A fully recombinant ELISA using in vivo biotinylated antibody fragments for the detection of potato leafroll virus.
A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for potato leafroll virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible V3HCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 degrees C or -20 degrees C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents.
Al-Mrabeh
Ahmad
A
Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.
Ziegler
Angelika
A
Cowan
Graham
G
Torrance
Lesley
L
D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D052679:Luteoviridae / Q000302:isolation & purification*; D010935:Plant Diseases / Q000821:virology*; D011198:Solanum tuberosum / Q000821:virology*; D013194:Staining and Labeling
D007128:Immunoglobulin Fragments