Anti–Pepper mild mottle virus Polyclonal Antibody : Production, Characteristics and Diagnostic Application

Abstract

Pepper mild mottle virus (PMMoV) is a plant virus in the genus Tobamovirus infecting peppers which are economic plants. The virus can be transmitted from plant to plant mechanically and by seed, causing serious yield loss. Since no chemical control is available therefore screening for disease–free seed by serological assay is recommended for control measure. Production high quality antibody is very important in this case. The objective of this study is to produce a specific polyclonal antibody against PMMoV by using its recombinant coat protein (PMMoV–CP) as an antigen. The virus isolated from Kamphaeng Saen district, Nakhon Pathom Province was used as a template for PMMoV coat protein (PMMoV–CP) gene cloning. The PMMoV–CP gene, 474 bp, was ligated into pQE80–L expression vector and produced the recombinant PMMoV–CP at 17.5 kDa which was then immunized into a New Zealand White rabbit subcutaneously and intramuscularly. The antiserum had been collected weekly for the period of eight weeks. Their titers ranged from 25,600 – 204,800 with the highest titer presented in 8^th week. Indirect PTA–ELISA using the antiserum at a dilution 1:1000 could detect PMMoV in the infected leaf saps up to the dilution 1:5,120. Specificity test was performed and the result showed high specificity to five tobamoviruses including PMMoV, Tobacco mosaic virus (TMV), Odontoglossum ringsot virus (ORSV), Tomato mosaic virus (ToMV) and Ribgrass mosaic virus (RMV), but showed no cross reaction to other viruses tested including Cucumber green mottle mosaic virus (CGMMV) in the genus Tobamovirus; Potato virus Y (PVY), Chilli veinal mottle virus (ChiVMV) and Papaya ringspot virus (PRSV) in the genus Potyvirus; Tomato necrotic ring virus (TNRV), Watermelon silver mottle virus (WSMoV) and Melon yellow spot virus (MYSV) in the genus Tospovirus; Tomato leaf curl New Delhi virus (ToLCNDV) in the genus Begomovirus and Cucumber mosaic virus (CMV) in the genus Cucumovirus. Examination of the antiserum reactivity was carried out by comparison with the commercial antibodies from Agdia and the results from every sample were corresponding.