Effective application of DAS-ELISA for detection of grapevine leafroll associated closterovirus-3 using a polyclonal antiserum developed from recombinant coat protein

A polyclonal antiserum (As163) specific to grapevine leafroll associated cl osterovirus-3 (GLRaV-3) was developed using a recombinant coat protein expr essed in E. coli from a cDNA clone identified after immunoscreening of a cD NA library. Specificity of the antiserum to GLRaV-3 was shown by Western bl ot and immunosorbent electron microscopy. With this antiserum, an effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for GLRaV-3 detection. To evaluate the sensitivity of the antise rum in DAS-ELISA for virus detection, different combinations of antibodies were compared. Although best results were obtained when As163 was used for coating and a monoclonal antibody (MabNY1.1) was used as an enzyme conjugat e, good results were also obtained when As163 was used both for coating and as an enzyme conjugate. Using this As163-Mab system in DAS-ELISA, we confi rmed the presence of GLRaV-3 in a diverse collection of leafroll infected v ines.