A Fluorescence-Based High-Throughput Platform for Glycotyping the Hematopoietic Cell Lineage

基于荧光的造血细胞谱系糖分型高通量平台

• 批准号: 10248374
• 负责人: Hannes Erich Buelow
• 金额: $ 42.75万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248374
• 关键词: Address; Affinity; Antibodies; Binding; Biological Assay; Biological Markers; Blood; Blood Cells; Cell Adhesion; Cell Differentiation process; Cell Line; Cell Lineage; Cell surface; Cells; Characteristics; Chimeric Proteins; Communities; Complement; Complex; Coupled; Data; Development; Diagnostic; Differentiation Antigens; Disease; Engineering; Epitopes; Erythroid Cells; Evaluation; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; Future; Glycosaminoglycans; Goals; Growth; Hematopoiesis; Hematopoietic; Heparitin Sulfate; Immunoglobulin Fragments; Immunoglobulin G; Label; Laboratories; Libraries; Lymphoid Cell; Malignant Neoplasms; Methods; Modification; Molecular; Molecular Profiling; Mus; Myeloid Cells; Nature; Oligosaccharides; Organism; Pathology; Pattern; Physiology; Polysaccharides; Population; Positioning Attribute; Production; Proteins; Protocols documentation; Reagent; Research; Resolution; Scheme; Sorting - Cell Movement; Standardization; Structure; Surface; System; Techniques; Technology; Testing; Therapeutic; Tissues; Transplantation; Weight; analytical method; base; cell motility; cell type; computerized tools; experimental study; in vivo; insight; member; novel; novel diagnostics; novel therapeutic intervention; polysulfated glycosaminoglycan; population based; progenitor; software development; sortase; stem; tool

PIs: Buelow – Steidl Project Summary The surface characteristics of cells in multicellular organisms are a direct reflection of their function. Sensitive methods for detecting and defining novel surface characteristics of cells offer great opportunities for gaining new mechanistic insights and developing new therapeutic strategies. Heparan sulfate (HS) glycans are ubiquitous on the cell surface of metazoans and are expressed with extraordinary temporal and spatial resolution. Since HS are amongst the most diverse molecules in nature, as a result of complex modification patterns of the glycans, they could potentially provide the means to strongly discriminate between different cell and tissue types. However, current analytical methods for HS glycan structure determination are expensive, technically challenging and often low resolution, limiting their discriminatory power and widespread use. The development of novel tools to define and exploit the structural characteristics of HS on the surface of cells could open new avenues with important diagnostic and, possibly, therapeutic potential. We will develop a high- throughput, multiplexed fluorescence-based platform to define the HS surface characteristics using a set of HS-specific single chain variable fragment (scFvs) antibodies. We will first engineer the set of HS-specific single-chain variable fragment antibodies with various immunotags as well as directly label them with fluorescent tags using Sortase based approaches. Next, we will systematically determine the HS motifs that are recognized by the scFv antibodies using a combination of microarrays with defined heparan sulfate oligosaccharides and novel computational tools, which we will develop to define the HS motifs recognized by these reagents. Finally, in proof of principle experiments, we will apply our tool box of immuno reagents against defined HS motifs as an orthogonal system to the well-established cluster of differentiation (CD) system to characterize and define hematopoietic cell lineages. We will leverage our laboratories' unique capabilities in protein production and hematopoiesis research to generate, test, and disseminate the fluorescently labeled scFvs reagents and protocols to the research community. We anticipate that our robust, efficient and economical assays will lead to more widespread and standardized analyses of HS characteristics, which will lay the foundation for the development of novel diagnostic and therapeutic approaches in the blood and other systems in the future.

PIS：比洛-斯泰德尔 项目摘要 多细胞生物体中细胞的表面特征是其功能的直接反映。 检测和确定电池新的表面特征的灵敏方法为 获得新的机制洞察力和开发新的治疗策略。硫酸乙酰肝素(HS)多糖是 普遍存在于后生动物的细胞表面，并以非凡的时间和空间表达 决议。由于HS是自然界中种类最多的分子之一，因此经过复杂的修饰 糖链的模式，它们可能提供了强烈区分不同细胞的手段 和组织类型。然而，目前用于HS多糖结构测定的分析方法成本较高， 技术上具有挑战性，分辨率往往较低，限制了它们的歧视性力量和广泛使用。这个 开发新的工具来确定和开发细胞表面HS的结构特征 可以开辟新的途径，具有重要的诊断和治疗潜力。我们将开发一种高- 吞吐量，基于多路荧光的平台，使用一组定义HS表面特性 HS特异性单链可变区(ScFvs)抗体。我们将首先设计一套特定于HS的 具有多种免疫标记的单链可变区抗体及其直接标记 使用基于山梨酸酶的方法的荧光标记。接下来，我们将系统地确定HS主题 使用带有定义的硫酸乙酰肝素的微阵列组合，由scFv抗体识别 寡糖和新的计算工具，我们将开发它们来定义由 这些试剂。最后，在原理实验的证明中，我们将应用我们的免疫试剂工具箱来对抗 将HS基序定义为与成熟的分化簇(CD)系统正交的系统，以 描述和定义造血细胞谱系。我们将利用我们实验室的独特能力 蛋白质生产和造血学研究以产生、测试和传播荧光标记的 单链抗体试剂和方案提供给研究界。我们期待着我们强大、高效和 经济分析将导致对HS特征进行更广泛和标准化的分析，这将 为开发血液和其他疾病的新诊断和治疗方法奠定基础 未来的系统。