Generation of a specific monoclonal recombinant antibody against 'Candidatus Phytoplasma aurantifolia' using phage display technology

Witches' broom disease of lime (WBDL) is a destructive disease caused by 'Candidatus Phytoplasma aurantifolia' and is a limiting factor for lime production in Southern Iran. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. Lack of natural resistance against phytoplasma diseases has highlighted the importance of alternative approaches and recombinant antibody mediated resistance is among them. The immunodominant membrane protein (IMP) is a major protein present on the surface of phytoplasma cells and is important for both diagnostics and interactions with plant hosts and insect vectors. Generation of specific recombinant antibodies with high binding ability against IMP proteins is beneficial for both diagnostic purposes and development of recombinant antibody-mediated resistance against disease. Phage display is a powerful technology for generation of specific recombinant antibodies such as single chain variable fragment antibodies (scFv). This study describes generation of a specific scFv fragment through panning of naive phage display libraries. For this aim, the gene encoding the IMP protein was isolated and cloned into a bacterial expression vector and recombinant IMP protein was expressed in bacterial cells and purified through affinity chromatography. Purified recombinant IMP protein was used for panning of naive Tomlinson I and J scFv phage display libraries. Following three rounds of panning for selection and amplification of specific binders, capability of individual clones for production of specific scFvs was evaluated by an ELISA assay. The preliminary results showed generation of specific scFv recombinant antibodies with strong binding ability against the IMP protein. Complementary studies revealed that the scFvs are able to bind native IMP and could detect the presence of phytoplasma in infected plants. Further immunoassay analysis confirmed that generated scFvs are able to detect epitopes along the IMP amino acid residues. As far as we know, this is the first succesful application of phage display libraries for generation of scFv recombinant antibodies against phytoplasma cells.