Selection of phage antibodies to surface epitopes of Phytophthora infestans

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage librar y displaying single-chain Fv (scFv) antibody fragments. The library was sub panned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Bind ing of monoclonal phage antibodies was demonstrated by enzyme-linked immuno sorbent assay (ELISA) and diversity was established by BstNI restriction en zyme digest patterns. Antibodies were subcloned as fusions at the C-terminu s of maltose binding protein (MBP) and expressed as soluble proteins in Esc herichia coli. These antibody fusion proteins bound to P. infestans germlin gs and to mycelial homogenates from various Phytophthora species. The bindi ng activities to mycelial homogenates of fungal species not belonging to th e order Peronosporales were substantially lower. Several phage-displayed sc Fvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophtho ra demonstrates the potential of phage antibody technology in isolating ant ibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions. (C) 1999 Elsevier S cience B.V. All rights reserved.