title: Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.
abstract: Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.
authors: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
authors: Nandlal
authors: N
authors: Choudhary
authors: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
authors: Avijit
authors: A
authors: Roy
authors: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
authors: A
authors: A
authors: Govindarajulu
authors: USDA-APHIS-PPQ, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
authors: M K
authors: MK
authors: Nakhla
authors: USDA-APHIS-PPQ, Center for Plant Health Science and Technology, Director's Office, Riverdale, MD 20737, USA.
authors: L
authors: L
authors: Levy
authors: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA. Electronic address: rhby@ufl.edu.
authors: R H
authors: RH
authors: Brlansky
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000302:isolation & purification; D000956:Antigens, Viral / Q000032:analysis / Q000276:immunology; D036022:Capsid Proteins / Q000032:analysis* / Q000276:immunology; D002957:Citrus / Q000821:virology*; D007118:Immunoassay / Q000379:methods; D051379:Mice; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins
keywords: Citrus leprosis virus C; DAS-ELISA; DBIA; IC-RT-PCR; Monoclonal antibody