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

基本信息

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
项目类别：
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

项目摘要

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：Buelow - Steidl 项目摘要多细胞生物中细胞的表面特征是其功能的直接反映。检测和定义细胞的新表面特征的敏感方法为您提供了巨大的机会获得新的机械见解并制定新的治疗策略。硫酸乙酰肝素（HS）聚糖是多余的后生动物表面无处不在，并以非凡的临时和空间表达解决。由于HS是自然界中最长的分子之一，因此由于复杂的修饰而导致聚糖的模式，它们可能会提供强烈区分不同细胞的手段和组织类型。但是，当前用于HS聚糖结构确定的分析方法很昂贵，在技术上挑战，通常是低分辨率的，限制了它们的歧视能力和宽度的使用。这开发新的工具来定义和探索细胞表面上HS的结构特征我们将发展一个高级吞吐量，基于荧光的荧光平台，使用一组一组定义HS表面特征 HS特异性的单链可变片段（SCFV）抗体。我们将首先设计一组HS特定的单链可变片段抗体，具有各种免疫原子，并将其直接标记为使用基于分子酶的方法的荧光标签。接下来，我们将系统地确定HS主题使用SCFV抗体使用与定义的硫酸乙酰肝素的组合来识别SCFV抗体寡糖和新颖的计算工具，我们将开发这些寡糖，以定义由HS图案识别的HS主题这些试剂。最后，为了证明主要实验的证明，我们将应用我们的免疫试剂工具盒定义的HS图案是与公认的分化集群（CD）系统的正交系统表征并定义造血细胞谱系。我们将利用实验室在蛋白质产生和造血研究以生成，测试和传播标记的荧光 SCFV试剂和研究界的协议。我们预计我们的稳健，高效和经济测定将导致对HS特征进行更多宽度和标准化分析，这将为在血液和其他方面开发新型诊断和治疗方法的发展奠定了基础将来的系统。