Detection of Sclerotinia sclerotiorum Using a Monomeric and Dimeric Single-Chain Fragment Variable (scFv) Antibody WILLIAM YAJIMA, † MUHAMMAD H. RAHMAN, † DIPANKAR DAS, § MAVANUR R. SURESH, § AND NAT N. V. KAV* ,† Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5, and Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors’ knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test. KEYWORDS: Canola; phage display; phytopathogen; scFv; Sclerotinia sclerotiorum INTRODUCTION Sclerotinia sclerotiorum (Lib.) de Bary is a well-known necrotrophic phytopathogenic fungus with over 400 different potential plant hosts (1, 2). The wide range of host plants includes numerous agriculturally important crops such as canola, in which the fungus is capable of causing the disease sclerotinia stem rot (3). Canola is grown in Europe and Asia as well as in North America, where, in Canada, the harvested acreage exceeded 14.6 million acres, leading to over 8.7 million tonnes of canola produced in 2007 (4). If stem rot inoculum is present in adequate quantities and environmental conditions are favor- able for infection, yield loss per plant can be as high as 50% (5). Risk assessment tools or disease management strategies that can be used to minimize the deleterious effects of S. sclerotiorum include the use of disease-forecasting protocols such as a petal test kit and a checklist or survey, appropriate seeding to prevent high crop density, and fungicide application (6-11). Currently, there are only canola cultivars that are partially resistant to S. sclerotiorum infection, and there are few genetic sources of resistance for breeders, which highlights the importance of effective disease forecasting and management procedures (12, 13). In addition, the diverse range of plants that can act as hosts as well as the longevity of sclerotia can limit the effectiveness of crop rotations (14). Furthermore, novel tests to detect the presence of S. sclerotiorum in a particular field or during a particular growing season may supplement currently used disease-forecasting systems and provide canola growers with another tool to assess stem rot risk and thus determine the need for fungicide to limit the damage caused by this fungus. Previous attempts at using antibodies to detect S. sclerotiorum were based on polyclonal antibodies (15, 16). Antibody produc- tion requires the immunization of animals followed by collection of serum containing the antibodies generated by the immune systems of the animals. A potential drawback of polyclonal antibody production is the limited amount of serum that can be collected from individual animals, which necessitates continual immunization. Furthermore, the polyclonal antibodies in the collected serum are diverse in terms of which epitopes are recognized and, hence, may have a broader cross-reactivity. An alternative involves the production and use of monoclonal antibodies using hybridoma technology (17). Unlike polyclonal antibody production, monoclonal antibodies can be produced so that each clone will produce antibodies that can bind to a specific epitope of a specific antigen. The nature of monoclonal antibody production also theoretically enables the continual production of specific antibodies provided that the hybridoma cells are properly maintained. Alternatively, recombinant anti- body technology can be utilized to generate single-chain variable * Address correspondence to this author at 3-18M Agriculture/ Forestry Centre, University of Alberta, Edmonton, AB, Canada T6G 2P5 [telephone (780) 492-7584; fax (780) 492-4265; e-mail nat@ualberta.ca]. † Department of Agricultural, Food and Nutritional Science. § Faculty of Pharmacy and Pharmaceutical Sciences. J. Agric. Food Chem. 2008, 56, 9455–9463 9455 10.1021/jf801768g CCC: $40.75  2008 American Chemical Society Published on Web 09/19/2008