THE PLANT CELL, Vol 8, Issue 4 673-685, Copyright © 1996 by American Society of Plant Biologists
An Antibody Fab Selected from a Recombinant Phage Display Library Detects Deesterified Pectic Polysaccharide Rhamnogalacturonan II in Plant Cells
MNV. Williams, G. Freshour, A. G. Darvill, P. Albersheim and M. G. Hahn
Complex Carbohydrate Research Center, 220 Riverbend Road, University of Georgia, Athens, Georgia 30602
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/k 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.
