Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection

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 Lcys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalisation of gold with the scFvC Lcys fusion proteins elaborated. Successful covalent attachment of functional scFvC Lcys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC Lcys 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 Lcys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.