(728f) 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.