This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.
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