Herstellung von Antikörpern gegen Venturia inaequalis zur Entwicklung von Biosensoren

Venturia inaequalis, the causative agent of apple scab, is one of the most serious diseases in apple orchards. Management of apple scab is focused on the prevention of primary infection by ascospores in the spring. As there is a demand for highly specific and realtime monitoring of the ascospore discharge, the development of a specific bionsensor was launched. The major objective of this thesis was the generation of ascospore-specific antibodies for the development of an antibody based biosensor. A recombinant phage diplay antibody library was generated from immunized mice and screened against Venturia ascospores. The binding of selected antibodies was monitored by flow cytometry, ELISA and fluorescence microscopy. ScFv portions of six different phage antibodies were expressed in E. coli and purified by IMAC (immobilized metal affinity chromatography). All antibodies showed high affinity to Venturia inaequalis ascospores but no cross-reactivity to Venturia conidiospores and mycelium. They also showed no reactivity to Plectosphaerella cucumerina, Alternaria brassicicola, Botrytis cinerea and Verticillium dahliae spores. These antibodies will be used in the ongoing development of a biosensor for the online detection of airborne Venturia inaequalis ascospores. A Western screening of a cDNA library of Venturia inaequalis with the above mentioned antibodies lead to the assumption, that the glycosylation of the recognized antigen plays an important role in antigen-antibody recognition. Lectin blocking and specific digestion of free sugar side chains gave further evidences