Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis.

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

Affinity purification of mAb from serum-containing hybridoma culture supernatant through a novel nanobody that discriminates mouse IgG from bovine IgG by recognizing the mouse kappa constant region (mCK).

When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.

Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display.

BACKGROUND: Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. METHODS: An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. RESULTS: A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M-1 and 2.08 × 107 M-1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. CONCLUSION: The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics.

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A.

While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

Identification of Anti-TNFα VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum).

Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs' epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications.

Generation, biochemical characterizations and validation of potent nanobodies derived from alpaca specific for human receptor of advanced glycation end product.

A detrimental role of the receptor for the advanced glycation end product (RAGE) has been identified in the immune response, and various pathological conditions and its V and C1 domains in the extracellular region of RAGE are believed to be the main ligand-binding domains. Consequently, specific inhibitors targeting those domains could be of clinical value in fighting against the pathological condition associated with RAGE over-activation. Single-domain antibodies, also called nanobodies (Nbs), are antibody fragments engineered from the heavy-chain only antibodies found in camelids, which offer a range of advantages in therapy. In this study, we report the development and characterization of the V-C1 domain-specific Nbs. Three Nbs (3CNB, 4BNB, and 5ENB) targeting V-C1 domain of human RAGE were isolated from an immunized alpaca using a phage display. All of these Nbs revealed high thermostability. 3CNB, 4BNB, and 5ENB bind to V-C1 domain with a dissociation constant (KD) of 27.25, 39.37, and 47.85 nM, respectively, using Isothermal Titration Calorimetry (ITC). After homodimerization using human IgG1-Fc fusion, their binding affinity improved to 0.55, 0.62, and 0.41 nM, respectively, using Surface Plasmon Resonance (SPR). Flow cytometry showed all the Fc fusions Nbs can bind to human RAGE expressed on the cell surface. Competitive ELISA further confirmed their V-C1-hS100B blocking ability in solution, providing insights into the applicability of Nbs in treating RAGE-associated diseases.

Nanobodies as powerful pulmonary targeted biotherapeutics against SARS-CoV-2, pharmaceutical point of view.

Background Since December 2019, the newly emerged SARS-CoV-2 virus continues to infect humans and many people died from severe Covid-19 during the last 2 years worldwide. Different approaches are being used for treatment of this infection and its consequences, but limited results have been achieved and new therapeutics are still needed. One of the most interesting biotherapeutics in this era are Nanobodies which have shown very promising results in recent researches. Scope of review Here, we have reviewed the potentials of Nanobodies in Covid-19 treatment. We have also discussed the properties of these biotherapeutics that make them very suitable for pulmonary drug delivery, which seems to be very important route of administration in this disease. Major conclusion Nanobodies with their special biological and biophysical characteristics and their resistance against harsh manufacturing condition, can be considered as promising, targeted biotherapeutics which can be administered by pulmonary delivery pharmaceutical systems against Covid-19. General significance Covid-19 has become a global problem during the last two years and with emerging mutant strains, prophylactic and therapeutic approaches are still highly needed. Nanobodies with their specific properties can be considered as valuable and promising candidates in Covid-19 therapy.

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Improving the yield of recalcitrant Nanobodies® by simple modifications to the standard protocol.

Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VHH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni2+ ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.

Generation and characterization of a laforin nanobody inhibitor.

OBJECTIVES: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor. DESIGN AND METHODS: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity. RESULTS: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity. CONCLUSIONS: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.

A comparative study and evaluation of anti-EGFR nanobodies expressed in Pichia pastoris and Escherichia coli as antitumor moieties.

Anti-EGFR nanobodies have been successfully applied as antitumor moieties in the photodynamic therapy and drug delivery systems. But the yields of nanobodies were still limited due to the volumetric capacity of the periplasmic compartments and inclusion bodies of Escherichia coli. A comparative study of Pichia pastoris and Escherichia coli was done through characterizing their products. Nanobody 7D12 and 7D12-9G8 were successfully expressed in Pichia pastoris with 6-13.6-fold higher yield. Both two types of nanobodies had internalization ability to be developed as antitumor moieties.

Development of a novel capillary electrophoresis-based approach for detection of anti-drug antibody responses.

Peggy Sue is a capillary-based western/immunoassay platform that can separate and characterize proteins by size or charge. A quick and automated immunogenicity assay was developed on Peggy Sue based on charge separation and compared with a traditional bridging method using preclinical samples from non-human primate studies. The results generated with the Peggy Sue assay were comparable to those of the bridging assays. The Peggy Sue platform has several advantages, including time efficiency, low sample consumption, and easy automation. The platform is especially ideal for further characterization of anti-drug antibody (ADA) specificity against complex biologics such as bispecific or multi-specific biotherapeutics as it is easy to conduct domain specificity assessment of observed ADA responses. Our evaluation suggests that the Peggy Sue platform is a promising tool for preclinical ADA analysis.

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer.

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects.

Secretory production of a camelid single-domain antibody (VHH, nanobody) by the Serratia marcescens Lip system in Escherichia coli.

Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.

Nanobodies mapped to cross-reactive and divergent epitopes on A(H7N9) influenza hemagglutinin using yeast display.

Influenza H7N9 virus continues to cause infections in humans and represents a significant pandemic risk. During the most recent 5th epidemic wave in 2016/17 two distinct lineages with increased human infections and wider geographical spread emerged. In preparation for any future adaptations, broadly reactive antibodies against H7N9 are required for surveillance, therapy and prophylaxis. In this study we have isolated a panel of nanobodies (Nbs) with broad reactivity across H7 influenza strains, including H7N9 strains between 2013 and 2017. We also describe Nbs capable of distinguishing between the most recent high and low pathogenicity Yangtze River Delta lineage H7N9 strains. Nanobodies were classified into 5 distinct groups based on their epitope footprint determined using yeast display and mutational scanning. The epitope footprint of Nbs capable of distinguishing high pathogenic (HP) A/Guangdong/17SF003/2016 from low pathogenic (LP) A/Hong Kong/125/2017 (H7N9) were correlated to natural sequence divergence in the head domain at lysine 164. Several Nbs binding to the head domain were capable of viral neutralisation. The potency of one nanobody NB7-14 could be increased over 1000-fold to 113 pM by linking two Nbs together. Nbs specific for distinct epitopes on H7N9 may be useful for surveillance or therapy in human or veterinary settings.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

A novel intracellular nanobody against HPV16 E6 oncoprotein.

Cervical cancer occurs as a result of the persistent infection of high-risk human papillomavirus (HPV). HPV16 oncoproteins E6 and E7 exert different and concerted pro-tumor actions in cell transformation and malignance maintenance in various m echanisms. Nanobody expressed as "intracellular antibodies" (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. In this work, phage-display approach was employed to select the high affinity HPV16 E6-specific nanobody, nanobody Nb9 against HPV16 E6 was selected. Nb9 has high affinity (Kaff =6.3 × 108 M-) and can specifically bind endogenous HPV16 E6 protein in HPV16 positive CaSki and SiHa cells. In Nb9 overexpressed SiHa and CaSki cells, nucleus localization of HPV16 E6 was inhibited, p53 inactivation was prevented and increased apoptosis was observed. Moreover, tumor growth was inhibited in mouse xenograft model. Taken together, our results suggested that nanobody Nb9 could be a useful inhibitor for HPV16 E6 function and particularly appropriate for the treatment of HPV-associated disease.

Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p.

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are ß-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Šresolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated 10 distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA.

The Zika virus was introduced in Brazil in 2015 and, shortly after, spread all over the Americas. Nowadays, it remains present in more than 80 countries and represents a major threat due to some singularities among other flaviviruses. Due to its easy transmission, high percentage of silent cases, the severity of its associated complications, and the lack of prophylactic methods and effective treatments, it is essential to develop reliable and rapid diagnostic tests for early containment of the infection. Nonstructural protein 1 (NS1), a glycoprotein involved in all flavivirus infections, is secreted since the beginning of the infection into the blood stream and has proven to be a valuable biomarker for the early diagnosis of other flaviviral infections. Here, we describe the development of a highly sensitive nanobody ELISA for the detection of the NS1 protein in serum samples. Nanobodies were selected from a library generated from a llama immunized with Zika NS1 (ZVNS1) by a two-step high-throughput screening geared to identify the most sensitive and specific nanobody pairs. The assay was performed with a sub-ng/mL detection limit in the sera and showed excellent reproducibility and accuracy when validated with serum samples spiked with 0.80, 1.60, or 3.10 ng/mL of ZVNS1. Furthermore, the specificity of the developed ELISA was demonstrated using a panel of flavivirus' NS1 proteins; this is of extreme relevance in countries endemic for more than one flavivirus. Considering that the nanobody sequences are provided, the assay can be reproduced in any laboratory at low cost, which may help to strengthen the diagnostic capacity of the disease even in low-resource countries.