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.

抽象的 分泌蛋白是生物系统中局部和全身细胞信号的基础。 涉及这些蛋白质的网络的调节和失调在 疾病的健康，衰老和发展。因此，研究这些蛋白质及其网络 对于我们对生物学的理解至关重要。毫不奇怪，这组蛋白质构成 药物的大部分靶标，也代表有吸引力的靶标 用于诊断和未来的精密医学工作。不幸的是，其中许多分泌 蛋白质难以检测。现有的技术用于研究这些分泌的蛋白质 但是，鉴于它们的特殊性，他们要么表现不佳，要么非常昂贵和/或复杂。 包含多种细胞类型。尽管需要研究分泌蛋白的需求无处不在，但 不是一项广泛可用（廉价）并且能够提供的技术 单细胞分辨率同时了解多种分泌蛋白的信息，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激子荧光（PF），我们的专有，Ultrabright 荧光纳米构造旨在增强基于荧光的性能 免疫测定。我们已经建立了高敏性，多重免疫测定平台 利用这项技术，该技术目前正在与外部合作者进行测试。这 平台包括廉价的高性能，单重荧光微板/幻灯片 读取器优化用于我们的PFS。我们将扩展此现有平台以适应PF- 增强的细胞分泌测定法，我们称为Pfluorospot。将导致的升级平台 从拟议的工作中，将是一个真正多才多艺的强大工具，用于测量 蛋白质。 在我们的初步工作中，我们已经成功地以商业规模制造，并且很高 可重复性链霉亲和素偶联的PF，在频谱上等效于FITC，CY3和CY5 （PF490，PF550和PF650）。我们还创建了三个完全独特的PF 激发/发射光谱：两个具有激发光谱，例如FITC，并像CY3一样发射 Cy5（分别为PF490_550和PF490_650），并且具有像CY3一样激发频谱 发出例如CY5（PF550_650）。这些独特的PF是按原型量表合成的 在这项工作中扩大规模，因为它们将允许3个激光器分开询问6个激光器 目标。为了证明适用于细胞分泌测定的适用性，链霉亲和素Pf550和 将PF650与小型抗体结合在一起，并用于执行双色Pfluorospot。 这些最初的pfluorospot实验表明，可以在其中获得其他信息 相对于ELISPOT/Fluorospot的测定，执行更简单，更明显。是 可能可视化单个细胞的粘附细胞并更好地定位分泌细胞。这是 对于理解异质性，在共文化中的空间效应以及 多功能性，我们建议向读者添加第四激光以适应细胞标记。 此外，我们现有的读者和 表现出高灵敏度，高分辨率和简短的阅读时间。 我们认为，我们的Pfluorospot平台和读者将成为首选的秘密平台，以及 将使更多更多的生物学研究人员能够测量重要的分泌蛋白质。