Research Project
8744384
Glycosylation is one of the most frequently occurring post-translational modifications of proteins. Changes in expression levels of certain glycoproteins and altered glycosylation patterns have been detected in most of the major human cancer types and at different stages of tumor progression. Early detection of glycosylat ion changes is therefore crucial for cancer diagnosis and treatment. O-Glycosylation acts antagonistically to phosphorylation, changing the activity and function of the protein. An extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in oncogenes and tumor suppressors plays an important role in tumorigenesis. Currently, no site-specific affinity reagents for O-glycosylation are commercially available. Most of the currently used methods require expensive instruments and are time consuming. Thus, there is a need to develop well characterized high affinity site-specific reagents to study the function of carbohydrate modifications of proteins in cancer biology. In the Phase I proposal, we generated thirty three polyclonal chicken IgY antibodies, using O-GlcNAc modified peptide antigens derived from cancer-relevant proteins. In this Phase II proposal, we will generate renewable high-affinity monoclonal scFv IgYs against 33 O-glycosylated antigens and we will evaluate the performance of each purified monoclonal IgY in Western blotting, immune-precipitation and imaging.
