Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

High-throughput, high-level production of PS-tag-fused single-chain Fvs by microplate-based culture.

In the present study, we investigated a microplate-based culture (MBC) of Escherichia coli for the high-throughput, high-level production of PS-tag-fused scFvs (scFv-PS) in insoluble form. The Overnight Express™ Autoinduction System (OE system) was adopted to skip the laborious induction step of the addition of IPTG. ScFv and scFv-PS began to be expressed after 6h by conventional flask culture (FLC) and by MBC when utilizing the OE system, and similar specific productivity levels were attained during cultivation. In MBC, an important factor that directly affected the production levels was rotational speed during cultivation, suggesting that the mass transfer of oxygen was rate-limiting. In a comparison of the productivity of flask cultures utilizing the 2YT-IPTG and OE systems, MBC utilizing the OE system was the highest, with approximately 1mg of insoluble scFv-PS obtained from each well under optimal conditions (1400 rpm). The results of SDS-PAGE and a cross-contamination check indicated that very similar cultivation conditions were attained in each well, without cross-contamination. Thus, MBC using the OE system is very useful for the high-throughput, high-level production of scFv-PS, which can be activated on the surface of hydrophilic PS plates by solid-phase refolding. Therefore, the production of a variety of specific scFv-PSs for cytokines and biomarkers will make possible the construction of sensitive and low-cost antibody microarrays, which will be very useful in clinical diagnosis and biochemical research.

Preparation of scFv-immobilized quartz crystal microbalance sensor by PS-tag-mediated solid-phase refolding.

The activation of a single-chain Fv antibody on the surface of a quartz crystal microbalance (QCM) sensor chip was investigated in order to develop an economical and sensitive immuno-QCM sensor system for use in clinical diagnosis. On the bare gold surface of a QCM sensor chip, approximately 60nm of hydrophilic polystyrene (phi-PS) thin film that specifically binds the polystyrene-binding peptide (PS-tag) was prepared by spin-coating and O(2)-plasma irradiation. When the adsorption of PS-tag-fused anti-ED-B scFv (scFv-PS) onto the phi-PS surface was directly monitored, the maximum density of scFv-PS attained was 1.56µg/cm(2), 1.6-times higher than that of scFv. 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 QCM sensor chip rapidly allowed the detection of clear signals for antigen at the range of 0.1-10µg/ml, while no signal was detectable for 10µg/ml BSA as a negative control. The scFv-PS-immobilized QCM sensor 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.

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.