Recombinant VHHs Research Articles

COVID-19 impedimetric biosensor based on polypyrrole nanotubes, nickel hydroxide and VHH antibody fragment: specific, sensitive, and rapid viral detection in saliva samples

SARS-CoV-2 rapid spread required urgent, accurate, and prompt diagnosis to control the virus dissemination and pandemic management. Several sensors were developed using different biorecognition elements to obtain high specificity and sensitivity. However, the task to achieve these parameters in combination with fast detection, simplicity, and portability to identify the biorecognition element even in low concentration remains a challenge. Therefore, we developed an electrochemical biosensor based on polypyrrole nanotubes coupled via Ni(OH)2 ligation to an engineered antigen-binding fragment of heavy chain-only antibodies (VHH) termed Sb#15. Herein we report Sb#15-His6 expression, purification, and characterization of its interaction with the receptor-binding domain (RBD) of SARS-CoV-2 in addition to the construction and validation of a biosensor. The recombinant Sb#15 is correctly folded and interacts with the RBD with a dissociation constant (KD) of 27.1 ± 6.4 nmol/L. The biosensing platform was developed using polypyrrole nanotubes and Ni(OH)2, which can properly orientate the immobilization of Sb#15-His6 at the electrode surface through His-tag interaction for the sensitive SARS-CoV-2 antigen detection. The quantification limit was determined as 0.01 pg/mL using recombinant RBD, which was expressively lower than commercial monoclonal antibodies. In pre-characterized saliva, both Omicron and Delta SARS-CoV-2 were accurately detected only in positive samples, meeting all the requirements recommended by the World Health Organization for in vitro diagnostics. A low sample volume of saliva is needed to perform the detection, providing results within 15 min without further sample preparations. In summary, a new perspective allying recombinant VHHs with biosensor development and real sample detection was explored, addressing the need for accurate, rapid, and sensitive biosensors.

High Expression Achievement of Active and Robust Anti-β2 microglobulin Nanobodies via E.coli Hosts Selection.

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-β2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-β2 microglobulin (β2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10−8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-β2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (VHH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.

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Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen–VHH complexes. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins.

Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk

Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 104 colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

BackgroundRecombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.Methodology/Principal FindingsAntibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10−10 M.Conclusions/SignificanceA broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beads

Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen–VHH complexes. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins.

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein.

Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=30.04, b=37.15, c=83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Head‐to‐tail fusions of camelid antibodies can be expressed in planta and bind in rumen fluid

We have compared the accumulation of recombinant variable heavy-chain portions [VHH (variable heavy-chain antibody from camelids)] of camelid antibodies in a variety of subcellular compartments produced in planta. The VHH coding sequences were optimized for expression in thale cress (Arabidopsis thaliana) and placed individually or as fused tandem heterodimers in synthetic plant-organelle-targeting cassettes designed to target the protein to either the cytoplasm, ER (endoplasmic reticulum), protein storage vacuole or chloroplast. Accumulation of individual VHHs was only detected in plants transformed with the ER-targeting cassette, whereas accumulation of the tandem VHHs was detected for all cassettes and was the highest with the ER cassette [0.1-0.7% (w/w) of total soluble proteins]. The ability of the plant-produced tandem VHH to reduce TNFalpha (tumour necrosis factor alpha) cytotoxicity was found to be comparable with previously characterized recombinant VHHs. In vitro antigen binding and functional stability in rumen fluid were determined on both prokaryotically expressed and plant-expressed tandem VHHs. The plant-produced VHH did not appear to be any more stable in rumen fluid than other soluble plant proteins; however, it was able to bind equally well to the antigen in the presence or absence of rumen fluid.

Stable recombinant alpaca antibodies for detection of Tulip virus X

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at −20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C.