Monoclonal antibody-based diagnostic assays for pepper mild mottle virus

Abstract

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.