Novel solid-phase refolding method for preparation of scFv-immobilized polystyrene plates with high-antigen-binding activity.
In the present study, we demonstrated site-specific immobilization and solid-phase refolding of single-chain Fv antibodies on hydrophilic polystyrene (phi-PS) plates that was mediated by novel polystyrene binding peptides (PS-tags: RIIIRRIRR), which were originally isolated and optimized in previous studies. Three PS-tag-fused scFvs, namely scFv-PS, scFv-(PS), and scFv-PSII, which were over-expressed in the insoluble fraction of Escherichia coli cells were denatured and site-specifically immobilized onto hydrophilic PS plates in the presence of 0.5-4 M urea and 0.1% Tween 20. The antigen-binding activity of the scFvs was efficiently recovered by washing the surface of the plate with PBS that contained 0.1% Tween 20 (PBST). The solid-phase refolding mediated by PS-tag was successfully applied to several scFvs such as mouse anti-CRP antibodies and an anti-RNase antibody, although further investigation of the versatility of scFv-PSII is needed. The maximal density of PS-tag-fused scFvs was increased more than 15-fold compared with a whole monoclonal antibody (mAb) immobilized on Maxisorp and, consequently, the sensitivity of PS-tag-fused scFvs for CRP in a sandwich ELISA was increased 25-fold. Thus, the novel, solid-phase, refolding method mediated by a PS-tag will be very useful for preparation of solid supports coated with recombinant antibody fragments, which can be used in immunoassays and immuno-separation.
Kumada
Yoichi
Y
Department of Chemistry and Materials Technology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan. kumada@chem.kit.ac.jp
Shiritani
Yuki
Y
Hamasaki
Kyoko
K
Nakagawa
Aya
A
Sasaki
Eiju
E
Kishimoto
Michimasa
M
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D011137:Polystyrenes / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification
D000941:Antigens; D011137:Polystyrenes; D057127:Single-Chain Antibodies