(342a) Immobilization of Recombinant Antibody Fragment Utilizing Material-Binding Peptides for Sensitive Immunoassay and Immuno-Sensors

Recombinant antibody fragments such as Fab, scFv and VHH are recently considered as next generation of diagnostic reagents because they can be immobilized with high density on the surfaces of materials, and consequently, antigen molecules would be efficiently captured by these ligand antibodies. However, such antibody fragments have not been utilized as a ligand antibody in enzyme immunoassay (EIA) because of difficulty in site-specific immobilization of antibody fragments with high remaining antigen-binding activities due to conformational change of antigen-binding domains after immobilization. In order to immobilize recombinant antibody fragments with high remaining activity, site-specific attachment of antibody fragments through the material-binding peptides is necessary. In this study, we originally identified material-binding peptides that can strongly interact with the surface of polystyrene (PS), polymethyl methacrylate (PMMA), and silicon nitride (SiN). By introducing these peptides to the C-terminus of recombinant antibody fragments, it was possible to increase both density as well as remaining activity of the fragments after passive adsorption to the solid materials. We also investigated efficient refolding method for peptide-tagged antibody fragments and thus, all of peptide-fused antibody fragments could be refolded with over 90 % of recovery, followed by site-specific immobilization. Our developed methods for refolding and immobilization of material-binding peptide-fused antibody fragments are considerably useful for preparation of high-performance of antibody-immobilized solid-support for immunoassay and immune-sensors.