Development of a Carbohydrate Microarray

碳水化合物-蛋白质相互作用对于许多生物学过程是必不可少的。 过程，如炎症、细菌和病毒粘附以及转移。因此，在本发明中， 人们对鉴定碳水化合物结合蛋白很感兴趣， 开发配体以调节它们的活性。碳水化合物-蛋白质相互作用分析 是由很多因素造成的首先，碳水化合物极难分离 或合成。因此，在许多情况下只能获得少量。第二、 用于研究结合的传统方法不是高通量的并且需要大量的 材料第三，碳水化合物和蛋白质之间的单价相互作用是众所周知的。 弱.为了获得高亲合力，碳水化合物结合蛋白含有多个结合位点 并形成多价复合物。因此，碳水化合物必须以多价形式存在， 方式和碳水化合物的间距和方向在 识别.碳水化合物微阵列含有许多不同的碳水化合物，或聚糖， 以空间限定的排列固定在固体支持物上。使用数组，可以 评估凝集素、抗体、细胞或病毒与阵列上所有碳水化合物的结合 同步该阵列提供碳水化合物的多价呈现，并且 小型化形式仅需要皮克量的每种碳水化合物用于测定。我们有 成功地产生了碳水化合物微阵列和测定以检测结合。来构建我们的 我们将碳水化合物附着在载体蛋白上，如牛血清白蛋白， 新糖蛋白这些新糖蛋白，以及天然糖蛋白， 使用机器人微阵列仪制备环氧涂层的显微镜载玻片以产生微阵列。 新糖蛋白可用作用于其它测定的多价试剂，例如ELISA，如 多价抑制剂和作为多价免疫原。最近，我们专注于扩大 以及改进碳水化合物微阵列。一个目标是增加聚糖的数量 在数组上。去年，我们将阵列的大小从125个阵列扩展到204个阵列 件.由于碳水化合物在阵列表面上的间距和方向是关键的， 对于形成多价复合物，第二个目标是探索改变 介绍我们的阵列组件。我们通过调节碳水化合物的浓度 每个白蛋白分子上附着的碳水化合物的数量。我们已经证明， 改变密度的方法可用于评价凝集素、单克隆抗体、 抗体和血清抗体。我们目前正在研究不同密度的 通过混合或掺杂在未修饰的白蛋白中来制备新糖蛋白。

Carbohydrate-protein interactions are essential for a wide range of biological processes such as inflammation, bacterial and viral adhesion, and metastasis. As a result, there has been significant interest in identifying carbohydrate binding proteins and developing ligands to modulate their activity. Analysis of carbohydrate-protein interactions is complicated by a number of factors. First, carbohydrates are extremely difficult to isolate or synthesize. Therefore, only small amounts can be obtained in many cases. Second, traditional methods for studying binding are not high-throughput and require large amounts of material. Third, monovalent interactions between carbohydrates and proteins are notoriously weak. To achieve a high avidity, carbohydrate-binding proteins contain multiple binding sites and form multivalent complexes. As a result, carbohydrates must be presented in a multivalent fashion and the spacing and orientation of the carbohydrates play a critical role in recognition. Carbohydrate microarrays contain many different carbohydrates, or glycans, immobilized on a solid support in a spatially-defined arrangement. Using an array, one can evaluate binding of a lectin, antibody, cell, or virus to all the carbohydrates on the array simultaneously. The array provides a multivalent presentation of carbohydrates, and the miniaturized format requires only picogram amounts of each carbohydrate for an assay. We have successfully generated a carbohydrate microarray and assay to detect binding. To construct our array, we attach carbohydrates to a carrier protein, such as bovine serum albumin, to produce neoglycoproteins. These neoglycoproteins, as well as natural glycoproteins, are printed onto epoxide coated microscope slides using a robotic microarrayer to produce microarrays. Neoglycoproteins can be used as multivalent reagents for other assays such as ELISAs, as multivalent inhibitors, and as multivalent immunogens. Recently, we have focused on expanding and improving the carbohydrate microarray. One objective is to increase the number of glycans on the array. Over the last year, we have expanded the size of our array from 125 to 204 array components. Since spacing and orientation of carbohydrates on the array surface is critical for formation of a multivalent complex, a second objective is to explore methods for varying presentation of our array components. We have varied the carbohydrate density by modulating the number of carbohydrates attached to each molecule of albumin. We have shown that this method for varying density is useful for evaluating recognition of lectins, monoclonal antibodies, and serum antibodies. We are currently investigating varying the density of neoglycoproteins by mixing or doping in unmodified albumin.