Preparation of polyclonal antiserum to beet necrotic yellow vein virus and its application for immunodiagnostics

Abstract

Sugar beet rhizomania, caused by the beet necrotic yellow vein virus (BNYVV), was recorded in several areas of Lithuania. From symptomatic sugar beet leaves the virus was mechanically transmitted to the indicator plants for maintenance, purification and storage. The virus was maintained in Chenopodium quinoa which served as a propagation host for BNYVV. Transmission of the virus to indicator plants was confirmed by the DAS-ELISA, immunosorbent electron microscopy (IEM) and PCR methods. BNYVV purified by PEG and density gradient centrifugation was used for the production of polyclonal antiserum suitable for immunodiagnostics by ELISA and Western blot analysis. The polyclonal antibody titer obtained in indirect ELISA reactions with a purified virus suspension was found to be 1 : 1600. For the immunoenzyme system formation polyclonal antibodies were conjugated to horseradish peroxidase (HPR). The developed system allowed BNYVV detection in the range of 0.3–0.15 μg/ml. Keywords: beet necrotic yellow vein virus, ELISA, horseradish peroxidase, Western blot