Ž . Journal of Immunological Methods 228 1999 97–108 www.elsevier.nlrlocaterjim Recombinant technology Selection of phage antibodies to surface epitopes of Phytophthora infestans Kevin C. Gough a, ) , Yi Li a , Tristan J. Vaughan b , Andrew J. Williams b , William Cockburn a , Garry C. Whitelam a a Department of Biology, Adrian Building, UniÕersity of Leicester, UniÕersity Road, Leicester, LE1 7RH, Leicestershire, UK b Cambridge Antibody Technology, Unit B3, The Science Park, Melbourn, Cambridgeshire, SG8 6JJ, UK Received 21 January 1999; received in revised form 30 April 1999; accepted 10 May 1999 Abstract Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were Ž . 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 Ž . ELISA and diversity was established by Bst NI restriction enzyme digest patterns. Antibodies were subcloned as fusions at Ž . 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 hostrpathogen interactions. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phage antibodies; Phytophthora infestans; Surface epitopes AbbreÕiations: scFv, single-chain Fv; PBS, phosphate buffered saline; CFU, colony forming unit; PCR, polymerase chain reac- tion; ELISA, enzyme-linked immunosorbent assay; EDTA, eth- ylenediaminetetraacetic acid; IPTG, isopropyl b-D-thiogalacto- pyranoside; MBP, maltose binding protein; SDS, sodium dodecyl sulphate ) Corresponding author. Tel.: q44-116-2523339; Fax: q44- 116-2522791; E-mail: kcg3@le.ac.uk 1. Introduction Interest in the plant pathogen Phytophthora infes- tans has intensified in recent years due to dramati- cally increasing losses due to late blight in many Ž . parts of the world Daly, 1996 . Infection typically begins when sporangia land on plant tissue and 0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 99 00080-0