Selection of phage antibodies to surface epitopes of Phytophthora infestans

• Published in: Journal of immunological methods Vol. 228; no. 1; pp. 97 - 108
• Main Authors: Gough, Kevin C, Li, Yi, Vaughan, Tristan J, Williams, Andrew J, Cockburn, William, Whitelam, Garry C
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 31-08-1999; Elsevier
• Subjects: Animals; Antibodies, Fungal; Antibody Affinity; Antigens, Fungal - genetics; Antigens, Surface - genetics; Base Sequence; Biological and medical sciences; DNA Primers - genetics; Enzyme-Linked Immunosorbent Assay; Epitopes - genetics; Fundamental and applied biological sciences. Psychology; Microbiology; Microscopy, Fluorescence; Mycology; Pathogenicity, host-agent relations, miscellaneous strains, epidemiology; Peptide Library; Phage antibodies; Phytophthora - genetics; Phytophthora - immunology; Phytophthora - pathogenicity; Phytophthora infestans; Plants - microbiology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Surface epitopes; EDTA, ethylenediaminetetraacetic acid; Phage antibodies; scFv, single-chain Fv; Phytophthora infestans; PBS, phosphate buffered saline; PCR, polymerase chain reaction; CFU, colony forming unit; MBP, maltose binding protein; SDS, sodium dodecyl sulphate; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl β- d-thiogalactopyranoside; Surface epitopes; Performance evaluation; Purification; Plant pathogen; Antigenic determinant; Antibody; Escherichia coli; Phycomycetes; Host agent relation; Method; In vitro; Fungi; Characterization; Phage display; Bacteria; Enterobacteriaceae; Thallophyta
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00080-0

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen,
Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by
BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the
C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in
Escherichia
coli. These antibody fusion proteins bound to
P. infestans germlings and to mycelial homogenates from various
Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs 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
Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.