Protein Engineering of the Soluble Metal-dependent Formate Dehydrogenase from Escherichia coli.

Formate is the most targeted C1 building block and electron carrier in the post-petroleum era. Formate dehydrogenase (FDH), which catalyzes the production or degradation of formate, has acquired considerable attention. Among FDHs, a metal-dependent FDH that carries a complex active center, molybdenum-pterin cofactor, can directly transfer electrons from formate to other redox proteins without generating NAD(P)H. Previously, we reported an expression system for membrane-bound metal-dependent FDH from E. coli (encoded by the fdoG-fdoH-fdoI operon) and succeeded in its conversion to a soluble protein. However, this protein exhibited a too low stability to be purified and analyzed biochemically. In this study, we tried to improve the stability of heterologously expressed FDH through rational and irrational approaches. As a result, a mutant with the highest specific activity was obtained through a rational approach. This study not only yielded a promising FDH enzyme with enhanced activity and stability for industrial applications, but also offered relevant insights for the handling of recombinant large proteins.

A Novel Mucidosphaerium sp. Downregulates Inflammatory Gene Expression in Skin and Articular Cells.

CONTEXT: Hot-spring therapy is occasionally used for the treatment of inflammatory diseases. Microorganisms might contribute to the anti-inflammatory functions seen in thermal mud therapies. Natural microorganisms, derived from traditional spa resorts, could be useful as a preventive strategy for alternative medical applications. OBJECTIVE: The aim of the study was to find effective microalgae from prominent hot springs to use for the treatment of inflammatory diseases. DESIGN: The research team performed an in-vitro study. Microalgae, derived from Beppu hot springs, were isolated and homogeneously cultured. SETTING: The study took place at the Saravio Central Institute at Saravio Cosmetics in Oita, Japan and the Department of Bioscience and Biotechnology in the Graduate School of Agriculture at Shinshu University in Nagano, Japan. INTERVENTION: For identification, the 18S ribosomal RNA genes of microalgae were investigated by DNA sequencing and homology search, together with microscopic observation. OUTCOME MEASURES: To examine the pharmacological activities of the algal extracts, real-time polymerase chain reactions were performed, using either primary dermal fibroblasts (DFs), dermal papilla cells (DPCs), or fibroblast-like synoviocytes (FLSs). To test the antioxidant activity, both the oxygen radical absorbance capacity and the generation of intracellular reactive oxygen species (ROS) were evaluated. RESULTS: A novel strain of green algae, Mucidosphaerium sp., was isolated from a Beppu hot spring. The algal extract downregulated gene-expression levels of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor- alpha (TNF-α), in various primary cells pre-exposed to IL-1ß. The protein level of the risk factors was concomitantly reduced. In addition, the algal extract suppressed the IL-1ß-induced upregulation of cyclooxygenase-2, nerve growth factor, and matrix metalloproteinase-1 (MMP-1) and MMP-3 in DFs. It also inhibited that of MMP-1, -3, and -9 in FLSs. Moreover, the extract inhibited total MMP protease activities. The microalgae decreased the intracellular reactive oxygen species (ROS) level in FLSs with an antioxidant activity of 178.3 ± 0.9 µmol of trolox equivalent/g. CONCLUSIONS: The present study showed that the novel Mucidosphaerium sp., derived from a Beppu hot spring, suppressed inflammatory reactions in both cutaneous and articular cells, partly due to its antioxidative properties. The novel algal strain may be a useful tool as an alternative medicine for skin and joint inflammatory disorders.

Overexpression of endogenous 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in cyanobacterium Synechocystis sp. PCC6803 accelerates protein aggregation.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for the production of precursors of all isoprenoids. In a previous study, we had examined the overexpression of an endogenous DXS in a Synechocystis sp. PCC6803 mutant (DXS_ox), and found that the dxs mRNA level was 4-fold higher than that in the wild-type (WT) strain. However, the DXS protein level was only 1.5-fold higher, leading to the assumption that the level might be regulated by post-transcriptional events. In this study, we have additionally introduced an exogenous isoprene synthase (IspS; which can release MEP pathway products from the cell as gaseous isoprene) into the WT and DXS_ox strains (WT-isP and DXSox-isP strains, respectively), and their detailed DXS expression profiles were investigated from the induction phase through to the late-logarithmic phase. In the induction phase, the isoprene productivity of the DXSox-isP strain was slightly but significantly (1.4- to 1.8-fold) higher than that of the WT-isP strain, whereas the levels were comparable in the other phases. Interestingly, the ratios of soluble:insoluble DXS protein were remarkably low in the DXSox-isP strain during the induction phase to the early-logarithmic phase, resulting in a moderate level of soluble DXS. All our results suggested that the high translation rate of DXS disturbs the refolding process of DXS. To enhance the concentration of the active DXS in cyanobacteria, the enhancement of the DXS maturation system or the introduction of exogenous and robust DXS proteins might be necessary.

Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.

Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

BACKGROUND: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. METHODS: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). RESULTS: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. CONCLUSIONS: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.

Enhancement of L-cysteine production by disruption of yciW in Escherichia coli.

Using in silico analysis, the yciW gene of Escherichia coli was identified as a novel L-cysteine regulon that may be regulated by the transcriptional activator CysB for sulfur metabolic genes. We found that overexpression of yciW conferred tolerance to L-cysteine, but disruption of yciW increased L-cysteine production in E. coli.

Simultaneous retention of thermostability and specific activity in chimeric human alkaline phosphatases.

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75°C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.

Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120.

Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.

Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP(+)-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

A protein-protein interaction assay based on the functional complementation of mutant firefly luciferases.

We report a novel bioluminescent protein-protein interaction (PPI) assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named "Donor", which virtually lacks oxidative activity, while the other, named "Acceptor", is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure proteins. When the interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.

Expression of an antibody-enzyme complex by the L-chain fusion method.

In this report, we describe a novel method for directly preparing enzyme-labeled antibodies harvested from IgM-producing hybridoma cells. We constructed expression vectors for antibody light (L) chain-enzyme fusion proteins by linking either the genes for the murine lambda L chain or its constant region (C(L)) with one of two proteins, either the secreted placental alkaline phosphatase or Gaussia luciferase (Gluc). When the vectors were transfected into anti-NP (4-hydroxy-3-nitrophacetyl) IgM-producing myeloma cells, secretion of the IgM-enzyme complex from the gene-transfected cells was confirmed by a direct enzyme-linked immunosorbent assay with an immobilized antigen. Furthermore, when human hybridoma HF10B4, a cell line that produces anti-human lung cancer IgM, was transfected with the vector containing L-Gluc, a significantly stronger signal was obtained for the human lung carcinoma SBC-1 cells than for cervical HeLa cells. Because successful production of an active IgM-enzyme complex containing a heterologous L chain-enzyme fusion was observed, the L-chain fusion method will be a generally applicable method for preparing various IgM-enzyme complexes.

Open-sandwich molecular imprinting: making a recognition matrix with antigen-imprinted antibody fragments.

A novel antibody-polymer conjugation method termed open-sandwich molecular imprinting (OS-MIP) has been proposed to produce a specific recognition matrix in the presence of a target antigen. The resultant carboxymethyldextran matrix conjugated with two separate antibody variable region fragments imprinted with the cognate antigen showed higher antigen-binding capacity than non-imprinted ones and was successfully used to sensitively monitor multiple antigen binding/desorption events by a surface plasmon resonance biosensor. Furthermore, when each fragment was labeled with different fluorophores before conjugation, the fluorescence signals of the matrix made by OS-MIP clearly showed an antigen concentration dependent increase in Förster resonance energy transfer between the two dyes. By using a combination of various methods for detecting interaction, OS-MIP will be a useful platform for detecting various targets from small molecules to proteins with high sensitivity and specificity.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Activation of circularly permutated ß-lactamase tethered to antibody domains by specific small molecules.

Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 ß-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.

Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4.

To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.

Micro open-sandwich ELISA to rapidly evaluate thyroid hormone concentration from serum samples.

BACKGROUND: Thyroxine (T4) is the most commonly measured thyroid hormone for the diagnosis of thyroid function. To elucidate a rapid and sensitive assay for T4, we made a microfluidics-based noncompetitive immunodetection chip system using anti-T4 antibody fragments obtained from a phage display library. RESULTS: Based on the open-sandwich ELISA principle that detects antigen-dependency of the interaction between the two antibody variable regions V(H) and V(L), we could detect less than 1 ng/ml of T4. The assay was also successfully applied to evaluate total T4 concentration in the serum of healthy individuals. CONCLUSION: This would be the first micro open-sandwich ELISA constructed with antibody fragments directly selected from immunized mice. The system will be applied to the sensitive detection of many diagnostic markers.

Expression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cells.

Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional V(H)-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent V(H)/V(L) interaction for antigen quantitation.

Antibody affinity maturation in vitro using unconjugated peptide antigen.

Selection of antibody library in vitro is almost always performed on a certain solid-phase with immobilized antigen. However, for the selection of small molecule binders, conjugation of the antigen to a carrier molecule is indispensable, which often leads to the selection of unwanted binders such as conjugate-binders or those with insufficient specificity. Here we describe a rapid and efficient way to improve the affinity of an anti-small molecule antibody without antigen derivatization. The method is based on the open-sandwich (OS) principle, which utilizes the antigen-dependent stabilization of antibody variable domain Fv. We used an anti-osteocalcin C-terminal peptide Fv that showed a good response but with moderate sensitivity in OS ELISA as a model. By selecting PCR-randomized V(H)-displaying phages for superior binders to the immobilized V(L) fragment in the presence of limited amount of antigen peptide, V(H) mutants that show superior detection sensitivity in OS ELISA were obtained, and were characterized to retain improved antigen-binding affinity. Furthermore, saturation mutagenesis of a mutant resulted in further improvement in sensitivity. This 'OS-selection' will be the first to select anti-small molecule antibodies without using conjugated antigens, and especially useful in the affinity maturation of antibodies whose Fv has limited stability in the absence of antigen.