Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay.

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL-VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery.

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

[A Novel System for Discovering High-affinity Antibody Mutants That Enables Immunoassays with Higher Sensitivities -Development and Application of Clonal Array Profiling (CAP)].

Antibody engineering is a powerful method used to generate high-affinity antibodies that enables sensitive immunoassays. It is commonly performed with the following steps: First, antibody fragments (e.g., single-chain Fv fragments; scFvs) with various mutations are displayed on the surface of filamentous bacteriophages to generate a diverse scFv-phages library. Then, rare clones with improved affinities are selected from the library via "panning" against target antigens immobilized on solid phases. However, this process often fails because of biased proliferation of scFv-phage clones and competition with large excesses of clones with weaker affinities. To overcome these limitations, we developed a clonal array profiling (CAP) method in which scFv-phage members in a library are individually examined for their antigen-binding ability. The advantage of CAP over conventional panning was evident in a comparative study that explored a library of anti-cortisol scFvs. CAP isolated five scFv mutants with >30-fold enhanced affinity (Ka) compared with wild-type scFv, enabling >11-fold more sensitive immunoassays. In contrast, no clones showing >5-fold higher Ka were found via panning. Considering the unique features of the primary structures of the improved scFvs found via CAP, we constructed new anti-cortisol scFv libraries where amino acid substitutions or insertions were introduced into the heavy-chain framework region 1 (VH-FR1). As expected, we obtained 21 mutants with >15-fold enhanced affinities. This VH-FR1-targeting mutagenesis also succeeded in generating affinity-matured scFvs against psilocybin, a hallucinogenic compound found in some mushrooms, which could be applied for developing on-site identification systems for hallucinogenic mushrooms, e.g. as immunochromatography devices.

Retrieving Dissociation-Resistant Antibody Mutants: An Efficient Strategy for Developing Immunoassays with Improved Sensitivities.

Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, "panning" has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17-31-fold improved Ka from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; Ka, 3.6 × 108 M-1), but their Ka did not exceed 1.1 × 1010 M-1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with "ordinary affinities," dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33-56-fold increased Ka (1.2-2.0 × 1010 M-1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18-51-fold higher sensitivity than the assay performed using wt-scFv.

Evaluation of multi-specificity of antibody G2 using its single-chain Fv and its covalently linked antigen peptides.

The antibody G2 specifically binds to four peptides with different amino acid sequences: Pep18mer, Pep8, Pep395, and PepH4P6. To elucidate the multi-specificity of G2, we generated a G2 single-chain Fv (scFv) antibody and analyzed its binding thermodynamics and kinetics to antigen peptides. Our results clearly showed that the recognition of PepH4P6 was similar to that of Pep18mer, to which G2 could obtain binding ability through the deletion of Pro95 at light chain on the affinity maturation process. The covalent linking of peptides could increase the thermal stability of G2 scFv due to intramolecular antigen binding. In the effects of respective peptides, the increased thermal stability of G2 scFv linked to Pep8 was significant, possibly due to the rapid dissociation. Binding experiments of G2 scFv linked to peptides to other peptides showed decreased association rates relative to those of antigen-free G2 scFv while the dissociation rates were almost unchanged.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography.

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56 × 10-10 to 4.04 × 10-8 M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19 mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9 mg-scFv/cm3. The resin exhibited 7.69 mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17ß by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17ß (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli.

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

Fabrication of Fragment Antibody-Enzyme Complex as a Sensing Element for Immunosensing.

Antibody-enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method.

Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein.

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15-13 and 12-22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15-13 and 12-22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm-baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15-13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12-22 Fab has a weaker binding affinity (KD ~ µM) with the RABVG compared to the 15-13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15-13 and 12-22 antibodies as another potential biopharmaceutical for targeting rabies. The 15-13 and 12-22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the µM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.

Using Engineered Mammalian Cells for an Epitope-Directed Antibody Affinity Maturation System.

Antibodies have been attracting attention as therapeutic tools owing to their high affinity and specificity. To develop potent antibodies, affinity maturation, epitope regulation, and using target antigens in native form are pivotal requirements. Here we describe a method to conduct epitope-directed affinity maturation of antibodies using engineered mammalian cells. This method utilizes protein chimeras that transduce cell death signaling in response to antibody binding. As the competition of antibody binding inhibits the cell death signaling, only affinity-matured antibodies retaining the same epitope as an original one can be selected using cell survival as readout.

Gold nanoparticles and tilt pairs to assess protein flexibility by cryo-electron microscopy.

A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.

NanoLuc luciferase as a suitable fusion partner of recombinant antibody fragments for developing sensitive luminescent immunoassays.

Enzyme-linked immunosorbent assays (ELISAs) are essential for monitoring various biomarkers. Competitive and noncompetitive (sandwich) assay formats are used to determine hapten and macromolecule levels, respectively. Both formats require more sensitive detection of reporter enzymes for greater assay sensitivities. We previously reported the utility of wild-type Gaussia luciferase (wtGLuc) as a fusion partner with antibody single-chain Fv fragments (scFvs) for developing sensitive luminescent ELISAs. Here, we evaluated utility of NanoLuc luciferase (NLuc), a recently developed luciferase, as fusion partner with scFvs from the view of comparison with wtGLuc and a mutant of alkaline phosphatase (ALP'). Thyroxine (T4) and T4-labeled albumin were chosen as model haptenic and macromolecular antigens, respectively. An in-house-prepared anti-T4 scFv was fused with NLuc, wtGLuc, or ALP'. The scFv-NLuc fusion protein showed 47-fold and 29-fold lower limit of detection [LOD; 59 zmol (per assay)] than the wtGLuc- and ALP'-fusions, respectively. In a competitive T4 ELISA, the NLuc-fusion showed 9.3- and 6.3-fold lower LOD, (0.67 pg) than the wtGLuc- and ALP'-fusions, respectively, with a higher specificity in clinical applications. A typical colorimetric ELISA using a peroxidase-labeled second antibody showed 70-fold higher LOD than NLuc-based ELISA. Another advantage of the NLuc-fusion was shown in the sandwich assays; the LOD of T4-labeled albumin (5.0 fmol) was >6-fold lower than that of the other luminescent ELISAs. In an additional sandwich assay developed to count bacteriophage particles, NLuc enabled more sensitive determination than wtGLuc, whereas ALP' showed nearly equivalent performance. Its slowest alteration rate for light intensity after starting the enzyme reaction should enable robust batch-by-batch assay operations. Thus, we concluded that scFv-NLuc fusions serve as suitable probes in various types of immunoassays and may facilitate higher sensitivities with practical specificities.

Rapid and homogeneous electrochemical detection by fabricating a high affinity bispecific antibody-enzyme complex using two Catcher/Tag systems.

Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogeneous electrochemical detection by fabricating a high-affinity bispecific AEC (bsAEC) using two Catcher/Tag systems. Recently, we reported a convenient and universal method to fabricate AECs using the SpyCatcher/SpyTag system. The resultant anti-epidermal growth factor receptor (anti-EGFR) AEC worked efficiently as a sensing element; however, the sensitivities did not meet the clinically required detection range of the soluble ectodomain of EGFR (sEGFR). To induce high affinity even to monomeric targets like sEGFR, we designed a convenient fabrication method for bsAEC using two Catcher/Tag systems, which did not express cross-reactivity. The anti-EGFR bsAEC was successfully prepared by constructing glucose dehydrogenase with two different catcher domains at the N- and C-terminus and by combining two corresponding Tag-fused anti-EGFR single-chain Fvs (scFvs), which recognize different epitopes on sEGFR. As expected, bsAEC showed a higher affinity than that of bivalent AEC with two identical anti-EGFR scFvs at low concentrations of sEGFR, and met the clinically required detection range of sEGFR. Further, by combining magnet beads, we established a rapid and wash-free homogeneous electrochemical detection method. This study offers new insights into the fabrication of universal POCT devices.

Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay.

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.

Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins.

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and-most importantly-it enables high-throughput procedures.

Screen printed electrode-based biosensor functionalized with magnetic cobalt/single-chain antibody fragments for cocaine biosensing in different matrices.

On-site detection of substance abuse is an important approach in the preventive and intervention protocols implementations. It is known that the traditional methods are heavy, time-consuming, and need a high level of logistical requirements. As such, biosensors represent great potential to simplify and improve substance abuse detection. In this study, we have designed a functionalized screen-printed electrode (SPE) electrochemical biosensor with cobalt oxide nanoparticles and single-chain antibody fragments (scFvs) for cocaine detection. Different electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry were used to examine the functionality of the designed biosensor. Furthermore, SEM observations were performed to observe the surface changes after functionalization. The results showed that the linearity ranged between 5.0 and 250 ng/mL and a detection limit of 3.6 ng/mL (n = 6). These results were compared to results obtained from Q-TOF/MS where four different matrices (serum, sweat, urine, and saliva) were spiked with 100 ng/mL cocaine and were analyzed by both methods (Biosensor and Q-TOF/MS). Results showed a higher performance of the biosensor compared to traditional methods. In addition, the selectivity of the biosensor was shown in the presence of different interferents where the designed platform showed a specific response to only cocaine. In conclusion, the designed biosensor proposes great potential for portable and on-site substance abuse detection in addition to boasting the capability of reuse of the SPE and thus, reducing the costs related to such applications.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.