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: 10705734
• 负责人: Scott L Crick
• 金额: $ 78.66万
• 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/10705734
• 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; Protein Secretion; Proteins; Reader; Reading; Regulation; Reproducibility; Research Personnel; Resolution; Role; Sampling; Set protein; Signal Transduction; Slide; Stains; Streptavidin; System; Techniques; Technology; Testing; Visualization; Work; antibody conjugate; automated analysis; biological systems; cell type; cost; cyanine dye 5; design and construction; empowerment; enzyme linked immunospot assay; exosome; experimental study; manufacture; nano; novel; optical spectra; plasmonics; precision medicine; prototype; scale up; secretory protein; tool; verification and validation

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全身细胞信号传导的基础。 涉及这些蛋白质的网络的调节和失调在以下方面起着关键作用： 健康、衰老和疾病的发展。因此，研究这些蛋白质及其网络 对我们理解生物学至关重要毫不奇怪，这组特殊的蛋白质构成了 大部分药物的目标，也代表了有吸引力的目标 用于诊断和未来的精准医疗。不幸的是，许多这些秘密 蛋白质很难检测。现有的技术可以用来研究这些分泌蛋白 但是它们要么表现不佳，要么极其昂贵和/或由于其特定用途而复杂。 含有多种细胞类型。尽管研究分泌蛋白的需求无处不在， 不是一种广泛可用（廉价）且能够提供 同时在单细胞分辨率上获得关于多种分泌蛋白的信息， 在目前的项目中解决这一需求。 我们技术的核心是Plasmonic Fluor（PF），这是我们专有的超亮荧光 设计用于增强基于荧光的 免疫测定。我们已经建立了一个高灵敏度的多重免疫分析平台 利用这项技术，目前正在与外部合作者进行测试。这 平台包括一个便宜的，高性能的，单激光荧光微孔板/载玻片 阅读器优化与我们的PF一起使用。我们将扩展现有的平台，以容纳PF- 我们称之为荧光点的增强细胞分泌试验。升级后的平台将导致 从拟议的努力将是一个真正的通用和强大的工具，用于衡量 proteins. 在我们的初步工作中，我们已经成功地以商业规模生产， 与FITC、Cy 3和Cy 5光谱等效的链霉亲和素结合PF的重现性 （分别为PF 490、PF 550和PF 650）。我们还创造了三个完全独特的PF 激发/发射光谱：两个具有类似FITC的激发光谱，发射类似Cy 3， Cy 5（分别为PF490_550和PF490_650），以及一种具有类似Cy 3的激发光谱，但 发射类似Cy 5（PF550_650）。这些独特的PF是在原型规模合成的，我们的目标是 因为他们将允许使用3个激光器来询问6个独立的 目标的在对细胞分泌测定的适用性的证明中，链霉亲和素-PF 550和 将PF 650以小规模与抗体缀合并用于进行双色荧光斑点。 这些最初的PFluorospot实验表明，在这些实验中可以获得额外的信息。 相对于ELISP 0 T/FluoroSpot，这是一种更简单、更快速的检测方法。这是 可以使粘附细胞的单个细胞可视化并更好地定位分泌细胞。这是 对于理解异质性，共同文化中的空间效应， 多功能性，我们建议在我们的阅读器中添加第四个激光器以容纳细胞标记物。 此外，用我们现有的读数器读取单色荧光斑点测定， 显示出高灵敏度、高分辨率和短读取时间。 我们相信我们的Pfluorospot平台和阅读器将成为分泌组学的首选平台， 将使更多的生物研究人员能够测量重要的分泌蛋白质。