Production of polyclonal antiserum against Cowpea mild mottle virus coat protein and its application in virus detection

Abstract

Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to yield losses in the main producing regions of Brazil. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein gene of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of the purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/ToF mass spectrometer analyses. A rabbit was immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antiserum was demonstrated by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection.

Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination

“…Symptoms were recorded until 28 days post-inoculation (dpi). Systemic top leaves were used for indirect ELISA tests (Clark et al, 1986) using a polyclonal antiserum (Carvalho et al, 2013) to confirm CPMMV infection in these plants. Additionally, plants showing only local symptoms on inoculated leaves were used for indirect ELISA.…”

Comparison of Bemisia tabaci Infestation, Virus Infection, and Yield in Conventional and Transgenic Bean Golden Mosaic Virus-Resistant Common Bean Elite Lines

“…In addition, 10 leaves were selected randomly from each plot and taken to the laboratory for confirmation of the presence of Cowpea mild mottle virus via ELISA. An antiserum specific for Cowpea mild mottle virus was graciously supplied by Carvalho as well as the specific ELISA methodology (Carvalho et al 2013b). The initial identification of the presence of Cowpea mild mottle virus was performed by electron microscopy (JEM 1011, JEOL USA, Inc., Peabody, Massachusetts, USA) and by serology (indirect ELISA) using antiserum produced by an isolate of Cowpea mild mottle virus infecting soybean plants (Faria et al 2016).…”

Cowpea mild mottle virus (Carlavirus, Betaflexiviridae): a review

“…In January of 2014, we collected 200 soybean samples with symptoms of systemic necrosis in fields of the municipalities of Vacaria, Capão Bonito do Sul and Lagoa Vermelha (all in RS). All samples were tested by indirect ELISA (Clark et al 1986) using a polyclonal antiserum (Carvalho et al 2013) to confirm CPMMV infections and none of the samples were positive (L.G. Zanardo and C.M.…”