A Novel Multiplexed Assay for Rapid Antibody Screening

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胰腺细胞和胰岛细胞分化，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，通过生产具有匹配抗体对的原型多重分析方法，用于定量2种细胞分化的两个重要标记（Nkx2.2和MafB），以及在测试和开发的后期阶段的9种附加分析方法（淀粉酶2B， FoxA2， GCK, HNF6, Isl1, NeuroD1, Neurog3， Pax4和Pax6），证明开发胰腺细胞分化的多重蛋白质定量分析方法的可行性。在II期，Primorigen将完成现有的多达11个2细胞分化标记（I期）的检测小组，并开发一个新的小组，用于鉴定和表征成熟的胰岛细胞群，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腺β细胞的下游发育和成熟提供更完整的解决方案，并将通过获得对检测的性能和特异性的独立证实来验证。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 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.