title: Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display.
abstract: The rotavirus non-structural NSP4 protein causes membrane destabilization as well as an increase in intracellular calcium levels in eukaryotic cells and induces diarrhea in young mice, acting as a viral enterotoxin. In this study the phage display technique was used to generate a panel of single-chain variable fragment (scFv) antibodies specific for the NSP4 protein of the human rotavirus strain Wa from a human semi-synthetic scFv library. After several rounds of panning and selection on NSP4 adsorbed to polystyrene tubes, individual scFv were isolated and characterised by fingerprinting and by sequencing the VH and VL genes. The isolated scFv antibodies specifically recognize NSP4 in enzyme immunoassay and in Western blot. Four truncated forms of the NSP4 protein were constructed which allowed us to map the binding region of the selected scFv antibodies to the C-terminal portion of NSP4. The isolated scFv antibodies constitute valuable tools to analyse the mechanisms of NSP4 functions.
authors.affiliation: Department of Microbiology, School of Medicine, University of Valencia, Hospital Clinico Universitario, Avda. Blasco Ibañez 17, 46010 Valencia, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Rodríguez-Díaz
authors.forename: Vicente
authors.initials: V
authors.lastname: Monedero
authors.forename: Gaspar
authors.initials: G
authors.lastname: Pérez-Martínez
authors.forename: Javier
authors.initials: J
authors.lastname: Buesa
mesh_terms: D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D004768:Enterotoxins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000302:isolation & purification; D019151:Peptide Library; D012401:Rotavirus / Q000276:immunology*; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D004768:Enterotoxins; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D017361:Viral Nonstructural Proteins