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

项目总结 抗体是高度特异的、多样化的、被广泛检测的生物标志物，用于确定最近或既往 接触病原体，测量疫苗的保护作用，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种或一种小的抗体。 一次反应的次数，因此与个人反应的规模和多样性不相称 抗体反应。一种使用小样本量更全面地询问这种反应性多样性的工具 将使新一代系统免疫学、疾病关联性和流行病学研究成为可能 监视系统。在这里，我们建议优化并显著扩展我们为高度- 多路、可重复且廉价的分析方法，可实现对抗体的灵敏和高分辨率分析 1微克L血液中100,000种抗原的反应性。我们的方法利用了快速、全面的- 一种制备100,000个DNA条形码多肽(‘PepSeq’)的体外方法 用DNA测序法检测血清抗体。作为概念验证，我们将在这里重点介绍 以所有已知可感染人类的病毒(即人类病毒)为目标的检测。病毒是一种理想的用例 这项技术，因为病毒代表着对免疫系统的令人难以置信的多样化和无处不在的挑战，并且 由于它们的基因组很小，完整的病毒体可以用最少的基因组在单个文库中覆盖 多样性的丧失。我们用这种病毒检测的初步数据证实了这种方法的可行性。这里, 我们将优化多种样本类型的检测程序，以提高灵敏度和特异度， 同时降低成本。我们还将建立特定于同种类型的分析的标准化协议，使 实现抗原特异性单细胞表征的技术，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界使用这项技术。贯穿始终 在这个过程中，我们将生成一组抗病毒抗体图谱，包括纵向图谱- 这些数据将通过ImmPort门户网站提供给社区。如果成功，这个项目将 交付：(I)优化的检测标准操作程序和资料库，用于全面评估泛病毒免疫。 样本量，(2)一大套可公开获得的抗病毒免疫数据集，以及(3)用于 可直接推广到其他靶点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.