Abstract
Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis.
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