(320c) Site-Specific Immobilization and Orientation Control of Scfv-Fc Antibodies for Ultra-Sensitive Detection of Influenza Virus

Recombinant antibody fragments such as single-chain Fv antibodies (scFvs) and nano bodies (VHHs) have been expected as next generation of ligand proteins to capture target antigen molecules on solid surfaces in a variety of immuno-diagnostics. Such antibody fragments could be efficiently selected from a phage library, and could be modified to a variety of formats including Fab, scFv-Fc, and even full length of IgG. Especially, rabbit scFv-Fc that scFv is genetically-fused with Fc region of IgG has advantages for use in immunological detection of influenza virus due to their stability and strong binding ability compared with scFvs. Here, we developed and characterized new types of scFv-Fcs genetically-fused with Avi-tag which is special amino acid sequence recognized by biotin ligase. The scFv-Fc-Avi-tag was successfully produced by baculovirus-insect cell system with co-expression of biotin-ligase by use of pFastBacDual vector. Site-specific biotinylation at C-terminus of scFv-Fc-Avi-tag was confirmed by western blotting as well as sandwich ELISA that streptavidin was coated on a plate. Both scFv-Fc and scFv-Fc-Avi-tag in a culture supernatant were bound to Protein A-coupled affinity resin and mono-biotinylated scFv-Fc-Avi-tag was specifically eluted from Protein A-coupled resin at pH 2.5, while non-biotinylated scFv-Fc-Avi-tag was washed out at pH 3.0. Consequently, antigen-binding activity of scFv-Fc-Avi-tag immobilized via streptavidin-biotin interaction became significantly higher than those of conventional scFv-Fc physically-adsorbed, and also biotin-conjugated scFv-Fc. Ten-fold lower concentration of antigen was detected by use of scFv-Fc-Avi-tag-immobilized plate, in comparison with that immobilized with conventional mouse antibodies. These results clearly indicated that orientation of scFv-Fc immobilized on a solid support was significantly improved and thus, active antigen-binding domains of scFv-Fc-Avi-tag might be exposed with higher probability.

We further investigated immobilization of scFv-Fc-Avi-tag on nitrocellulose for application to immuno-chromatography. Streptavidin was indirectly immobilized on the surface of nitrocellulose that biotin-oligo-DNA was covalently coupled by UV irradiation in order to avoid conformational change of streptavidin. Under the optimal condition, 10-times lower concentration of NP antigen was detected with one-tenth amount of scFv-Fc-Avi-tag, compared with that the conventional scFv-Fc-adsorbed NC membrane.

Thus, scFv-Fc-Avi-tag developed in this study would be significantly useful as a ligand antibody on a variety of solid surface and it would contribute development of sensitive and economical immuno-diagnostics.