title: Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.
abstract: Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.
authors.affiliation: Plant-pathogen interactions programme, Scottish Crop Research Institute, Invergowrie Dundee DD2 5DA, Scotland, UK. ltorra@scri.ac.uk
authors.forename: L
authors.initials: L
authors.lastname: Torrance
authors.forename: A
authors.initials: A
authors.lastname: Ziegler
authors.forename: H
authors.initials: H
authors.lastname: Pittman
authors.forename: M
authors.initials: M
authors.lastname: Paterson
authors.forename: R
authors.initials: R
authors.lastname: Toth
authors.forename: I
authors.initials: I
authors.lastname: Eggleston
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D017798:Comovirus / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D022801:Complementarity Determining Regions / Q000235:genetics; D003545:Cysteine; D005822:Genetic Vectors; D006046:Gold / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D008301:Maleimides / Q000138:chemical synthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000914:Antibodies, Viral; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008301:Maleimides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C043592:maleimide; D006046:Gold; D003545:Cysteine