HEPARAN SULFATE LIGAND REQUIREMENTS OF PHAGE DISPLAY ANTIBODIES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 货车Kuppepyruvate实验室采用噬菌体展示技术产生抗HS单链可变片段抗体，其选择性地识别HS寡糖基序。 重要的是，许多抗体显示出各种组织切片的独特染色模式，并且具有区分健康和患病组织的能力，后者包括肿瘤和肾脏病理组织。 然而，抗体技术的主要限制是缺乏对抗体识别的HS表位的硫酸化模式的了解。预计BTBR技术可以解决这一重要缺陷。该资源将开发的集成方法将用于鉴定噬菌体展示衍生的单链抗体的配体需求。 在这种方法中，HS将通过化学和/或酶促方法部分片段化，并且通过尺寸排阻色谱法和SAX分级分离得到的寡糖混合物。 以高亲和力与抗体结合的片段将通过亲和纯化分离，并通过质谱方法确定化合物的化学结构。 推定的配体和结构类似物将通过模块化方法制备，并且所得化合物将用于建立结构活性关系（SAR）。 此外，将通过计算方法建立推定的寡糖配体，并且还将对所得化合物进行化学合成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Van Kuppevelt laboratory has adopted phage display technology to generate anti-HS single chain variable fragment antibodies, which selectively recognize HS oligosaccharide motifs. Importantly, many of the antibodies show unique staining patterns of various tissue sections and have an ability to distinguish between healthy and diseased tissue, the latter including tumorous and nephropathological tissue. However, a major limitation of the antibody technology is a lack of knowledge of sulfation patterns of HS epitopes that are recognized by the antibodies. It is to be expected that the BTBR technology can address this important deficiency. The integrated approach that will be developed by this resource will be employed to identify ligand requirements of the phage display derived single chain antibodies. In this approach, HS will be partially fragmented by chemical and/or enzymatic approaches and the resulting mixtures of oligosaccharides fractionated by size exclusion chromatography and SAX. Fragments that bind with high affinity to an antibody will be isolated by affinity purification and the chemical structures of the compounds determined by mass spectroscopic approaches. Putative ligands and structural analogs will be prepared by a modular approach and the resulting compounds will be employed to establish structure activity relationships (SAR). In addition, putative oligosaccharide ligand will be established by computational approaches and the resulting compounds will also be subjected to chemical synthesis.