New Multiplexed Quantitative Detection of Pluripotency and Germ Layer Proteins

多能性和胚层蛋白的新型多重定量检测

• 批准号: 8130968
• 负责人: Bradley H. Garcia
• 金额: $ 61.39万
• 依托单位: PRIMORIGEN BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8130968
• 关键词: Address; Amino Acid Sequence; Antibodies; Binding; Biological Assay; Blood Cells; Cell Culture Techniques; Cell Line; Cell Therapy; Cells; Cessation of life; Collaborations; Communities; Companions; Computer software; Custom; Data; Data Analyses; Data Reporting; Data Set; Detection; Development; Doctor of Philosophy; Ectoderm; Endoderm; Enzyme-Linked Immunosorbent Assay; Evaluation; Feedback; Foxes; Funding; Generations; Genetic; Germ Layers; Goals; Government; Growth; Hospitals; Human; Human Genome; Image; Image Analysis; Immunoassay; Letters; Mesoderm; Metabolism; Methods; Molecular Profiling; Pattern; Peptide Sequence Determination; Performance; Persons; Phase; Principal Investigator; Printing; Proteins; Proteome; Pyroxylin; Reagent; Regenerative Medicine; Regulation; Reproduction; Research; Research Personnel; Run-On Assays; Sampling; Screening procedure; Secure; Signal Transduction; Small Business Innovation Research Grant; Smooth Muscle Actin Staining Method; Solutions; Source; Specificity; Speed; Spottings; Staging; Stem cells; Surface; System; Technology; Testing; Therapeutic Studies; Touch sensation; Transplantation; Umbilical Cord Blood; Universities; Validation; Variant; Wisconsin; Work; assay development; base; beta Tubulin; c-Myc Staining Method; claudin 6; cost; data acquisition; design; experience; high throughput screening; human embryonal carcinoma cell; miniaturize; novel; phase 1 study; pluripotency; programs; protein profiling; public health relevance; research study; software development; stem cell differentiation; tool; user friendly software

DESCRIPTION (provided by applicant): Primorigen Biosciences will use Phase II SBIR funds to accelerate and expand the development of new regenerative medicine cell therapies by developing a novel high throughput, low cost protein quantification system for characterizing pluripotent cell lines and early germ layer differentiation. Primorigen successfully confirmed the technical feasibility of this proposal by achieving both major goals of the Phase I project, first demonstrating (and now routinely using) its proprietary Spots on Dots" frameless microarray platform for high-throughput antibody screening and characterization, and then demonstrating feasibility of developing a multiplexed protein quantitation assay for cell pluripotency by identifying matched pairs of antibodies (working sandwich assays) for four key pluripotency targets: Sox2, Oct4, Nanog and Rex1. In Phase II, Primorigen will expand the panel of antibodies for pluripotency targets and develop a second array consisting of antibodies for 9 markers of early germ layer differentiation. These combined tools will provide a more complete solution for tracking downstream development of pluripotent cells, and will be validated using a variety of appropriately differentiated stem cells, and by obtaining independent validation of the performance and specificity of the assays. In addition, Primorigen will collaborate with a software developer to produce a software/flatbed scanner package that enables standardized analysis and reporting of data from assays run on the Spots on Dots" platform in a rapid, 'one touch' format. PUBLIC HEALTH RELEVANCE: Proteins facilitate many of the cell's most basic functions of reproduction, metabolism, growth, and programmed death. In the past few years, thousands of new protein sequences and post- translational variants have been identified by the human genome and proteome projects. This protein jackpot has hastened the drive to understand protein-based regulation but it also challenges researchers to find better biophysical methods to quantitatively detect protein markers. As a result, there is demand for inexpensive, miniaturized, multiplexed high throughput assays that can generate thousands of protein detection data points per experiment. Primorigen's SBIR proposal will address this need for the cell therapy research community by developing well characterized antibody and protein assay content for detecting and quantifying human cell markers of pluripotency and early germ layer differentiation, and then formatting these in a high throughput, low-cost multiplexed immunoassay format in a frameless microarray that is supported by one touch user friendly software for data analysis and reporting.

描述(申请人提供)：Primorigen Biosciences将利用第二阶段SBIR资金，通过开发一种新的高通量、低成本的蛋白质定量系统来加速和扩大新的再生医学细胞疗法的开发，以表征多潜能细胞系和早期胚层分化。Primorigen通过实现第一阶段项目的两个主要目标，成功地确认了这项建议的技术可行性，首先展示(现在经常使用)其专有斑点在Dots“无框架微阵列平台上进行高通量抗体筛选和鉴定，然后通过确定针对四个关键多能性靶标：Sox2、Oct4、Nanog和REx1的匹配抗体对(工作夹心法)，证明开发一种用于细胞多能性的多重蛋白质定量检测的可行性。在第二阶段，Primorigen将扩大针对多能性靶标的抗体组合，并开发第二个阵列，由针对9个早期生殖层分化标记的抗体组成。这些组合工具将为跟踪多能细胞的下游发展提供更完整的解决方案，并将使用各种适当分化的干细胞进行验证，并通过获得对检测的性能和特异性的独立验证来进行验证。此外，Primorigen将与一家软件开发商合作，生产一套软件/平板扫描仪套装，以快速、‘一触式’的格式对在Dots“平台上的斑点”上运行的化验数据进行标准化分析和报告。 与公共健康相关：蛋白质促进了细胞的许多最基本的功能，如生殖、新陈代谢、生长和程序性死亡。在过去的几年里，人类基因组和蛋白质组计划已经鉴定了数千个新的蛋白质序列和翻译后变体。这一蛋白质大奖加速了理解基于蛋白质的调控的努力，但也挑战了研究人员寻找更好的生物物理方法来定量检测蛋白质标记物。因此，存在对廉价、小型化、多路高通量分析的需求，这种分析可以在每次实验中产生数千个蛋白质检测数据点。Primorigen的SBIR建议将通过开发具有良好特性的抗体和蛋白质分析内容来检测和量化多能性和早期生殖层分化的人类细胞标记物，然后将这些内容以高通量、低成本的多重免疫分析格式格式化成无框架微阵列，该微阵列由一键用户友好型数据分析和报告软件支持，从而满足细胞治疗研究界的这一需求。