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Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)

Kumada, Yoichi, Hamasaki, Kyoko, Shiritani, Yuki, Nakagawa, Aya, Kuroki, Daisuke, Ohse, Takuhito, Choi, Dong Hwan, Katakura, Yoshio, Kishimoto, Michimasa
Analytical and bioanalytical chemistry 2009 v.395 no.3 pp. 759-765
C-reactive protein, Escherichia coli, enzyme-linked immunosorbent assay, humans, hydrophilicity, hydrophobicity, monoclonal antibodies, peptides, polysorbates, polystyrenes, proteomics
Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.