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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：
9813902
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 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9813902
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.

pi：布洛-斯泰德尔