Search Results (205)

Mimotope-based immunoassays offer an eco-friendly alternative to chemically synthesized antigens for the quantitative analysis of small molecules, but their use for practical on-site and high-throughput residue monitoring remains limited. Herein, we report the selection, production, and application of a phage display–derived mimotope targeting an anti-uniconazole monoclonal antibody (UCZ-mAb), with the aim of developing two complementary immunoassays that enable sensitive, eco-friendly detection of UCZ residues in agricultural samples. A 12-mer phage-displayed peptide library was screened to identify UCZ-specific mimotopes, and a selected sequence was genetically fused to SpyTag and expressed in Escherichia coli to generate a SpyTagged mimotope. Leveraging the SpyCatcher/SpyTag self-assembly system, the SpyTagged mimotope was directionally conjugated onto SpyCatcher-functionalized time-resolved fluorescence beads (TRFBs) and subsequently used as a signal-labeled competitive antigen in a lateral flow immunoassay (LFIA) designed for rapid on-site screening. In parallel, a wash-free magnetic separation immunoassay (MSIA) suitable for green, high-throughput screening in routine laboratories was established using self-assembled mimotope-TRFB probes. The LFIA and MSIA exhibited half-maximal inhibitory concentrations (IC₅₀) of 3.70–6.72 μg/kg and 16.4–18.3 μg/kg, respectively, in real samples. Spiked-sample recoveries ranged from 91.1 to 107.8% for LFIA and 92.6–115.7% for MSIA, demonstrating acceptable accuracy and precision. These results indicate that the SpyTagged mimotope–based LFIA and MSIA provide complementary, reliable, and sensitive platforms for on-site screening and high-throughput monitoring of UCZ residues in agricultural samples, while avoiding the drawbacks associated with traditional chemical antigen synthesis. Full article

Enzyme-Linked-Immunosorbent-Spot (ELISpot) is a highly sensitive technique capable of detecting low-level immune responses, offering critical insights into therapy-induced immune activation. Our mouse interferon-gamma (mIFN-γ) ELISpot assay was originally based on a monoclonal capture antibody and a rabbit polyclonal detection antibody. The objective of our study was to replace the polyclonal detection antibody with a monoclonal alternative, using a llama immune library and phage display technology. A llama was immunized with recombinant mIFN-γ, and an immune VHH library was constructed. The library underwent two rounds of panning using the recombinant antigen. Subsequently, 190 clones were screened by Enzyme-Linked-Immunosorbent Assay (ELISA), yielding 27 specific binders to mIFN-γ. Sequence analysis revealed 24 unique clones grouped into four families based on their CDR3-VH sequences. One representative clone from each family was reformatted as VHH-Human Fragment Crystallizable (VHH-hFc) fusion and produced recombinantly for testing in the ELISpot assay. The purified candidates were evaluated in pairs on native mIFN-γ from mouse splenocytes. Two candidates, H3 and G4, were selected for further trial. Comparative analysis of ELISpot performance showed that G4 is a promising substitute for the original rabbit polyclonal antibody, enhancing the overall performance of the mIFN-γ ELISpot assay. This study highlights the potential of VHH antibodies in ELISpot applications and supports their use as a robust, reproducible alternative to polyclonal antibodies. Full article

Background: Antibodies that cross the blood–brain barrier (BBB) by targeting receptor-mediated transport (RMT) systems can allow efficient drug delivery to the central nervous system (CNS). In order to improve brain uptake of antibodies, their binding properties have been engineered, but it is not always clear what antibody properties dictate BBB transport efficiency. In this study, we therefore developed and employed an in vitro phenotypic screen and a quantitative transcytosis assay in an attempt to identify improved variants of a previously identified BBB transcytosing antibody known as 46.1. Methods: First, a random mutagenic 46.1 antibody phage display library was screened for improved transcytosis through a human induced pluripotent stem cell (iPSC)-derived BBB model. These screens yielded antibody variants that enriched over multiple screening rounds; however, when produced as soluble antibodies, the variants did not display improved in vitro transcytosis over the wild-type (WT) 46.1 antibody. As a second strategy, we performed a targeted histidine point mutation of a solvent-exposed residue in each complementarity-determining region (CDR) and evaluated the in vitro transcytosis capacity of the variants. Results and Conclusions: In this way, we identified a 46.1 variant, R162H, with modestly improved in vitro transcytosis properties. These results show that the iPSC-derived BBB screening insights and evaluation strategies presented here could facilitate the engineering and optimization of lead antibodies for CNS delivery. Full article

Monoclonal antibodies (mAbs) represent one of the most successful classes of biopharmaceuticals, with more than 100 approved for treating oncological, immunological, and infectious diseases. Antibody discovery and development have been driven by diverse methodologies. Classical strategies such as mouse hybridoma technology, phage display, transgenic mouse models, and single B cell isolation have enabled the generation of high-affinity therapeutic antibodies. Beyond binding affinity, recent innovations in combinatorial antibody libraries have facilitated the selection of functional antibodies within cellular environments, revealing their ability to act as agonists or antagonists and influence signal transduction pathways. These insights expand therapeutic applications by enabling modulation of complex cellular responses. Recent breakthroughs in artificial intelligence, involving antibody generation supported by rapidly growing antibody sequence and structure databases, are transforming computational protein design. This review highlights five major approaches (hybridoma technology, phage display, transgenic mouse models, and single B cell isolation, de novo antibody design) for antibody discovery and development. These approaches offer innovative strategies designed to accelerate the discovery process and enhance therapeutic outcomes for human diseases. Full article

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples. Full article

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues. Full article

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC₅₀ values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans. Full article

Background/Objectives: Calsequestrin (CSQ) is a calcium-binding protein that is highly soluble and can serve as a solubility-enhancing fusion tag in recombinant protein expression. Its unique property of calcium-induced precipitation followed by EDTA-mediated resolubilization enables efficient purification. However, the broader application of CSQ-tagged proteins in research have been hampered by the lack of reliable anti-CSQ detection reagents. This study aimed to develop single-domain antibodies (sdAbs) against CSQ for use in diverse immunoassays and cell-based analyses. Methods: Single-domain antibodies were selected from phage-displayed chicken VH libraries generated from CSQ-immunized chickens. After biopanning, CSQ-specific VH sdAb clones were isolated and expressed as VH–human kappa light chain constant region (VH-Cκ) fusion proteins in E. coli. The PE06 clone was chosen for further characterization and conjugated to horseradish peroxidase (HRP) and Alexa Fluor 647 for assay applications. Results: PE06 VH-Cκ fusion protein demonstrated specific binding to CSQ-tagged proteins and enabled reliable detection in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and flow cytometry. These results validated its utility as a chemically defined detection reagent for CSQ fusion proteins expressed in E. coli. Conclusions: This study establishes a CSQ-specific chicken VH sdAb as a versatile detection tool for CSQ-tagged proteins. The approach expands the utility of CSQ as a protein fusion tag and enables the development of recombinant antibodies fused with CSQ, such as scFv-CSQ constructs, for broad application in research and assay systems. Full article

Background/Objectives: The complexity of diseases such as cancer and auto-immune disorders drives the need for unique, target-driven therapeutics. A broader arsenal to generate better biologics-based therapeutics is needed to provide more efficient and effective antibody generation technologies. The critical parameter for antibody generation is to generate as much candidate diversity to each target as possible. Method/Results: We present guidelines for having an efficient process using a fully synthetic human single-domain antibody (sdAb) phage display library. Critical milestones for success focused on library quality control (QC) assessments, evaluation of specific biopanning outputs, and construct designs that enabled efficient transition to mammalian expression. The synthetic VHO libraries produced epitope diversity better than an immunized sourced library with candidates possessing nM potencies and monodispersity > 90% via SEC. Conclusions: Synthetic human scaffold sdAb phage display libraries was constructed, biopanned, and selected candidates that could be directly transitioned for mammalian expression. The diverse VHO sets of candidates produced from many targets easily provided opportunities to make a multi-specific biological compound. Both synthetic and immunized phage selection campaign results suggested that these technologies complemented each other to generate therapeutic candidates. Finally, we demonstrated how diverse data produced from a process that used VHO synthetic libraries could accelerate drug discovery. Full article

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by tissue damage in multiple organs caused by autoantibodies and the resulting immune complexes. One possible way for complement system contribution to onset of autoimmune disorder could be realized by the impairment of C1q-mediated apoptotic clearance as part of human homeostasis. The capacity of C1q to bind early apoptotic cells could be decreased or even lost in the presence of anti-C1q antibodies. A monoclonal anti-idiotypic single-chain (scFv) antibody was selected from the phage library Griffin1” to recognize anti-C1q autoantibodies, purified from sera of lupus nephritis patients. Lupus-prone MRL/lpr mice were injected weekly with scFv A1 fragment-binding anti-C1q antibodies. The number of in vitro and ex vivo studies with collected cells, sera, and organs from the treated animals was performed. scFv treatment changed the percentage of different B-, T-, and NK-cell subpopulations as well as plasma cells and plasmablasts in the spleen and bone marrow. An increase in the levels of splenocyte proliferation, anti-C1q antibodies, and the number of plasma cells producing anti-dsDNA and anti-C1q antibodies were also observed in scFv-treated animals. High levels of proteinuria and hematuria combined with unstable levels of IL10 and IFNγ promote the development of severe lupus and shorten the survival of treated MRL/lpr mice. Therapy with the scFv A1 antibody resulted in BCR recognition on the surface of anti-C1q-specific B-cells and had a disease progression effect, enhancing lupus symptoms in the MRL/lpr mouse model of SLE. Full article

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development. Full article

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD₅₀) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD₅₀ TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD₅₀ TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A/H1N1 virus. Methods: A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition. Results: The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 μg/mL against the A/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays. Conclusions: This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A/H1N1 virus, as it may reduce the severity of their symptoms. Full article

Engineered scaffold-based proteins that bind to concrete targets with high affinity offer significant advantages over traditional antibodies in theranostic applications. Their development often relies on display methods, where large libraries of variants are physically contacted with the desired target protein and pools of binding variants can be selected. Herein, we use a novel combined artificial intelligence/physics-based computational framework and phage display approach to obtain ubiquitin based Affilin^® proteins targeting the human epidermal growth factor receptor 3 (HER3) extracellular domain, a relevant tumor target. As traditional antibodies against the receptor have failed so far, we sought to provide molecules in a smaller more versatile format to cover the medical need in HER3 related diseases. We demonstrate that the developed in silico pipeline can generate de novo Affilin^® proteins binding the biochemical HER3 target using a small training set of <1000 sequences. The classical phage display yielded primary candidates with low nanomolar affinities to the biochemical target and HER3-expressing cells. The latter could be further optimized by phage display and computational maturation alike. These combined efforts resulted in four HER3 ligands with high affinity, cell binding, and serum stability with theranostic potential. Full article