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- Title
An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.
- Authors
Cogotzi, Laura; Giampetruzzi, Annalisa; Nölke, Greta; Orecchia, Martin; Elicio, Vito; Castellano, Maria; Martelli, Giovanni; Fischer, Rainer; Schillberg, Stefan; Saldarelli, Pasquale
- Abstract
Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent CL-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The CL-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection.
- Subjects
GRAPEVINE leafroll virus; DIAGNOSIS of escherichia coli diseases; ENZYME-linked immunosorbent assay; ANTIBODY-enzyme conjugates; ANTIBODY diversity; VIRAL disease diagnosis; ELECTRON microscopy
- Publication
Archives of Virology, 2009, Vol 154, Issue 1, p19
- ISSN
0304-8608
- Publication type
Article
- DOI
10.1007/s00705-008-0263-y