New Multiplexed Quantitative Detection of Pluripotency and Germ Layer Proteins

描述（由申请人提供）：Primorigen Biosciences将利用SBIR II期资金，通过开发一种新的高通量，低成本的蛋白质定量系统来表征多能细胞系和早期生殖层分化，加速和扩大新的再生医学细胞疗法的发展。Primorigen通过实现第一阶段项目的两个主要目标，首先展示（现在常规使用）其专有的spot on Dots无框架微阵列平台，用于高通量抗体筛选和表征，成功地证实了该提案的技术可行性。然后通过鉴定四个关键多能性靶点：Sox2， Oct4， Nanog和Rex1的匹配抗体对（工作三明治法），证明开发细胞多能性的多重蛋白定量分析的可行性。在第二阶段，Primorigen将扩大针对多能性靶点的抗体范围，并开发由针对9种早期生殖层分化标记物的抗体组成的第二阵列。这些组合工具将为跟踪多能细胞的下游发育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。此外，Primorigen将与一家软件开发商合作，生产一种软件/平板扫描仪软件包，该软件包能够以快速的“一键式”格式，对spot on Dots”平台上运行的分析数据进行标准化分析和报告。

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.