Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).
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.
Kumada
Yoichi
Y
Department of Chemistry and Materials Technology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan. kumada@chem.kit.ac.jp
Hamasaki
Kyoko
K
Shiritani
Yuki
Y
Nakagawa
Aya
A
Kuroki
Daisuke
D
Ohse
Takuhito
T
Choi
Dong Hwan
DH
Katakura
Yoshio
Y
Kishimoto
Michimasa
M
D000595:Amino Acid Sequence; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D002097:C-Reactive Protein / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011137:Polystyrenes / Q000737:chemistry*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013997:Time Factors
D055910:Antibodies, Immobilized; D007135:Immunoglobulin Variable Region; D010455:Peptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D002097:C-Reactive Protein