Development of a novel and broadly applicable sandwich ELISA assay based on rabbit single-chain variable fragments and a modified Ig-binding domain of protein L fused to a polystyrene-binding peptide.

Most of currently available sandwich-type enzyme-linked immunosorbent assays (ELISA) require the use of full-length animal-derived antibodies which poses welfare criticisms and are often expensive to produce. There is therefore a strong demand for the development of more affordable and animal-free methods to produce antibodies for sandwich ELISA assay. To address these issues, we propose here the development of a new technology based on two complementary rabbit single-chain variable fragments (scFvs) and an Ig-binding domain of protein L (PpL1) fused to a polystyrene-binding peptide (PS-tag) that can be recombinantly produced in bacteria.

Albumin-based strategies to effectively prolong the circulation half-life of small immunomodulatory payloads in cancer therapy.

Small immunomodulatory payloads (IMMs) such as peptide vaccines and cytokines have the capability to activate and boost the immune response against cancer. However, their clinical use has often been hindered by their poor stability and short circulating half-lives. To enhance the pharmacokinetic properties of small IMMs and promote their trafficking and accumulation in lymphatic and tumor tissues, a large variety of strategies have been developed.

Generation of rabbit single-chain variable fragments with different physicochemical and biological properties by complementary determining region-grafting technology.

In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries.

Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories.

Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy.

Identification and characterization of rabbit scFv antibodies suitable for immuno-affinity separation of recombinant human kynureninase from Escherichia coli cell lysate.

In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.

Crystal structure of human serum albumin in complex with megabody reveals unique human and murine cross-reactive binding site.

The pharmacokinetic properties of small biotherapeutics can be enhanced via conjugation to cross-reactive albumin-binding ligands in a process that improves their safety and accelerates testing through multiple pre-clinical animal models. In this context, the small and stable heavy-chain-only nanobody NbAlb1, capable of binding both human and murine albumin, has recently been successfully applied to improve the stability and prolong the in vivo plasma residence time of multiple small therapeutic candidates. Despite its clinical efficacy, the mechanism of cross-reactivity of NbAlb1 between human and murine serum albumins has not yet been investigated.

Development and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies.

In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (k) as a sorting index, scFv clones with a dissociation constant (K) ranging from 4.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >10 variants led to no further improvements.

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, k, of six different candidates were less than 10 s and their dissociation constants, Ks, were ranged from 5.

Effective production of single-chain variable fragment (scFv) antibody using recombinant Escherichia coli by DO-stat fed-batch culture.

Dissolved oxygen (DO)-stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was applied to anti-CRP single-chain variable fragment (scFv) production using recombinant Escherichia coli. The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5 to 0.

Combination of Aptamer Amplifier and Antigen-Binding Fragment Probe as a Novel Strategy to Improve Detection Limit of Silicon Nanowire Field-Effect Transistor Immunosensors.

Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement.

Conformational Dynamics Contribute to Substrate Selectivity and Catalysis in Human Kynureninase.

Kynureninases (KYNases) are enzymes that play a key role in tryptophan catabolism through the degradation of intermediate kynurenine and 3'-hydroxy-kynurenine metabolites (KYN and OH-KYN, respectively). Bacterial KYNases exhibit high catalytic efficiency toward KYN and moderate activity toward OH-KYN, whereas animal KYNases are highly selective for OH-KYN, exhibiting only minimal activity toward the smaller KYN substrate. These differences reflect divergent pathways for KYN and OH-KYN utilization in the respective kingdoms.

Efficient and robust isolation of rabbit scFv antibodies using antigen-coupled multilamellar vesicles.

We demonstrated an efficient screening method for rabbit scFv antibodies using antigen-coupled multi-lamellar vesicles (Ag-MLVs) as solid supports. Model phages displaying mouse anti-human IgG scFv at a probability of 10-10% were successfully isolated by Ag-MLVs after 3 or less rounds of biopanning, whereas they could not be isolated using conventional antigen-coated immunotubes. This screening method was applied to isolate rabbit antigen-specific scFvs from 4 different phage libraries.

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.

Oriented immobilization of rBC2LCN lectin for highly sensitive detection of human pluripotent stem cells using cell culture supernatants.

Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E.

Efficient preparation and site-directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)-binding peptide (PMMA-Tag).

A PMMA-binding peptide (PMMA-tag) was genetically fused with the C-terminal region of an anti-human chorionic gonadotropin (hCG) single-domain antibody (VHH). It was over-expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one-step IMAC purification.

Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture.

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments.

Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.

Identification and characterization of peptide fragments for the direct and site-specific immobilization of functional proteins onto the surface of silicon nitride.

In this study, we successfully identified peptide fragments that have a strong affinity toward the surface of a silicon nitride (SiN) substrate. An E. coli soluble protein, which was preferentially adsorbed onto the surface of a SiN substrate was isolated by 2D electrophoresis, and it was identified as "elongation factor Tu (ELN)" via the peptide MS fingerprinting method.

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.

Production of antibody fragments in Escherichia coli.

Escherichia coli is a host widely used in the industrial production of recombinant proteins. However, the expression of heterologous proteins in E. coli often encounters the formation of inclusion bodies, which are insoluble and nonfunctional protein aggregates.