(84a) Site-Specific Immobilization of Recombinant Antibody Fragments for Biosensing

Recently, more attention has been paid to the construction of efficient and sensitive immunoassays and protein microarray systems that are based on ELISA methods. One of the crucially important aspects of the construction of protein-based biochips is site-specific immobilization of recombinant antibody fragments such as scFv, Fv and Fab onto the solid surface in a manner that maintains their conformation and biological activity to the greatest possible extent. We demonstrated site-specific immobilization and solid-phase refolding of small antibody fragments onto polystyrene supports mediated by the novel polystyrene binding peptides (PS-tags: RIIIRRIRR) that had been originally isolated and optimized in the previous studies. The solid-phase refolding mediated by PS-tag was applicable to a variety of scFvs, Fvs, and Fabs. Adsorption levels of recombinant antibody fragments were much higher that of whole antibody and their antigen-binding activities were highly preserved on the surface of PS plate. Consequently, they are applicable to not only immunoassay using a microtiter plate, but also a variety of biosensors based on surface plasmon resonance (SPR), surface plasmon fluorescence spectroscopy (SPFS) and quartz crystal microbalance (QCM).