Cell Mimic Microarrays for the Multivalent Pathogen Profiling & Characterization

描述（由申请人提供）：本研究项目旨在开发用于病原体定量分析和表征，病原体抑制剂筛选和疫苗开发的流体聚糖微阵列。该方法针对病原体攻击初始阶段的共同机制：通过受体蛋白和聚糖分子之间的多价相互作用识别并附着在宿主细胞上。聚糖的巨大变化和多价相互作用的复杂性使得大规模分析和分析技术的使用成为必要，特别是聚糖微阵列。提出的流体方法克服了当前聚糖微阵列技术的两个主要限制：缺乏流动性和难以定量控制聚糖密度。多价细胞表面相互作用通常需要流体细胞膜环境的移动性，并且是表面聚糖密度的强大功能。为了在分析和表征中定量应用聚糖微阵列，必须确保聚糖密度在广泛范围内的流动性和控制。第二阶段的具体目标是：目标1：使用血凝素（流感病毒的主要抗原）和树突状细胞受体DC-SIGN （HIV-1病毒上甘露糖部分的结合受体）作为模型系统，并建立二级相互作用在结合亲和力、亲和度和特异性中的作用。这些实验将建立流体微阵列在分析和表征复杂病原体-细胞表面相互作用方面的普遍适用性；目的2：使用几种对甘露糖具有不同亲和力和选择性的大肠杆菌菌株作为模型系统，并建立流体和密度梯度聚糖微阵列可用于定量分析同一物种菌株之间结合亲和力和多价性的可变性。量化这种变异对于理解和监测随机突变如何导致新的病原体威胁至关重要，最近爆发的禽流感和猪流感就是例证；目标3：建立优化流体聚糖微阵列的化学程序，包括支持的脂质双分子层斑点的空间限制，斑点区域外表面的有效阻断，微阵列干燥和再水化的可恢复性，以及内容聚糖微阵列的长期稳定性。这些实际问题必须解决，以发展流体聚糖微阵列作为一个可行的产品。这项研发计划的长期目标是开发一种有效的高通量工具来对抗病原体威胁。

DESCRIPTION (provided by applicant): This research project aims to develop fluidic glycan microarrays for the quantitative profiling and characterization of pathogens, and for the screening of pathogen inhibitors and the development of vaccines. The approach targets a common mechanism at the initial stage of pathogen attack: the recognition of and attachment onto host cells via multivalent interaction between receptor proteins and glycan molecules. The tremendous variation in glycans and the complexity in multivalent interaction have necessitated the use of large-scale profiling and analysis techniques, particularly glycan microarrays. The proposed fluidic approach overcomes two major limitations of current glycan microarray technology: the lack of mobility and the difficulty in quantitatively controlling glycan density. Multivalent cell surface interactions often require mobility of the fluidic cell membrane environment and are strong functions of surface glycan density. In order to quantitatively apply the glycan microarray in profiling and characterization, one must ensure mobility and control of glycan density over a broad range. The specific aims during phase-II are: Aim 1: using haemagglutinin, a predominant antigen on influenza viruses, and the dendritic cell receptor DC-SIGN, a binding receptor for mannose moieties on HIV-1 virus, as model systems and establish the roles of secondary interactions in binding affinity, avidity, and specificity. These experiments will establish the general applicability of the fluidic microarrays in profiling and characterizing complex pathogen-cell surface interactions; Aim 2: using several strains of E. coli with varying affinity and selectivity towards mannose as model systems and establishing that the fluidic and density gradient glycan microarray can be used to quantitatively profile the variability in binding affinity and multivalency among strains of the same species. Quantifying such variability is essential to the understanding and surveillance of how random mutations can lead to new pathogen threats, as exemplified by the recent outbreaks of avian flu and swine flu; Aim 3: To establish chemical procedures for the optimization of the fluidic glycan microarray, including spatial confinement of the supported lipid bilayer spots, efficient blocking of surfaces outside the spotted areas, recoverability in drying and rehydration of the microarrays, and long term stability of content glycan microarrays. These practical issues must be addressed in developing the fluidic glycan microarray as a viable product. The long-term goal of this R&D plan is to develop an effective high-throughput tool in the combat against pathogen threats.