An Antibody Fab Selected from a Recombinant Phage Display Library Detects Deesterified Pectic Polysaccharide Rhamnogalacturonan II in Plant Cells

• Published in: The Plant cell 1996-04, Vol.8 (4), p.673-685
• Main Authors: Myron N. V. Williams, Freshour, Glenn, Darvill, Alan G., Albersheim, Peter, Hahn, Michael G.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Bacteriophages; Base Sequence; Cell membranes; Cell walls; Coliphages; DNA Primers - chemistry; Epitopes; Fluorescent Antibody Technique, Indirect; Gene Library; Genes, Immunoglobulin; Immunoglobulin Fab Fragments - chemistry; Immunohistochemistry; Libraries; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Monoclonal antibodies; Pectins - chemistry; Pectins - immunology; Plant cells; Plants; Polysaccharides; Rhamnose
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.8.4.673

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/κ antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and that such RG-II esterification is absent near the plasma membrane.