A Novel Multiplexed Assay for Rapid Antibody Screening

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表征胰腺2细胞和胰岛细胞的分化，从而加速和扩大新的再生医学细胞疗法的开发。Primorigen成功地确认了这项建议的技术可行性，实现了第一阶段项目的两个主要目标，首先展示(现在经常使用)其专有斑点在Dots的无框架微阵列平台上进行高通量抗体筛选和鉴定，然后通过在测试和开发的后期阶段使用9种额外的分析方法(淀粉酶2B、FOXA2、GCK、HNF6、Isl1、Neurod1、Neurog3、Pax4和Pax6)证明了开发一种用于胰腺β细胞分化的多重蛋白质定量分析的可行性，该方法与匹配的抗体对一起用于定量两个细胞分化的两个重要标记物(Nkx2.2和MafB)。在第二阶段，Primorigen将完成现有的最多11个2细胞分化标记物的分析小组(第一阶段)，并开发一个新的小组，用于识别和表征成熟的胰岛细胞群体，包括1、2、4、5和胰腺多肽(PP)细胞类型。这些组合工具将为跟踪胰岛β细胞的下游发育和成熟提供更完整的解决方案，并将通过获得对这些分析的性能和特异性的独立证实来进行验证。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满足这一需求。这些检测方法对检测和量化胰岛和胰岛细胞分化成熟的标志物具有重要价值。分析将在一个高吞吐量、低成本的多路复用平台中格式化，该平台由一键用户友好的数据分析和报告软件支持。

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 pancreatic 2 cell and islet cell 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 pancreatic beta cell differentiation by producing a prototype multiplexed assay with matched antibody pairs for quantifying two important markers of 2 cell differentiation (Nkx2.2 and MafB) and with 9 additional assays (Amylase 2B, FoxA2, GCK, HNF6, Isl1, NeuroD1, Neurog3, Pax4 and Pax6) in advanced stages of testing and development. In Phase II, Primorigen will complete the existing panel of assays for up to 11 markers of 2 cell differentiation (Phase I) and develop a new panel for identifying and characterizing mature islet cell populations, including 1, 2, 4, 5, and Pancreatic Polypeptide (PP) cell types. These combined tools will provide a more complete solution for tracking downstream development and maturation of pancreatic beta cells, and will be validated by obtaining independent corroboration 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 by developing well-characterized antibody and protein assay content. The assays will be valuable for detecting and quantifying markers of pancreatic beta- and islet-cell differentiation and maturation. The assays will be formatted in a high throughput, low-cost multiplexed platform that is supported by one touch user friendly software for data analysis and reporting.