Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays

• Published in: Journal of virological methods Vol. 243; pp. 177 - 181
• Main Authors: Choudhary, Nandlal, Roy, Avijit, Leon, M.G., Wei, G., Nakhla, M.K., Levy, L., Brlansky, R.H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2017
• Subjects: Antibodies; CiLV-C2; Citrus - virology; Citrus leprosis disease; Citrus virus; Colombia; Enzyme-Linked Immunosorbent Assay - methods; Plant Diseases - virology; Reverse Transcriptase Polymerase Chain Reaction - methods; Rhabdoviridae - immunology; Rhabdoviridae - isolation & purification; Sensitivity and Specificity; TAS-ELISA; Antibodies; CiLV-C2; TAS-ELISA; Citrus virus; Citrus leprosis disease
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2017.02.012

•Putative coat protein gene p29 of Citrus leprosis virus C2 (CiLV-C2) was amplified.•p29 gene was cloned and putative coat protein was expressed in E. coli.•Purified recombinant coat protein of CiLV-C2 was used for monoclonal antibodies and polyclonal antisera production.•Monoclonal antibody E7 and polyclonal antiserum UF715 were selected for detection of CiLV-C2 in TAS-ELISA.•MAb-E7 were selected for CiLV-C2 detection in IC-RT-PCR and was found more sensitive to TAS-ELISA. The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.