(681a) 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

Abstract

             
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 Maxisorp^TM 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.