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 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.
Esteban
Olga
O
Departamento de Protección Vegetal y Biotecnología, Laboratorio de Virología e Inmunología, Instituto Valenciano de Investigaciones Agrarias, Apdo, Oficial, 46113 Moncada, Valencia, Spain.
García
Juan Antonio
JA
Gorris
María Teresa
MT
Domínguez
Elvira
E
Cambra
Mariano
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism; D051379:Mice; D016366:Open Reading Frames; D017801:Plum Pox Virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014026:Nicotiana / Q000166:cytology / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C061396:nuclear inclusion protein b, mosaic viruses; D012321:DNA-Directed RNA Polymerases