Monoclonal Antibody Toolkit for Plant Cell Walls

Specific Goals:

Generate, from hybridoma culture and from phage display single-chain antibody libraries, a series of monoclonal antibodies that recognize diverse carbohydrate structures within plant cell walls.

Characterize the epitope structures recognized by antibodies that each bind to unique glycostructures within the plant cell wall.

Make the monoclonal antibodies available to the scientific community.

Glycoconjugate Immunobiology

Most polysaccharides are weakly immunogenic and do not stimulate the production of high titers of antibodies. This is due in large part to the fact that the response to polysaccharides is believed to be T cell-independent. In contrast, proteins which are highly antigenic stimulate a T cell-dependent response. To generate a glycan that will stimulate a T cell-dependent response requires the use of glycoconjugate antigens in which an oligo- or polysaccharide is covalently coupled to a protein (Jennings and Sood [1994]. Synthetic glycoconjugates as human vaccines. In Neoglyconjugates: Preparation and Applications. Acadmeic Press, pp. 325-371).

An important consideration during the synthesis of glycoconjugate antigens is to minimize changes in structure and conformation of the individual components. Many plant polysaccharides have acid (furanose and keto residues) and base (O-acetyl, methyl esters) sensitive substituents. Such residues may be important for antigenicity. Thus, procedures to couple oligo- and polysaccharides to proteins must be sufficiently mild to retain the primary structure of the glycan. Moreover, immunogenicty is likely to be dependent on the three dimensional structure of the polysaccharide. Antibodies that recognize carbohydrates typically bind to structural motifs (epitopes) composed of one to six glycoses. However, the conformation of such short chain oligosaccharide epitopes may depend on their structural context within a larger oligo- or polysaccharide. Thus the choice of immunogen (short oligosaccharide, longer oligosaccharide, or polysaccharide) may influence whether or not antibodies capable of binding to the native polysaccharide in the wall are obtained..

A overview of our experimental approch for generating and characterizing monoclonal antibodies that recognize plant cell walls is shown in the figure below:

Follow the links below the image to obtain more information
Experimental plan - outline

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