2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of ...2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4–8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment.
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•Reliable step-wise workflow for anti-hapten nanobody discovery was presented.•Highly specific and sensitive nanobody against 2,4-D was obtained by optimized isolation procedure.•A sensitive one-step FLEIA for 2,4-D was developed based on nanobody-AP fusion protein.•The assay showed to be useful and applicable for monitoring 2,4-D pollution in environmental water.
Aflatoxin B1 (AFB1), one of the most toxic mycotoxins, is classified as a group I carcinogen and ubiquitous in various foods and agriproducts. Thus, accurate and sensitive determination of AFB1 is of ...great significance to meet the criteria of food safety. Direct detection of AFB1 is difficult by monoclonal antibody (mAb) with large molecular size (≈150 kD) since the target is too small to produce a detectable signal change. Herein, by combining the electrochemical properties of nanomaterials and the advantages of nanobodies, we developed a direct, highly selective and sensitive electrochemical immunosensor for small molecule detection. The proposed immunosensor had a wide calibration range of 0.01 to 100 ng mL−1 and a low detection limit of 3.3 pg mL−1 (S/N=3). Compared with the immunosensor prepared with mAb which was applied in the typical indirect immunoassay, the immunosensor in this work possessed two orders of magnitudes wider linear range and 10‐fold more sensitivity. The as‐obtained immunosensor was further successfully applied for sensing AFB1 in real samples. This proposed assay would provide a simple, highly sensitive and selective approach for the direct immunoassay of small molecule AFB1, and is extendable to the development of direct immunosensing systems for other small molecules detection by coupling nanocarbon and nanobody.
Direct small molecules immunoassay: By combining the electrochemical properties of nanomaterials and the advantages of nanobodies, a direct non‐competitive electrochemical immunoassay was constructed for the direct detection of AFB1. Compared with the immunosensor using mAb, this proposed assay would provide more excellent detection performance for detecting AFB1, and is extendable to the development of direct immunosensing systems for other small molecules detection by coupling nanocarbon and nanobody.
•Autocatalytic and oriented coupling of nanobody ligand through Spy-based strategy.•This strategy significantly improves ligand coupling efficiency and binding capacity.•Nanobody ligand can be ...immobilized without any pre-purification steps.•The resin is effective and stable throughout multiple cycles.•This strategy can serve as a potential platform for developing affinity resin.
The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.
Significance
Structure determination by cryo-EM is difficult or impossible to apply to proteins smaller than ∼100 kDa, excluding many membrane proteins and proteins of pharmaceutical importance from ...the analysis. Here, we report on a general method that allows structure determination of small proteins. The method is based on the availability of a nanobody to a target protein. The nanobody is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. We call the overall ensemble Legobody. The method is demonstrated for two small proteins that have sizes of ∼22 kDa.
We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.
Nanobodies (Nb) have a promising future as a part of next generation chemodrug delivery systems. Nb, or VHH, are small (15 kDa) monomeric antibody fragments consisting of the antigen binding region ...of heavy chain antibodies. Heavy chain antibodies are naturally produced by camelids, however the structure of their VHH regions can be readily reproduced in industrial expression systems, such as bacteria or yeast. Due to their small size, high solubility, remarkable stability, manipulatable characteristics, excellent in vivo tissue penetration, conjugation advantages, and ease of production, Nb have many advantages when compared against their antibody precursors. In this review, we discuss the generation and selection of Nbs via phage display libraries for easy screening, and the conjugation techniques involved in creating target-specific nanocarriers. Furthermore, we provide a comprehensive overview of recent developments and perspectives in the field of Nb drug conjugates (NDCs) and Nb-based drug vehicles (NDv) with respect to antitumor therapeutics.
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Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only ...available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera.
In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R
= 0.914) was found between the percent competitive inhibition values of cELISA and HI titers.
A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.
Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main ...purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as β9 and β10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0ng/mL with LOD of 0.4ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA.
•A bioluminescent-based immunoassay was created to detect prostate-specific antigen.•The new immunoassay relies on two nanobodies and a tri-part split nanoluciferase.•Recombinant proteins were expressed, purified, and used for detecting PSA.•The new test provides a quick and user-friendly in vitro assay for PSA.
Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of ...conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions.We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.
Purpose
The aim of the present paper is to review the role of HER2 antibodies, affibodies and nanobodies as vehicles for imaging and therapy approaches in breast cancer, including a detailed look at ...recent clinical data from antibody drug conjugates and nanobodies as well as affibodies that are currently under development.
Results
Clinical and preclinical studies have shown that the use of monoclonal antibodies in molecular imaging is impaired by slow blood clearance, associated with slow and low tumor uptake and with limited tumor penetration potential. Antibody fragments, such as nanobodies, on the other hand, can be radiolabelled with short-lived radioisotopes and provide high-contrast images within a few hours after injection, allowing early diagnosis and reduced radiation exposure of patients. Even in therapy, the small radioactively labeled nanobodies prove to be superior to radioactively labeled monoclonal antibodies due to their higher specificity and their ability to penetrate the tumor.
Conclusion
While monoclonal antibodies are well established drug delivery vehicles, the current literature on molecular imaging supports the notion that antibody fragments, such as affibodies or nanobodies, might be superior in this approach.
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