Applying of Recombinant Major Coat Protein for Production of Specific Antibody and Efficient Detection of Citrus Tristeza Virus (CTV)

Background and Aims: Citrus tristeza virus (CTV) is the most economically important pathogen of citrus throughout the world. To avoid the ominous effect of disease in citrus growing area, producing of virus free plants and elimination of infected plants are imperative. For this aim obtaining simple and sensitive diagnosis tools is crucial. The main objective of present study is applying of recombinant protein technology for production of specific antibody and developing of serological assays for efficient detection of CTV within infected plants. Materials and Methods: The major coat protein (p25) of CTV was selected as a target for preparation of polyclonal antibody. The gene encoding p25 was recombinantly expressed in bacterial host and the protein was purified through affinity chromatography approach. The purified recombinant coat protein was used for immunization of rabbit. Specificity of the prepared serum against CP was confirmed through serological assay. The immunoglobulin molecules were purified from serum through staphylococcus protein A followed by conjugation to alkaline phosphatase (AP) and horse radish peroxidase (HRP) enzymes. The prepared antibodies and conjugates were used for detection of infected plants by double antibody sandwich- enzyme linked immunosorbent assay (DAS-ELISA) and dot-blot immune binding assay (DIBA). Results: The p25 protein was expressed in bacterial host. The SDS-PAGE results confirmed high purity and integrity of CTV major coat protein with the expected size of about 29 kDa. The indirect ELISA results revealed that the antibody titer was around 1:65000. The IgG molecules purified through protein A column and SDS-PAGE results confirmed purity of the prepared antibody. The concentration of IgG was quantified by comparison to standard protein, BSA, which was estimated around 1 mg/ml. The results obtained from DAS-ELISA and DIBA assays proved that prepared antibodies could be effectively applied for detection of CTV infected plants. Conclusions: The prepared antibody and conjugates were powerful tools for detection of infected plants. To the best of our knowledge this is the first work for applying of peroxidase enzyme in developing of ELISA assay against CTV.