Site-Specific Immobilization and Activation of ScFv Antibodies by Poylstyrene-Binding Peptide for Biomarker Analysis

Recently, for biomarker analysis, more attention has been paid to the construction of sensitive and economical immunoassay and protein microarray systems that are based on ELISA methods. One of the crucially important aspects in the development of protein-based bio-assay systems is to immobilize ligand antibody with high density and high antigen-binding activity to the greatest possible extent. Furthermore, the use of recombinant antibody fragments such as single-chain Fv (scFv) antibodies in such ELISA system is a next subject to increase in density of antibody molecule immobilized and decrease in production cost of diagnostic agent. In this study, we developed site-specific immobilization and solid-phase refolding methods for single-chain Fv antibodies mediated by the novel polystyrene binding peptides (PS-tags: RIIIRRIRR) that had been originally isolated and optimized in the previous studies. PS-tag-fused scFvs over-expressed in the insoluble fraction of E. coli cells were denatured and site-specifically immobilized onto a hydrophilic PS plate. Their antigen-binding activities were efficiently recovered by solid-phase refolding. This refolding method mediated by PS-tag was applicable to a variety of scFvs for biomarker analyses such as mouse anti-CRP antibody, anti-CEA antibody, human anti-ED-B antibody and so on. In comparison with Maxisorp^TM immobilized with whole monoclonal antibody (Mab), more than 25 times higher density of PS-tag-fused scFvs were attained and consequently, 10 times higher sensitivity for CRP was detected in sandwich ELISA. The solid-phase refolding of PS-tag-fused scFvs and antigen detection were successfully demonstrated on the surfaces of SPR and QCM sensor chips that were spin-coated with polystyrene thin layer.