New Multiplexed Quantitative Detection of Pluripotency and Germ Layer Proteins

描述（由申请人提供）：Primorigen Biosciences将使用第二阶段SBIR资金，通过开发一种新型高通量、低成本的蛋白质定量系统来表征多能细胞系和早期胚层分化，加速和扩大新的再生医学细胞疗法的开发。Primorigen通过实现第一阶段项目的两个主要目标，成功地证实了该提案的技术可行性，（现在常规使用）其专有的点上点”无框架微阵列平台，用于高通量抗体筛选和表征，然后证明通过鉴定匹配的抗体对来开发用于细胞多能性的多重蛋白质定量测定的可行性（工作夹心测定）用于四个关键多能性靶标：Sox 2、Oct 4、Nanog和Rex 1。在第二阶段，Primorigen将扩大针对多能性靶点的抗体组，并开发由针对9种早期胚层分化标志物的抗体组成的第二个阵列。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，并通过获得对测定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，开发一个软件/平板扫描仪包，以快速、“一触式”的格式对在Spots on Dots平台上运行的检测数据进行标准化分析和报告。 公共卫生相关性：蛋白质促进细胞的许多最基本的功能，如生殖、代谢、生长和程序性死亡。在过去的几年中，人类基因组和蛋白质组计划已经鉴定了数千种新的蛋白质序列和翻译后变体。这种蛋白质头奖加速了理解基于蛋白质的调控的动力，但它也挑战研究人员找到更好的生物物理方法来定量检测蛋白质标记。因此，需要廉价的、小型化的、多路复用的高通量测定，其可以在每个实验中产生数千个蛋白质检测数据点。Primorigen的SBIR提案将通过开发用于检测和定量多能性和早期胚层分化的人类细胞标志物的良好表征的抗体和蛋白质测定内容，然后在无框架微阵列中以高通量、低成本的多路复用免疫测定格式格式化这些内容，该微阵列由用于数据分析和报告的一键式用户友好软件支持，从而满足细胞治疗研究界的这一需求。

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.