Immunodetection of Venturia inaequalis Ascospores with Phage Antibodies

We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V-gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non-germinated ascospores. The binding of selected phage antibodies was monitored by enzyme-linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross-reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage-derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers