Development of monoclonal antibodies and serological assays specific for Barley yellow dwarf virus GAV strain

Abstract

BackgroundBarley yellow dwarf virus (BYDV) is one of the most devastating plant viruses and belongs to a ubiquitous plant virus group. In China, four BYDV strains (GPV, GAV, PAV and RMV) have been identified based on their specific aphid vectors and serological properties. Among the four identified strains, the GAV is the most common BYDV strain in China. To diagnose, forecast of BYDV GAV, two reliable serological assays for BYDV GAV detection were established.MethodsWe purified virion from a confirmed BYDV GAV source and used it as the immunogen to produce monoclonal antibodies against the virus. Using the hybridoma technology, three highly specific murine monoclonal antibodies were produced and two serological assays [antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA)] were established for the BYDV GAV detection.ResultsAll three monoclonal antibodies reacted strongly and specifically with the BYDV GAV strain in crude leaf extracts. Titers of the monoclonal antibodies in ascitic fluids were up to 10−7 by indirect-ELISA. These three monoclonal antibodies (18A1, 18A9 and 12A11) all belonged to the isotype IgG1, kappa light chain. The highest dilution points for the three antibodies during the ACP-ELISA using infected crude leaf extracts were 1:163,840, 1:81,920 and 1:81,920 (w/v, g · mL−1), respectively. Result of dot-ELISA showed a successful detection of BYDV GAV strain in 1:5,120 (w/v, g · mL−1) diluted wheat leaf crude extracts. Analysis of 22 field wheat leaf samples and 33 aphid samples from the Shaanxi Province in China, using the two newly developed assays confirmed the presence of BYDV GAV in about 80 % of the wheat samples and 18 % of the aphid samples.ConclusionsAll three monoclonal antibodies are highly sensitive and specific to the BYDV GAV. The two newly developed serological assays are simple and effective. These two assays, particularly the dot-ELISA, are useful for high throughput detection of BYDV GAV in host plants and aphid vectors.

Development of polyclonal antiserum against movement protein from Potato leafroll virus and its application for the virus detection

“…The positive detection of the MP with anti-MP antisera may indicate that the virus is in the multiplication stage. Polyclonal and monoclonal MP antibodies specific to Barley yellow dwarf virus (Xie et al 2007;Li et al 2015), polyclonal MP antibody specific to Citrus leprosis virus C (Calegario et al 2012), and polyclonal MP antibody specific to Grapevine fanleaf virus (Koolivand et al 2016) have been reported to detect the corresponding viruses, but most were effective only in laboratory use. Western blotting with anti-MP PLRV in our study showed that the antiserum could detect PLRV efficiently, although ratios of 1:10000 to 1:40000 were better for color development.…”

“…Most plant viruses contain MP genes in their genome that are associated with cell-to-cell movement of the virus through the plasmodesmata (Wolf et al 1989;Haupt et al 2005;Akamatsu et al 2007). Anti-MP antisera used to detect these MPs in different plant viruses have been reported (Xie et al 2007;Calegario et al 2012;Li et al 2015;Koolivand et al 2016). As far as we know, there is no report on the preparation of PLRV-MP antiserum used for the detection of PLRV and its MP.…”

Monoclonal Antibody-Based Serological Detection Methods for Wheat Dwarf Virus

“…After the forth immunization, spleen cells were obtained from the immunized mice and used to produce hybridoma as described (Liu et al 2016). Screening of hybridomas secreting anti-WDV MAbs and production of ascitic fluids were as described by Li et al (2015). Specificity of the resulting MAbs was determined by Western blot as described previously (Wu et al 2013).…”

“…To date, several WDV detection methods, including a squash blot (Bendahmane 1995), PCR or Real-time PCR (Achon et al 2006;Koklu et al 2007;Zhang et al 2010), nucleic acid spot hybridization (NASH) (Jin et al 2015), and a polyclonal antibody (PAb)-based enzyme-linked immunosorbent assay (ELISA) (Nygren et al 2015), have been reported. Compared with PCR, realtime PCR and nucleic acid spot hybridization, serological assays were considered to be more convenient assays for large-scale epidemiological studies and on-site detection of viruses during field surveys (Li et al 2015;Liu et al 2016;Chen et al 2017). It is generally accepted that a reliable serological detection assay depends largely on the specificity and sensitivity of the antibody used in the assay.…”

Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

“…In regards to the serological detection, only antiserum against CABYV prepared by purified virion was once used in Western blotting or ELISA [4]. Some recent studies showed that polyclonal antiserum against MP can detect the accumulation of virus from the family Luteoviridae [40][41][42][43]. However, there are no reports on either preparing polyclonal antiserum against MP of the three viruses or the serological relationships among them.…”