Camelid Heavy-chain Antibodies Research Articles

Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

Iterative In Silico Screening for Optimizing Stable Conformation of Anti-SARS-CoV-2 Nanobodies.

Nanobodies (Nbs or VHHs) are single-domain antibodies (sdAbs) derived from camelid heavy-chain antibodies. Nbs have special and unique characteristics, such as small size, good tissue penetration, and cost-effective production, making Nbs a good candidate for the diagnosis and treatment of viruses and other pathologies. Identifying effective Nbs against COVID-19 would help us control this dangerous virus or other unknown variants in the future. Herein, we introduce an in silico screening strategy for optimizing stable conformation of anti-SARS-CoV-2 Nbs. Firstly, various complexes containing nanobodies were downloaded from the RCSB database, which were identified from immunized llamas. The primary docking between Nbs and the SARS-CoV-2 spike protein receptor-binding domain was performed through the ClusPro program, with the manual screening leaving the reasonable conformation to the next step. Then, the binding distances of atoms between the antigen-antibody interfaces were measured through the NeighborSearch algorithm. Finally, filtered nanobodies were acquired according to HADDOCK scores through HADDOCK docking the COVID-19 spike protein with nanobodies under restrictions of calculated molecular distance between active residues and antigenic epitopes less than 4.5 Å. In this way, those nanobodies with more reasonable conformation and stronger neutralizing efficacy were acquired. To validate the efficacy ranking of the nanobodies we obtained, we calculated the binding affinities (∆G) and dissociation constants (Kd) of all screened nanobodies using the PRODIGY web tool and predicted the stability changes induced by all possible point mutations in nanobodies using the MAESTROWeb server. Furthermore, we examined the performance of the relationship between nanobodies' ranking and their number of mutation-sensitive sites (Spearman correlation > 0.68); the results revealed a robust correlation, indicating that the superior nanobodies identified through our screening process exhibited fewer mutation hotspots and higher stability. This correlation analysis demonstrates the validity of our screening criteria, underscoring the suitability of these nanobodies for future development and practical implementation. In conclusion, this three-step screening strategy iteratively in silico greatly improved the accuracy of screening desired nanobodies compared to using only ClusPro docking or default HADDOCK docking settings. It provides new ideas for the screening of novel antibodies and computer-aided screening methods.

Construction of Camelus dromedaries Immune Single Domain Antibodies Library for Development of Schistosoma mansoni Specific Nanobodies Using Phage Display Strategy

Background: Schistosoma mansoni poses a considerable global public health challenge. In Egypt, approximately 60% of the inhabitants in the Northern and Eastern areas of the Nile Delta are affected by this parasite, whereas the Southern region experiences a significantly lower infection rate of 6%. background: Schistosoma (S.) mansoni infect 60% of the population in the Northern and Eastern parts of the Nile Delta and only 6% in the Southern part. Therefore, seeking for cost effective, sensitive and specific diagnostic tool for rapid detection of S. mansoni is necessary. Variable domains of camelid heavy-chain antibodies (VHHs) which are known as nanobodies (Nb) are approximately 15 kDa in size with high affinity to their antigens. Phage display technology was used in construction of Nbs library based on the camelid VHH framework for selection of S. mansoni specific Nbs Aim: Construction of an immune phage display Nbs library based on the VHH framework for selecting S. mansoni-specific Nbs for seeking cost-effective, sensitive, and specific diagnostic tools for rapidly detecting Schistosoma mansoni. Methods: Camel was immunized using soluble adult worm antigens (SAWP) for the production of Variable domains of heavy chains of camelid heavy-chain only antibodies (VHHs), which are known as nanobodies (Nb). The PBMCs repertoires VHH sequences library have been constructed with a high percentage of insertion and right orientation using pADL-23c phagmid and M13 phage followed by three rounds of bio-panning against SAWP using phage display technique. Evaluations using polyclonal phage ELISA and other techniques have been carried out to reveal the successful enrichment of anti-SAWP Nbs (VHH) clones. Evaluation of the diagnostic potentiality of these Nbs was carried out using ELISA on human serum samples confirmed for S. mansoni infection. Receiver Operator of Characteristics (ROC) curve analysis was used for discrimination between S. mansoni infection and both negative controls and the Fasciola hepatica group. Results: Using monoclonal ELISA, Nbs of 22 clones out of 24 selected clones showed binding affinity to SAWP. The cutoff values of the produced anti-S. mansoni Nbs was > 0.19, leading to 80% sensitivity, 95% specificity, and 90% accuracy. Sequence analysis of three of these Nbs with high binding affinities showed diversity in their targets, considering their CDR3 aa sequences. result: Using monoclonal ELISA, Nbs of 22 clones out of 24 selected clones showed binding affinity to SAWP. Sequence analysis of three of these Nbs with high binding affinities showed diversity in their targets considering their CDR3 aa sequences. Conclusion: This study successfully produced high diversity, anti-S.mansoni VHHs enriched phage library and the generated nanobodies have high diagnostic potential for S. mansoni infection in human patients. conclusion: We had successfully constructed high diversity VHH immune library against S. mansoni SAWP which can be efficiently used to develop anti-S. mansoni Nbs for diagnosis.

VHH CDR-H3 conformation is determined by VH germline usage

VHHs or nanobodies are single antigen binding domains originating from camelid heavy-chain antibodies. They are used as diagnostic and research tools and in a variety of therapeutic molecules. Analyzing variable domain structures from llama and alpaca we found that VHHs can be classified into two large structural clusters based on their CDR-H3 conformation. Extended CDR-H3 loops protrude into the solvent, whereas kinked CDR-H3 loops fold back onto framework regions. Both major families have distinct properties in terms of their CDR-H3 secondary structure, how their CDR-H3 interacts with the framework region and how they bind to antigens. We show that the CDR-H3 conformation of VHHs correlates with the germline from which the antibodies are derived: IGHV3-3 derived antibodies almost exclusively adopt a kinked CDR-H3 conformation while the CDR-H3 adopts an extended structure in most IGHV3S53 derived antibodies. We do not observe any bias stemming from V(D)J recombination in llama immune repertoires, suggesting that the correlation is the result of selection processes during B-cell development. Our findings demonstrate a previously undescribed impact of germline usage on antigen interaction and contribute to a better understanding on how properties of the antibody framework shape the immune repertoire.

Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins

Introduction: Drosophila melanogaster is a model organism for studying developmental biology and human neural disorders. Nanobodies are the variable domains of the heavy chains of camelid heavy-chain antibodies (VHHs) with high affinity to their antigens and have applications in basic research, similar to traditional antibodies. In addition, nanobodies acting as functionalized antibodies or protein binders have become an additional valuable approach in Drosophila . This study aimed to develop a VHH library against Drosophila proteins and confirm its availability by retrieving some Drosophila protein-specific nanobodies from the library. Methods: An alpaca was first immunized with Drosophila embryo lysate and then its lymphocytes were isolated. Total RNA was extracted and cDNA was synthesized. The vhh sequences were amplified by two round PCR, which were then ligated to a phage display vector pADL-10b. The ligation products were transduced into SS320 competent cells to generate a VHH library. From this library, nanobodies against CG7544, Myc, and CyclinE was enriched and screened by phage display technology and ELISA. DNA sequences of identified nanobodies were cloned into pADL-10b-Flag-His for expression and purification in Escherichia coli SS320. Binding ability of purified nanobodies with corresponding antigens were determined by ELISA and surface plasmon resonance in vitro . Results: In this study, an immune VHH library against Drosophila embryo proteins was generated with a capacity of 3 × 10 7 . From this library, eight nanobodies against three Drosophila proteins, Myc, CyclinE, and CG7544, were identified and the DNA sequences of these nanobodies were obtained. These nanobodies were successfully expressed and purified from Escherichia coli SS320, and were demonstrated to bind corresponding antigens with high affinity in vitro . Moreover, the equilibrium constant between the highest enriched nanobodies and corresponding antigens were calculated. Conclusion: In summary, we report the availability of an immune VHH library and a highly efficient panning strategy for nanobodies against proteins in Drosophila .

Structural, nonclinical, and clinical features of ozoralizumab: A novel tumour necrosis factor inhibitor.

Tumour necrosis factor (TNF) inhibitors are currently the most widely used biological agents to treat rheumatoid arthritis. Ozoralizumab (OZR), a novel TNF inhibitor, is an antibody using variable heavy-chain domains of heavy-chain antibody (VHHs) and became the first VHH drug approved for the treatment of rheumatoid arthritis in September 2022. VHHs isolated from camelid heavy-chain antibodies can bind antigens with a single molecule. OZR is a trivalent VHH that consists of two anti-human TNFα VHHs and one anti-human serum albumin (anti-HSA) VHH. This review summarizes OZR's unique structural characteristics and nonclinical and clinical data. The clinical data outline the pharmacokinetics, efficacy, relationship between efficacy and pharmacokinetics, and safety of OZR, focusing on a Phase II/III confirmatory study (OHZORA trial).

Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C β-Lactamases.

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 β-lactamase behaved as inhibitors. The structure of the complex VHH cAbCMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The β-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of β-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

Numaswitch, a biochemical platform for the efficient production of disulfide-rich pepteins

The application of long-chained peptides (+30 aa) and relatively short proteins (<300 aa) has experienced an increasing interest in recent years. However, a reliable production platform is still missing since manufacturing is challenged by inherent problems such as mis-folding, aggregation, and low production yields. And neither chemical synthesis nor available recombinant approaches are effective and efficient. This in particular holds true for disulfide-rich targets where the correct isomer needs to be formed. With the technology Numaswitch, we have now developed a biochemical tool that circumvents existing limitations and serves as first production platform for pepteins, hard-to-be-produced peptides and proteins between 30 and 300 amino acids in length, including disulfide-rich candidates. Numaswitch is based on bifunctional Switchtag proteins that force the high-titer expression of pure inclusion bodies and simultaneously assist in the efficient refolding of pepteins into functional pepteins. Here, we demonstrate the successful application of the Numaswitch platform for disulfide-containing pepteins, such as an antimicrobial fusion peptide, a single-chain variable fragment (scFv), a camelid heavy chain antibody fragment (VHH) and the human epidermal growth factor.

Structural Characterization of Nanobodies during Germline Maturation.

Camelid heavy-chain antibody variable domains (VHH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VHH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VHH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VHH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.

A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen.

Rapid and sensitive detection of bacterial pathogens present in food and environmental samples is of crucial importance to ensure human health and safety. Here, we present a one-step label-free colorimetric strategy based on M13 bacteriophage-displayed nanobody (phage-Nb) derived from camelid heavy-chain antibodies specific to Vibrio parahaemolyticus (V. parahaemolyticus). The thiolation of phage-Nb (Phage-Nb-SH) on pVIII shell proteins induces the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between nanobody and bacteria prevents the aggregation of AuNPs, resulting in visible color change due to alteration of surface plasmon resonance properties. Based on this phenomenon, a simple and sensitive colorimetric immunosensor for V. parahaemolyticus was developed. The assay can be accomplished within 100min, and exhibits a visual detection limit of 104 cfu/mL and a quantitative detection limit of 103 cfu/mL, with no cross-reactivity towards other bacterial species. This strategy takes full advantages of both the high specificity of phage-Nbs and the optical properties of AuNPs, enabling simple and rapid detection of bacterial pathogens.

Structure-guided design of a potent Clostridiodes difficile toxin A inhibitor.

Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridiodes difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC 50]; ≥2,700-fold [IC 99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC 50) to 29,000-fold (IC 99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen.

Integrating Single Domain Antibodies into Field-Deployable Rapid Assays.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19.

Preclinical Efficacy of BCMA-Directed CAR T Cells Incorporating a Novel D Domain Antigen Recognition Domain.

Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA.

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments.

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents.

Nanobody-Based Assays for the Detection of Environmental and Agricultural Contaminants.

Compared with traditional polyclonal and monoclonal antibodies, nanobodies derived from camelid heavy-chain antibodies have several advantages including small size, unique structure and binding geometry, high stability, and robust expression yields in numerous systems. Nanobody-based assays can also exhibit superior performance for immunodetection. Here, we describe protocols for three nanobody-based immunoassays for the detection of small chemical contaminants in environmental or agricultural samples: enzyme-linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and bioluminescent enzyme immunoassay (BLEIA). These methods are based on hapten-specific nanobodies, nanobody-alkaline phosphatase fusion proteins, and nanobody-nanoluciferase fusion proteins, respectively.

Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling.

Detection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.