Characterizing Antibodies Antibodies secreted by stable , monoclonal hydridoma lines or made by single, selected phage will be grouped according to their reactivities with a panel of structurally defined plant cell wall polysaccharide antigens using a sandwich ELISA. Proteins A and L bind to scFvs and will be used in place of secondary antibodies to detect phage-derived antibodies. Antibodies will also be grouped according to the patterns of immunolabeling of root, stem, and leaf tissues of Arabidopsis, Medicago, tomato, and rice plants. These initial screens will allow us to group antibodies into reactivity classes . Such information will be used to refine the ongoing immunogen preparation and immunization stratergies. For example, if numerous antibodies recognize a single or small number of carbohydrate epitopes then we will target our immunogens away from the immunodominant structure. These initial reactivity screens will also identify polysaccharides that have the highest binding to a given antibody thereby providing a starting point for the investigation of epitope structure.

Characterizing Epitope Structures Determining the binding specificties of the monoclonal antibodies is essential to enhance the value of these tools for functional genomics and immunolocalization studies of plant cell walls. Recent improvements in the availability of homogeneous glycan and glycosyl hydrolases, in the methods for chemical synthesis of oligosaccharides, and the development of glyco-arrays will facilitate the characterization of carbohydrate epitopes. Selective fragmentation of polysaccharides using homogeneous glycosyl hydrolases (e.g. arabinofuranosidase, galactosidase) and glycanhydrolases (e.g. endo arabinanses and galactanases) in combination with competitive ELISAs will be used to determine which structural features of a polysaccharide are likely to be recognized by a given antibody. This information will then be used to target the chemical synthesis oligosaccharides that cover the structural variants for the portion of the polysaccharide recognized by the antibody. Alternatively, we will generating oligosaccharides from polysaccharides using a combination of enzymic and chemical methods. For example, xylogluan oligosaccharides that vary in the extent and nature of their side chain substituents and length of glucan backbone can be generated by a combination of endoglucanse and xylosidase treatments together with selective acid hydrolysis. A combination of chemical and enzymic methods will also be used to generate pectic oligosaccharides that include the arabinose- and galactose containing side chains of RG-I as well as the galacturonan and rhamnogalacturonan backbones of HG and RG-I. Advances in combinatorial approaches to oligosaccharide synthesis have resulted in the generation of oligosaccharide libraries which together with methods for the immobilization of oligosaccharides on solid surfaces have allowed the creation of oligosaccharide micro-arrays. Such glyco-arrays have considerable potential for antibody screening and epitope characterization. We have established a collaboration with Richard Alvarez (U. Oklahoma) who is actively developing glyco-arrays (Glyco-consortium glycoarrays). Plant cell wall-related oligosaccharides that we generate will be added to the glyco-arrays being developed in the Alvarez lab. We will screen all antibodies that we obtain againsts the Alvarez glyco-array to identify oligosaccharides on the array that are recognized by our antibodies

Polysaccharide Isolation Fragmentation of Polysaccharides Coupling of Glycans to Proteins

Generating Hybridoma Cell Lines scFv phage-display libraries ELISA

Characterizing Antibodies Characterizing Epitope Structures

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A National Science Foundation-funded (Grant No DBI-0421683) research project at The Complex Carbohydrate Research Center of The University of Georgia