An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.
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/kappa 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 tha such RG-II esterification is absent near the plasma membrane.
Williams
M N
MN
Complex Carbohydrate Research Center, University of Georgia, Athens 30602, USA.
Freshour
G
G
Darvill
A G
AG
Albersheim
P
P
Hahn
M G
MG
D000818:Animals; D001483:Base Sequence; D003090:Coliphages; D017931:DNA Primers / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D015723:Gene Library; D005803:Genes, Immunoglobulin; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry / Q000276:immunology*; D012210:Rhamnose
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D010368:Pectins; D012210:Rhamnose