Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.
Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.
Harper
K
K
Scottish Crop Research Institute, Dundee, UK.
Toth
R L
RL
Mayo
M A
MA
Torrance
L
L
D000469:Alkaline Phosphatase / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000138:chemical synthesis; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D017793:Luteovirus / Q000276:immunology*; D019151:Peptide Library; D018515:Plant Leaves / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000821:virology*; D020349:Surface Plasmon Resonance
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase