Nanobody Library Research Articles

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models’ performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

Selective Modulation of Activated Protein C Activities By an Exosite-Specific Nanobody Library

Background Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Antibodies that can selectively modulate these APC activities could contribute to the understanding of the regulation of these physiological mechanisms and the development of therapeutics for the pathologies associated with these pathways Aim We sought to construct an antibody library consisting of small-sized nanobodies that could target neo-epitopes on the exosites of the activated form of protein C that are potentially inaccessible or whose three-dimensional structure is not recognized by conventional antibody formats. These APC-specific nanobodies will be used to determine the effect of the blockade of different exosites on APC pleiotropic activities. Methods A nanobody phage library specific for APC exosites was derived from peripheral blood mononuclear cells of llamas immunized with active site-blocked APC (APC-PPACK). The nanobody library was panned for APC-specificity. Biolayer interferometry (BLI) was used to determine the KDs and to epitope bin nanobodies into different clusters. APC functional assays, including Protac-APTT clotting assays, histone H3 cleavage assay, and SEAP-PAR1 cleavage assay with HEK293 cells, were employed to characterize the effects of the nanobodies on APC's pleiotropic activities. Results Of the total 1128 monoclonal phage clones screened in the library, 725 clones were identified as APC-PPACK binders. These clones were sequenced and 158 were found to be unique clones. Among them, 21 nanobodies belonging to 18 families were further characterized by their binding diversity. Overall, 3 clusters of nanobodies were identified based on their competition for APC exosites in BLI studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and SEAP-PAR1 cleavage, were employed to understand the diversity of the nanobodies. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential of regulating specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, LP17) inhibited all 3 APC functions. Five nanobodies (LP1, LP5, LP10, LP16, LP20) inhibited only 2 of the 3 functions. Mono-function inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP 9, LP11). LP 9 and LP11 are 2 of the nanobodies within the library that inhibited APC by >90% in a clotting assay (Table 1). The monospecific inhibition of APC anticoagulant activity could induce a pro-hemostatic effect in hemophilia and traumatic coagulopathy conditions. The molecular basis of APC's cytoprotective functions is based on its biased agonism of PAR1 by cleaving at R46. Interestingly, in R46/R41 cleavage ratio studies on SEAP-PAR1 cell lines, LP11 increased the APC pro-cytoprotective cleavage of R46 by up to 87%. The novel property of LP11 in enhancing R46 cleavage may potentially provide additional cytoprotection signaling and may potentially further benefit the outcome of these bleeding conditions. Conclusions A library of APC exosite-specific llama nanobodies yielded 21 nanobodies that can selectively modulate APC anticoagulation and cytoprotection pathways. The non-competing nature of nanobodies from each cluster allows the possibility of combining nanobodies from different clusters to better understand and study the biology of APC. These nanobodies represent a tool kit of 13 categories of antibodies based on their cluster and activity profile and offer the opportunity to develop novel therapeutics for the treatment of an array of APC-associated indications including acute bleeding, hemophilia, ischemia, and sepsis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phage-displayed nanobody-based fluorescence-linked immunosorbent assay for the detection of Cry3Bb toxin in corn

Cry toxins have been widely used as biopesticides and applied in transgenic crops for the control of pests, raising public concerns for unforeseen environmental and health risks. In this study, five phage-displayed nanobodies against Cry3Bb toxin were selected from a naive nanobody library by screening. Using purified phage-displayed nanobody as capture antibody, a novel double-antibody sandwich fluorescence-linked immunosorbent assay (FLISA) based on quantum dot beads (QBs) was established for the determination of Cry3Bb toxin. The developed method exhibited a linear working range of 31.25–500 ng/mL and a low detection limit of 8.45 ng/mL. The recoveries of Cry3Bb toxin from spiked corn samples were ranging from 93.1% to 116.8% with a coefficient of variation lower than 9.4%. Molecular modeling results showed that key amino acids in the CDR3 region of nanobody P27 combine with Cry3Bb toxin through hydrogen bonds and hydrophobic interactions. These results indicated that the phage-displayed nanobody-based QBs-FLISA may provide a possible tool for detecting Cry3Bb toxin in corn.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.

Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library.

The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\\ml and 9.6 ng\\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies

Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.

Screening of nanobody against Microcystis from a human phage display nanobody library

Screening of nanobody against Microcystis from a human phage display nanobody library

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library

To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library. We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA. A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies. A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

Tracking and Inhibiting Atypical Vascular Inflammation

Mitogen‐activated protein kinase (MAPK) p38 drives the onset and progression of many clinically challenging diseases. However, three decades of research have failed to yield clinically viable, selective MAPK p38 therapeutics. The extensive and ongoing studies have principally focused on therapeutics targeting the ATP‐pocket or active site of p38. These have failed in part due to the ubiquitous expression and dichotomous role of p38, leading to blockade of both physiological and pathological signaling pathways. A promising alternative strategy is to target either the substrates of p38 or the activators that drive p38‐induced pathological signaling. Our prior studies identified an atypical mechanism for G protein‐coupled receptor mediated activation of pathological p38 signaling during vascular inflammation. Several critical problems remain: firstly, how does atypical signaling drive inflammation, and secondly how can we stop pathological p38 activity? To further define how atypical p38 signaling induces inflammation we developed a fluorescent resonance energy transfer (FRET) biosensor platform to map atypical p38 signaling. This system revealed that GPCR induced p38 signaling is initiated via an endosomal axis. Blockade of receptor internalization shifts the spatial bias to that seen for MKK3/6‐dependent signaling with enhanced nuclear p38 activity. We predict that this endosomal axis drives pathological atypical p38 activity and the progression of vasculopathic disease progression. Critically, atypical p38 activity is initiated by a direct interaction between the adaptor protein TAB1 and two binding sites on p38. Blockade of the TAB1‐p38 interaction through mutation of select residues on TAB1 can disrupt the TAB1‐p38 interaction and prevent atypical p38 signaling. Modeling studies show that small molecule inhibitors that bind to the TAB1 binding motif on p38 can block this interaction. As an alternative strategy we focused on the TAB1 peptides that directly bind to p38. Using a phage‐display library of humanized nanobodies and a yeast two‐hybrid screen we have developed an array of 40 TAB1 blocking intrabodies (IntraT‐ab’s) as the first generation of selective atypical p38 inhibitors. We show that intraT‐ab’s bind TAB1, selectively blocking GPCR induced p38 (endosomal) signaling while leaving classical MKK3/6 dependent signaling intact. IntraT‐ab’s and our FRET platform provide the first opportunity to examine in detail the specific downstream mechanisms, targets and physiological consequences of atypical p38 signaling. Intracellular delivery/expression of intraT‐ab’s could also provide a therapeutic strategy for reducing p38 signaling in multiple diseases, including acute lung injury, retinopathies and neuroinflammation.

High‐throughput CRISPR for Generating Therapeutic Nanobodies

Engineered antibodies have had a widespread impact throughout biomedical sciences, from their use as fundamental research tools to their employment as therapeutics. Closely related nanobodies, derived from heavy‐chain only antibodies, have begun gaining much attention as alternatives with improved pharmacological properties. Containing only the antigen recognition components, nanobodies maintain the exceptional specificity of conventional antibodies but their simplistic design leads to increased stability and decreased toxicity. In recent years, nanobodies have proven especially useful for studying G protein‐coupled receptors (GPCRs), a class of membrane receptors responsible for activating intracellular proteins (e.g., Gα proteins) and initiating subsequent signaling pathways. Most GPCR‐targeting nanobodies to date have been designed as crystallographic chaperones for stabilizing the GPCR‐Gα interaction for in‐depth structural insights. However, harnessing the intrinsic specificity provided by nanobodies for therapeutic purposes has been limited to few receptors. In this study, we present a unique platform for discovering functionally selective nanobodies for a broad range of GPCRs. Using a yeast model proven invaluable for discovering GPCR ligands and structure‐stabilizing nanobodies, we engineered a system for rapidly identifying nanobody‐based therapeutic candidates. In this system, each cell uniquely contains 1) a tethered nanobody displayed outside the cell membrane, 2) a human GPCR, 3) one of ten Gα proteins for characterizing GPCR‐Gα interactions, and 4) a fluorescent reporter induced upon GPCR/Gα activation. By combining all components into a single cell, we can directly access the therapeutic potential of each nanobody‐GPCR combination by monitoring the (in)activity of relevant signaling pathways inspired from this interaction. To date, we have completed the initial engineering stages of this platform and showcased its utility through pharmacological activation of various GPCRs (e.g., SSTR5, AGTR1, CXCR4, and C5aR) via tethered peptide/protein agonists. Next, we will use high‐throughput CRISPR to generate an extensive nanobody library for rapidly identifying and functionally quantifying GPCR‐targeting nanobodies. Using this approach for a large variety of GPCRs, this platform will provide a vast collection of nanobody‐based therapeutic candidates, and with minimal modifications may be expanded for identifying therapeutics for other classes of membrane receptors.

Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken

BackgroundSalmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention of zoonotic infectious disease and controlling the risk of transmission of S. Enteriditidis critically requires the evolution of rapid and sensitive detection methods. The detection methods based on nucleic acid and conventional antibodies are fraught with limitations. Many of these limitations of the conventional antibodies can be circumvented using natural nanobodies which are endowed with characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed to select the special nanobodies against S. Enteriditidis for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect S. Enteritidis in the practical sample. The nanobody-horseradish peroxidase fusions can help in eliminating the use of secondary antibodies labeled with horseradish peroxidase, which can reduce the time of the experiment. Moreover, the novel sandwich ELISA developed in this study can be used to detect S. Enteriditidis specifically and rapidly with improved sensitivity.ResultsThis study screened four nanobodies from an immunized nanobody library, after four rounds of screening, using the phage display technology. Subsequently, the screened nanobodies were successfully expressed with the prokaryotic and eukaryotic expression systems, respectively. A sandwich ELISA employing the SE-Nb9 and horseradish peroxidase-Nb1 pair to capture and to detect S. Enteritidis, respectively, was developed and found to possess a detection limit of 5 × 104 colony forming units (CFU)/mL. In the established immunoassay, the 8 h-enrichment enabled the detection of up to approximately 10 CFU/mL of S. Enteriditidis in milk samples. Furthermore, we investigated the colonization distribution of S. Enteriditidis in infected chicken using the established assay, showing that the S. Enteriditidis could subsist in almost all parts of the intestinal tract. These results were in agreement with the results obtained from the real-time PCR and plate culture. The liver was specifically identified to be colonized with quite a several S. Enteriditidis, indicating the risk of S. Enteriditidis infection outside of intestinal tract.ConclusionsThis newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.Graphic

Development and comparison of three 89Zr-labeled anti-CLDN18.2 antibodies to noninvasively evaluate CLDN18.2 expression in gastric cancer: a preclinical study.

Isoform 2 of claudin 18 (CLDN18.2) is overexpressed in gastric cancer and may be a promising imaging target. In this study, we constructed three anti-CLDN18.2 antibodies and compared them in preclinical experiments. Screening from anti-CLDN18.2 nanobody library, we constructed three antibodies, anti-CLDN18.2 VHH (recombinant single-chain antibody fused with poly-histidine-tag), anti-CLDN18.2 VHH-ABD (recombinant single-chain antibody fused fused with albumin binding domain), and anti-CLDN18.2 VHH-Fc (recombinant single-chain antibody fused with IgG1-Fc) and radiolabeled with 89Zr. Affinity assay, in vitro stability, immunoactivity, blood pharmacokinetics, in vivo and ex vivo biodistribution study, specificity study, and immunohistochemical analysis were performed to assess these radiotracers. The EC50 were 12.21nM, 2.48nM, and 0.14nM for anti-CLDN18.2 VHH, anti-CLDN18.2 VHH-ABD, and anti-CLDN18.2 VHH-Fc, respectively. 89Zr-anti-CLDN18.2 VHH demonstrated the lowest tumor uptake in PET imaging. Both 89Zr-anti-CLDN18.2 VHH-ABD and 89Zr-anti-CLDN18.2 VHH-Fc demonstrated high tumor accumulation, with highest ID%/g of 25.78 ± 5.60 at 24h post-injection with 89Zr-anti-CLDN18.2 VHH-ABD and 49.43 ± 9.86 at 72h post-injection with 89Zr-anti-CLDN18.2 VHH-Fc. The specificity of 89Zr-anti-CLDN18.2 VHH-Fc targeting CLDN18.2 was further confirmed by blocking study. The ex vivo biodistribution results were consistent with in vivo biodistribution data. For 89Zr-anti-CLDN18.2 VHH-ABD, tumor uptake was 21.46 ± 1.78 ID%/g at 12h and 13.73 ± 2.22 ID%/g at 108h. For 89Zr-anti-CLDN18.2 VHH-Fc, the tumor accumulation was 25.28 ± 3.83 ID%/g at 12h and 40.13 ± 9.50 ID%/g at 108h. Immunohistochemistry of the xenograft tissue revealed high and homogenous CLDN18.2 expression in CO-SNU620 tumor. Both anti-CLDN18.2 VHH-ABD and anti-CLDN18.2 VHH-Fc can be efficiently and stably radiolabeled with 89Zr for noninvasive imaging and quantification of CLDN18.2 expression in gastric cancer, of which 89Zr-anti-CLDN18.2 VHH-ABD seems to be the optimal choice balancing tumor uptake and liver background. They can provide essential information to select patients who are likely to benefit from CLDN18.2-targeted treatment.

Development and characterization of single domain monoclonal antibody against programmed cell death ligand-1; as a cancer inhibitor candidate.

Objective(s):One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI.Materials and Methods:A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry. Results:Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth.Conclusion:The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.

Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity.

Nanobodies (Nbs), the single‐domain antigen‐binding fragments of dromedary heavy‐chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha‐synuclein (α‐syn). Phage display screening of the library allowed the identification of a nanobody, Nbα‐syn01, specific for α‐syn. Unlike previously developed nanobodies, Nbα‐syn01 recognized the N‐terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα‐syn01 and the engineered bivalent format BivNbα‐syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα‐syn01 and its bivalent format recognized preferentially α‐syn fibrils compared to the monomeric form. Nbα‐syn01 and BivNbα‐syn01 were also able to inhibit α‐syn‐seeded aggregation in vitro and reduced α‐syn‐seeded aggregation and toxicity in cells showing their potential to reduce α‐syn pathology. Moreover, both nanobody formats were able to recognize Lewy‐body pathology in human post‐mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα‐syn01 binds the N‐terminal region of the α‐syn aggregated form. Overall, these results highlight the potential of Nbα‐syn01 and BivNbα‐syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.

Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody

Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 1013 pfu mL−1 phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL−1 and IC50 was 0.0588 ng mL−1 in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay.

Selection and Modelling of a New Single-Domain Intrabody Against TDP-43.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated to deteriorating motor and cognitive functions, and short survival. The disease is caused by neuronal death which results in progressive muscle wasting and weakness, ultimately leading to lethal respiratory failure. The misbehaviour of a specific protein, TDP-43, which aggregates and becomes toxic in ALS patient’s neurons, is supposed to be one of the causes. TDP-43 is a DNA/RNA-binding protein involved in several functions related to nucleic acid metabolism. Sequestration of TDP-43 aggregates is a possible therapeutic strategy that could alleviate or block pathology. Here, we describe the selection and characterization of a new intracellular antibody (intrabody) against TDP-43 from a llama nanobody library. The structure of the selected intrabody was predicted in silico and the model was used to suggest mutations that enabled to improve its expression yield, facilitating its experimental validation. We showed how coupling experimental methodologies with in silico design may allow us to obtain an antibody able to recognize the RNA binding regions of TDP-43. Our findings illustrate a strategy for the mitigation of TDP-43 proteinopathy in ALS and provide a potential new tool for diagnostics.

Identification and characterization of inhibitory nanobody against p38δ

p38δ is a member of p38 mitogen-activated protein kinases (MAPKs) family that displays cell- and tissue-specific expression patterns. Recent studies demonstrate that p38δ is centrally involved in several pathologic events, such as diabetes, neurodegeneration diseases, inflammatory diseases, and cancer, and suggest that it may be a potential target for diagnosis and therapy of specific diseases. A nanobody is a new type of antibody that exhibits high antigen-binding activity, solubility, stability, and easy production. This study utilized phage display to isolate nanobodies specifically against p38δ from a fully synthetic nanobody library. Two of them, nanobodies Nb13-6 and Nb13-1, display high binding activity to p38δ, less cross-reactivity with other p38 MAPKs, and high thermal and pH stabilities. Modeling and docking analysis indicated that Nb13-6 is mostly linked to the activation loop of p38δ. Furthermore, detailed studies revealed that Nb13-6 inhibited the protein kinase activity of p38δ and the growth of cancer cells. Therefore, this study provides p38δ-specific nanobodies that are promisingly exploited for diagnosing and treating p38δ-associated diseases.

Nanobodies as binding-chaperones stabilize the recombinant Bombyx mori acetylcholinesterase and protect the enzyme activity in pesticide detection

In our previous study, the recombinant type II acetylcholinesterase from Bombyx mori (rBmAChE) presented outstanding sensitivity to pesticides, which exhibited great potential in pesticides detection. However, the poor stability of rBmAChE and also the unclear mechanism of its sensitivity hindered the applications in on-site testing of pesticides residues. In this study, we constructed an immune nanobody library, in which we obtained 48 rBmAChE-specific nanobodies. Among them, Nb4 and Nb9 were verified as the most prominent enhancers of the enzyme activity and stabilizers under thermal stress, which indicated their usage as protective reagents for rBmAChE. The simultaneously addition of the two Nbs enhanced the thermal-stability of rBmAChE against exposure to 50–70 °C, and also remained 100% residual activity after 30 days storage at − 20 °C or 4 °C, whereas 80% and 62% at − 80 °C and 25 °C. The homologous modeling and docking of Nb4 and Nb9 to rBmAChE indicated the stabilization of Nb4 to the peripheral anion site (PAS) of rBmAChE while Nb9 protected the C-terminal structure. Substrate docking demonstrated the importance of electrostatic attraction during catalytic process, that might be enhanced by Nbs. As a result, Nb4 and Nb9 were proved to have great potential on rBmAChE applications due to their regulation on enzyme activity and protection against thermal-inactivation and long-term storage of rBmAChE.

Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.

The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of target-displaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry.