An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.
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 C(L)-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 C(L)-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.
Cogotzi
Laura
L
Institute of Plant Virology, CNR, Bari, Italy.
Giampetruzzi
Annalisa
A
Nölke
Greta
G
Orecchia
Martin
M
Elicio
Vito
V
Castellano
Maria Antonietta
MA
Martelli
Giovanni P
GP
Fischer
Rainer
R
Schillberg
Stefan
S
Saldarelli
Pasquale
P
D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology*; D029381:Closteroviridae / Q000276:immunology / Q000502:physiology*; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D027843:Vitis / Q000821:virology*
D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins