Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

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...

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Published inBiochemical and biophysical research communications Vol. 301; no. 1; pp. 167 - 175
Main Authors Esteban, Olga, Garcı́a, Juan Antonio, Gorris, Marı́a Teresa, Domı́nguez, Elvira, Cambra, Mariano
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 31.01.2003
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Summary: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-NIb 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.
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ISSN:0006-291X
1090-2104
DOI:10.1016/S0006-291X(02)02993-5