High performance and widely accessible secretomics assay platform

高性能且广泛使用的分泌组学检测平台

• 批准号: 10547532
• 负责人: Scott L Crick
• 金额: $ 94.35万
• 依托单位: AURAGENT BIOSCIENCE, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-17 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547532
• 关键词: Address; Aging; Antibodies; Biological; Biological Assay; Biology; Biomedical Research; Cell secretion; Cells; Coculture Techniques; Color; Communication; Complex; Computer software; Data; Detection; Development; Diagnostic; Disease; Fluorescein-5-isothiocyanate; Fluorescence; Future; Health; Heterogeneity; Human Cell Line; Immunoassay; Individual; Lasers; Measurement; Measures; Pattern; Performance; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Play; Procedures; Proteins; Reader; Reading; Regulation; Reproducibility; Research Personnel; Resolution; Role; Sampling; Set protein; Signal Transduction; Slide; Stains; Streptavidin; System; Techniques; Technology; Testing; Time; Work; antibody conjugate; automated analysis; base; biological systems; cell type; cost; cyanine dye 5; design; enzyme linked immunospot assay; exosome; experimental study; novel; optical spectra; plasmonics; precision medicine; prototype; scale up; secretory protein; tool

Abstract Secretory proteins are fundamental to local and systemic cellular signaling in biological systems. The regulation and dysregulation of the networks involving these proteins plays a critical role in health, aging, and development of disease. As such, study of these proteins and their networks is critical to our understanding of biology. Not surprisingly, this particular set of proteins constitutes a large proportion of the targets of pharmaceutical drugs, and also represents attractive targets for diagnostics and future precision medicine efforts. Unfortunately, many of these secreted proteins are difficult to detect. There are existing technologies for studying these secreted proteins but they either underperform, are extremely expensive and/or complex given their specific-utility. containing multiple cell types. Although the need to study secreted proteins is ubiquitous, there is not a technology that is widely available (inexpensive) and capable of providing information on multiple secreted proteins simultaneously at single-cell resolution, and we aim to address this need in the current project. At the core of our technologies is the Plasmonic Fluor (PF), our proprietary, ultrabright fluorescent nanoconstruct designed to enhance the performance of fluorescence-based immunoassays. We have already built a high-sensitivity, multiplexed immunoassay platform leveraging this technology, which is currently being tested with external collaborators. This platform includes an inexpensive, high performance, single-laser fluorescence microplate/slide reader optimized for use with our PFs. We will extend this existing platform to accommodate PF- enhanced cellular secretion assays we call PFluorospot. The upgraded platform that will result from the proposed effort will be a truly versatile and powerful tool for the measurement of proteins. In our preliminary work, we have successfully manufactured at commercial scale and with high reproducibility streptavidin-conjugated PFs that are spectrally equivalent to FITC, Cy3, and Cy5 (PF490, PF550, and PF650, respectively). We have also created three PFs with entirely unique excitation/emission spectra: two which have excitation spectra like FITC and emit like Cy3 and Cy5 (PF490_550 and PF490_650, respectively), and one which has an excitation spectrum like Cy3 but emits like Cy5 (PF550_650). These unique PFs were synthesized at a prototype scale which we aim to scale up in this effort because they will allow 3 lasers to be used to interrogate 6 separate targets. In a demonstration of applicability to cellular secretion assays, streptavidin-PF550 and PF650 were conjugated to antibodies at a small scale and used to perform dual-color PFluorospot. These initial PFluorospot experiments show that additional information can be obtained in these assays relative to ELISPOT/FluoroSpot, and are simpler and significantly faster to perform. It was possible to visualize individual cells for adherent cells and better localize secreting cells. This is particularly important for understanding heterogeneity, spatial effects in co-culture, and polyfunctionality, and we propose to add a 4th laser to our reader to accommodate cell markers. Additionally, a single-color PFluorospot assay was read with our existing reader and demonstrated high sensitivity, high resolution, and short read times. We believe our Pfluorospot platform and reader will become the go-to secretomics platform, and will empower many more biological researchers to measure important, secreted proteins.

摘要 分泌蛋白是生物系统中局部和系统细胞信号传递的基础。 涉及这些蛋白质的网络的调节和失调在 健康、衰老和疾病的发展。因此，对这些蛋白质及其网络的研究 对我们理解生物学是至关重要的。毫不奇怪，这组特殊的蛋白质构成了 很大比例的药物靶标，也代表了有吸引力的靶标 用于诊断和未来的精准医学工作。不幸的是，其中很多都是秘密的 蛋白质很难被检测到。目前已有研究这些分泌蛋白的技术。 但考虑到它们的特定效用，它们要么表现不佳，要么极其昂贵和/或复杂。 包含多种细胞类型的。尽管研究分泌蛋白质的需要无处不在，但有 不是一种广泛可用(廉价)且能够提供 在单细胞分辨率下同时获得多个分泌蛋白的信息，我们 旨在解决当前项目中的这一需求。 我们技术的核心是等离子体氟(PF)，这是我们的专有超亮 设计用于增强基于荧光的性能的荧光纳米结构 免疫分析。我们已经建立了一个高灵敏度、多元化的免疫分析平台 利用这项技术，这项技术目前正在与外部合作者进行测试。这 平台包括价格低廉、高性能的单激光荧光微板/幻灯片 阅读器为使用我们的PFS进行了优化。我们将扩展这一现有平台，以适应PF- 增强的细胞分泌分析我们称之为PFluorospot。升级后的平台将带来 将是一种真正通用和强大的工具，用于测量 蛋白质。 在前期工作中，我们已经成功地实现了工业规模和高性能的生产。 与FITC、Cy3和Cy5光谱等同的链霉亲和素标记的PFS的重复性 (分别为PF490、PF550和PF650)。我们还创建了三个完全独一无二的PFS 激发/发射光谱：其中两个具有类似FITC的激发光谱和类似Cy3和Cy3的发射 Cy5(分别为PF490_550和PF490_650)，以及具有与Cy3类似的激发光谱但 像Cy5(PF550_650)一样发射。这些独特的PFS是在原型规模上合成的，我们的目标是 在这一努力中扩大规模，因为他们将允许使用3束激光来审问6个独立的 目标。在细胞分泌物分析的适用性演示中，链霉亲和素-PF550和 将PF650与抗体进行小规模偶联，进行双色荧光斑点检测。 这些初步的PFluorospot实验表明，可以在这些 与Elispot/FluoroSpot相比，分析更简单，执行速度也更快。确实是 可以显示单个细胞的贴壁细胞，并更好地定位分泌细胞。这是 对于理解异质性、共培养中的空间效应以及 多功能，我们建议在我们的阅读器上增加第四个激光器来适应细胞标记。 此外，我们用现有的阅读器读取了单色PFluorospot检测结果，并 表现出高灵敏度、高分辨率和短读取时间。 我们相信我们的Pflospot平台和阅读器将成为首选的秘密组学平台，并且 将使更多的生物研究人员能够测量重要的、分泌的蛋白质。