Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10^-3-10^-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.