Development of a Carbohydrate Microarray

碳水化合物-蛋白质相互作用对于广泛的生物过程如炎症、细菌和病毒粘附以及转移是必不可少的。因此，人们对鉴定碳水化合物结合蛋白和开发调节其活性的配体产生了极大的兴趣。碳水化合物-蛋白质相互作用的分析由于许多因素而变得复杂。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。其次，研究结合的传统方法不是高通量的，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非常弱。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的方式存在，并且碳水化合物的间隔和取向在识别中起着关键作用。 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。使用阵列，可以同时评估凝集素、抗体、细胞或病毒与阵列上所有碳水化合物的结合。该阵列提供了碳水化合物的多价呈现，并且小型化格式仅需要皮克量的每种碳水化合物用于测定。我们已经成功地产生了碳水化合物微阵列和测定来检测结合。为了构建我们的阵列，我们将碳水化合物连接到载体蛋白（如牛血清白蛋白）上，以产生新糖蛋白。 这些新糖蛋白以及天然糖蛋白使用机器人微阵列仪打印到环氧涂层的显微镜载玻片上以产生微阵列。新糖蛋白可用作用于其它测定（例如ELISA）的多价试剂、多价抑制剂和多价免疫原。最近，我们专注于扩大和改善碳水化合物微阵列。一个目的是增加阵列上聚糖的数量。在过去一年中，我们将阵列组件的大小从204个扩展到220个。由于阵列表面上的碳水化合物的间距和方向对于形成多价复合物是至关重要的，第二个目标是探索用于改变我们的阵列组件的呈现的方法。在过去的一年里，我们开发了一种方法，通过混合或掺杂未修饰的白蛋白来改变新糖蛋白的密度。这种方法能够更好地分析血清抗体亚群，并有助于鉴定凝集素的多价抑制剂。

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 204 to 220 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. Over the last year, we developed a method to vary the neoglycoprotein density by mixing or doping in unmodified albumin. This approach enables better profiling of serum antibody subpopulations and facilitates the identification of multivalent inhibitors for lectins.