample volume would enable a new generation of studies in systems immunology, disease association, and epidemiological surveillance. Here, we propose to optimize and significantly extend an approach we have developed for highly- multiplexed, reproducible and inexpensive assays that enable sensitive and high-resolution analysis of antibody reactivity across 100,000s of antigens from <1µL of blood. Our approach takes advantage of a rapid, fully-in- vitro method for generating 100,000s of DNA-barcoded peptides (`PepSeq') as probes for the highly-multiplexed interrogation of serum antibodies using DNA sequencing. As a proof-of-concept, we will be focusing here on an assay targeting all viruses known to infect humans (i.e., the human virome). The virome is an ideal use case for this technology, as viruses represent an incredibly diverse and ubiquitous challenge to the immune system, and because of their small genome sizes, the complete virome can be covered within a single library with minimal loss of diversity. Our preliminary data with this virome assay establishes the feasibility of this approach. Here, we will optimize the assay procedures for multiple sample types in order to increase sensitivity and specificity, while decreasing cost. We will also establish standardized protocols for isotype-specific profiling, adapt the technology to enable antigen-specific, single-cell characterization, and build a suite of open access data analysis and visualization tools to facilitate the use of this technology by the broader research community. Throughout this process, we will generate a panel of anti-virome antibody profiles, including a cohort profiled longitudinally – this data will be made available to the community through the ImmPort portal. If successful, this project will deliver: (i) an optimized assay SOP and library for comprehensive evaluation of pan-viral immunity using a small sample volume, (ii) a large set of publicly-available anti-virome immunity datasets, and (iii) a framework for multiplexed serological assay development that can be directly extended to other targets.