A monoclonal antibody (mAb 2A) able to react against the RNA replicase Nib from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral Nib in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for Nib was similar to that of the parental mAb and the region YLEAFY from PPV-Nlb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against Nib was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity. (C) 2003 Elsevier Science (USA). All rights reserved.