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

Abstract

Homogalacturonan (HG) is a multi‐functional pectic polysaccharide of primary cell walls involved in calcium cross‐linking and gel formation, and the regulation of ionic status and porosity of the cell wall matrix, and is a source of oligosaccharins functioning in development and defence. Phage display monoclonal antibodies with specificity for de‐esterified stretches (‘blocks’) of pectic HG have been isolated from a naive phage display library without the need for immunization of animals or conjugation of an oligosaccharide to protein. These antibodies, designated PAM1 and PAM2, bind specifically 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 indicated that the antibodies were specific to de‐esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of antibody binding to a series of oligogalacturonides indicated that optimal binding required in the region of 30 de‐esterified GalA residues. The recognition of such a large epitope by these antibodies allows the HG block architecture of primary cell walls to be identified and localized for the first time. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a ‘single pot’ phage display approach.