270783 Preparation of Sensitive Immuno-Sorbent and Immuno-Sensors by Solid-Phase Refolding of PS-Tag-Fused Scfvs
Preparation of sensitive immuno-sorbent and immuno-sensors by solid-phase refolding of PS-tag-fused scFvs
Yoichi Kumada, Kyoto Institute of Technology
In this study, we successfully developed a method of site-specific immobilization and activation of single-chain Fv antibodies (scFv) onto polystyrene supports mediated by the novel polystyrene binding peptides (PS-tags: RIIIRRIRR). PS-tag-fused scFvs (scFv-PS) over-expressed in the insoluble fraction of E. coli cells were denatured and site-specifically immobilized onto a hydrophilic PS plate in the presence of 0.5 ~ 2 M urea. Their antigen-binding activities were efficiently recovered by washing the PS plate with PBST. The solid-phase refolding mediated by PS-tag was applicable to a number of scFvs such as mouse anti-CRP, RNase, TSH, CEA, CA19-9, AAT, huIgG and huIgA antibody. In comparison with MaxisorpTM immobilized with whole monoclonal antibody (Mab), more than 10 times higher density of PS-tag-fused scFvs were attained and consequently, 10 ~ 100 times higher sensitivity was detected in sandwich ELISA. Furthermore, the activation of a single-chain Fv antibody on the surface of sensor chip for QCM as well as SPR sensors was also investigated in order to develop economical and sensitive immuno-sensor systems for use in clinical diagnosis. On the bare gold surface of sensor chips, approximately 20 ~ 60 nm of hydrophilic polystyrene (phi-PS) thin film that specifically binds the polystyrene-binding peptide (PS-tag) was prepared by spin-coating and O2-plasma irradiation. When the adsorption of scFv-PS onto the phi-PS surface was directly monitored, the maximum density of scFv-PS attained was 1.56 mg/cm2. The specific antigen-binding activity of scFv-PS after solid-phase refolding increased with the density of immobilized scFv-PS, and, consequently, activity 1.7 times higher than that of scFv was retained. The scFv-PS-immobilized sensor chips rapidly allowed the detection of clear signals for antigen with high sensitivity, while no signal was detectable for BSA as a negative control. The scFv-PS-immobilized sensors developed in the present study will therefore be very useful for the rapid and highly sensitive detection of biomarkers, and should be applicable to clinical diagnosis.
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