SUMMARY

Azole fungicides are widely used in agriculture. For food safety reasons it is necessary to control levels of azole pesticide residues in environmental and agricultural samples. Chromatography is the most frequent analytical method for the determination of azole fungicide residues in food. However, chromatographic methods require time-consuming sample treatment and expensive equipment. Consequently, complementary analysis techniques have been developed. One of the most successful techniques is the immunoassay, due to its simplicity and rapidity. Polyclonal and monoclonal based immunoassays for the determination of azole fungicides have been reported. In last years, advances in molecular biology have enabled the production of recombinant antibodies. Recombinant DNA technology has been highly applied for the detection of high molecular weight molecules; while the production of recombinant antibodies to low mass organic molecules, as pesticides, is rather less explored. 

Thus, the main aim of this thesis is the production of recombinant antibodies against the azole fungicides tetraconazole, hexaconazole and imazalil, and its application to the analysis of these fungicide residues in agrofood samples. 

On the one hand, molecular biology techniques were applied to obtain recombinant scFv (single chain fragment variable) from hybridomas producing monoclonal antibodies against tetraconazole, hexaconazole and imazalil. Subsequently, these fragments were expressed in bacteria using plasmid vectors. In addition, a screening system for selecting colonies that produce recombinant antibodies against the target fungicides was optimized. This system was based on simultaneous competitive and non-competitive ELISAs of recombinant antibody soluble fraction. Finally, an iterative enrichment protocol (panning) based on the phage display technology was applied to obtain recombinant antibodies against hexaconazol and imazalil. Using these techniques, recombinant antibodies against the three target pesticides were obtained. Each antibody was expressed as soluble scFv protein and as fusion scFv-pIII protein.

On the other hand, molecular biology techniques were used to obtain recombinant scFv libraries from lymphocytes of mice immunized against tetraconazole, hexaconazole and imazalil immunogens. In this case, the library construction and panning steps were optimized. Thereby, one recombinant antibody showing high affinity to tetraconazole was obtained, and it was expressed as soluble scFv and as scFv-pIII protein. 

Next, the analytical properties of immunoassays based on monoclonal and recombinant antibodies were compared. In all cases, monoclonal based immunoassays showed more sensitivity than recombinant based immunoassays. With respect to specificity, both monoclonal and recombinant antibodies showed similar recognition pattern of related compounds. 

Finally, fruit juices were fortified with tetraconazole, hexaconazole or imazalil and analyzed by monoclonal and recombinant based ELISAs. All the immunoassays allowed the rapid and simple determination of each fungicide with acceptable precision and recovery.