Cell wall antibodies without immunization: generation and use of de-esterified homogalacturonan block-specific antibodies from a naive phage display library

Homogalacturonan (HG) is a multi-functional pectic polysaccharide of primar y cell walls involved in calcium cross-linking and gel formation, and the r egulation of ionic status and porosity of the cell wall matrix, and is a so urce of oligosaccharins functioning in development and defence. Phage displ ay monoclonal antibodies with specificity for de-esterified stretches ('blo cks') of pectic HG have been isolated from a naive phage display library wi thout the need for immunization of animals or conjugation of an oligosaccha ride to protein. These antibodies, designated PAM1 and PAM2, bind specifica lly to de-esterified and un-substituted HG. Assays with a series of pectins de-esterified by the action of plant or fungal pectin methyl esterases ind icated that the antibodies were specific to de-esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of ant ibody binding to a series of oligogalacturonides indicated that optimal bin ding required in the region of 30 de-esterified GalA residues. The recognit ion of such a large epitope by these antibodies allows the HG block archite cture of primary cell walls to be identified and localized for the first ti me. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a 'sin gle pot' phage display approach.