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 partic les of Potato leafroll virus (PLRV) were obtained from two naive phage disp lay libraries. Phages were selected against PLRV particles or dissociated P LRV particles immobilised onto tubes. Individual PLRV-binding scFv were ide ntified by ELISA, after their expression either fused to the surface of pha ge particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphat ase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These p rocedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not wit h dissociated capsid protein, which suggests that they had reacted with dis continuous epitopes. Others reacted with dissociated capsid proteins and SD S-denatured protein, which suggests that they had reacted with continuous e pitopes. ScFv were also subcloned into pC(L) for expression as fusion prote ins with human kappa constant region (scFv-C-L). Expression of these constr ucts in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial cu lture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained wi th 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 pol yclonal antibodies. (C) 1999 Elsevier Science B.V. All rights reserved.