title: Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.
abstract: Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.
authors.affiliation: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
authors.forename: Nandlal
authors.initials: N
authors.lastname: Choudhary
authors.forename: Avijit
authors.initials: A
authors.lastname: Roy
authors.forename: Leon M
authors.initials: LM
authors.lastname: Guillermo
authors.forename: D D
authors.initials: DD
authors.lastname: Picton
authors.forename: G
authors.initials: G
authors.lastname: Wei
authors.forename: M K
authors.initials: MK
authors.lastname: Nakhla
authors.forename: L
authors.initials: L
authors.lastname: Levy
authors.forename: R H
authors.initials: RH
authors.lastname: Brlansky
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002957:Citrus / Q000821:virology*; D003062:Codon / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007159:Immunologic Tests / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D003062:Codon; D011994:Recombinant Proteins
keywords: CiLV-C; Citrus leprosis disease; Dot-blot immunoassay; ELISA; Expressed coat protein; Polyclonal antibody