Nanobodies: a promising approach to treatment of viral diseases

Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry. (AU)

Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models.

The weakened protective efficacy of COVID-19 vaccines and antibodies caused by SARS-CoV-2 variants presents a global health emergency, which underscores the urgent need for universal therapeutic antibody intervention for clinical patients. Here, we screened three alpacas-derived nanobodies (Nbs) with neutralizing activity from twenty RBD-specific Nbs. The three Nbs were fused with the Fc domain of human IgG, namely aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc, which could specifically bind RBD protein and competitively inhibit the binding of ACE2 receptor to RBD. They effectively neutralized SARS-CoV-2 pseudoviruses D614G, Alpha, Beta, Gamma, Delta, and Omicron sub-lineages BA.1, BA.2, BA.4, and BA.5 and authentic SARS-CoV-2 prototype, Delta, and Omicron BA.1, BA.2 strains. In mice-adapted COVID-19 severe model, intranasal administration of aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc effectively protected mice from lethal challenges and reduced viral loads in both the upper and lower respiratory tracts. In the COVID-19 mild model, aVHH-13-Fc, which represents the optimal neutralizing activity among the above three Nbs, effectively protected hamsters from the challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and BA.2 by significantly reducing viral replication and pathological alterations in the lungs. In structural modeling of aVHH-13 and RBD, aVHH-13 binds to the receptor-binding motif region of RBD and interacts with some highly conserved epitopes. Taken together, our study illustrated that alpaca-derived Nbs offered a therapeutic countermeasure against SARS-CoV-2, including those Delta and Omicron variants which have evolved into global pandemic strains.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy.

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.

Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction.

Protein-protein interactions are fundamental to life processes. Complementary computational, structural, and biophysical studies of these interactions enable the forces behind their specificity and strength to be understood. Antibody fragments such as single-chain antibodies have the specificity and affinity of full antibodies but a fraction of their size, expediting whole molecule studies and distal effects without exceeding the computational capacity of modeling systems. We previously reported the crystal structure of a high-affinity nanobody 59H10 bound to HIV-1 capsid protein p24 and deduced key interactions using all-atom molecular dynamics simulations. We studied the properties of closely related medium (37E7) and low (48G11) affinity nanobodies, to understand how changes of three (37E7) or one (48G11) amino acids impacted these interactions; however, the contributions of enthalpy and entropy were not quantified. Here, we report the use of qualitative and quantitative experimental and in silico approaches to separate the contributions of enthalpy and entropy. We used complementary circular dichroism spectroscopy and molecular dynamics simulations to qualitatively delineate changes between nanobodies in isolation and complexed with p24. Using quantitative techniques such as isothermal titration calorimetry alongside WaterMap and Free Energy Perturbation protocols, we found the difference between high (59H10) and medium (37E7) affinity nanobodies on binding to HIV-1 p24 is entropically driven, accounted for by the release of unstable waters from the hydrophobic surface of 59H10. Our results provide an exemplar of the utility of parallel in vitro and in silico studies and highlight that differences in entropic interactions between amino acids and water molecules are sufficient to drive orders of magnitude differences in affinity.

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering.

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

Isolation of nanobodies with potential to reduce patients' IgE binding to Bet v 1.

BACKGROUND: Recent studies showed that a single injection of human monoclonal allergen-specific IgG antibodies significantly reduced allergic symptoms in birch pollen-allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen-specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE-mediated responses. METHODS: A cDNA-VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope-specificity and affinity to Bet v 1. Furthermore, cross-reactivity to Bet v 1-homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen-induced basophil activation were investigated. RESULTS: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross-react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha-helix at the C-terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1-induced basophil activation. CONCLUSION: We identified high-affinity Bet v 1-specific nanobodies that recognize an important IgE epitope and reduce allergen-induced basophil activation revealing the first proof that allergen-specific nanobodies are useful tools for future treatment of pollen allergy.

Targeting small GTPases and their downstream pathways with intracellular macromolecule binders to define alternative therapeutic strategies in cancer.

The RAS superfamily of small GTPases regulates major physiological cellular processes. Mutation or deregulation of these small GTPases, their regulators and/or their effectors are associated with many diseases including cancer. Hence, targeting these classes of proteins is an important therapeutic strategy in cancer. This has been recently achieved with the approval of the first KRASG12C covalent inhibitors for the clinic. However, many other mutants and small GTPases are still considered as 'undruggable' with small molecule inhibitors because of a lack of well-defined pocket(s) at their surface. Therefore, alternative therapeutic strategies have been developed to target these proteins. In this review, we discuss the use of intracellular antibodies and derivatives - reagents that bind their antigen inside the cells - for the discovery of novel inhibitory mechanisms, targetable features and therapeutic strategies to inhibit small GTPases and their downstream pathways. These reagents are also versatile tools used to better understand the biological mechanisms regulated by small GTPases and to accelerate the drug discovery process.

A Methodological Approach Using rAAV Vectors Encoding Nanobody-Based Biologics to Evaluate ARTC2.2 and P2X7 In Vivo.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Anti-HER2 VHH Targeted Fluorescent Liposome as Bimodal Nanoparticle for Drug Delivery and Optical Imaging.

OBJECTIVES: The aim of this study was to formulate fluorescent-labeled targeted immunoliposome to visualize the delivery and distribution of drugs in real-time. METHODS: In this study, fluorescent-labeled liposomes were decorated with anti-HER2 VHH or Herceptin to improve the monitoring of intracellular drug delivery and tumor cell tracking with minimal side effects. The conjugation efficiency of antibodies was analyzed by SDS-PAGE silver staining. In addition, the physicochemical characterization of liposomes was performed using DLS and TEM. Finally, confocal microscopy visualized nanoparticles in the target cells. RESULTS: Quantitative and qualitative methods characterized the intracellular uptake of 110±10 nm particles with near 70% conjugation efficiency. In addition, live-cell trafficking during hours of incubation was monitored by wide-field microscopy imaging. The results show that the fluorescent- labeled nanoparticles can specifically bind to HER2-positive breast cancer with minimal off-target delivery. CONCLUSION: These nanoparticles can have several applications in personalized medicine, especially drug delivery and real-time visualization of cancer therapy. Moreover, this method also can be applied in the targeted delivery of contrast agents in imaging and thermotherapy.

Analysis of the molecular mechanism of SARS-CoV-2 antibodies.

A newly-emergent beta-coronavirus, SARS-CoV-2, rapidly has become a pandemic since 2020. It is a serious respiratory disease and caused more than 100 million of deaths in the world. WHO named it COVIA-19 and there is no effective targeted drug for it. The main treatment strategies include chemical medicine, traditional Chinese medicine (TCM) and biologics. Due to SARS-CoV-2 uses the spike proteins (S proteins) on its envelope to infect human cells, monoclonal antibodies that neutralize the S protein have become one of the hot research areas in the current research and treatment of SARS-CoV-2. In this study, we reviewed the antibodies that have been reported to have neutralizing activity against the SARS-CoV-2 infection. According to their different binding epitope regions in RBD or NTD, they are classified, and the mechanism of the representative antibodies in each category is discussed in depth, which provides potential foundation for future antibody and vaccine therapy and the development of antibody cocktails against SARS-CoV-2 mutants.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

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.

A Dual Target-Directed Single Domain-Based Fusion Protein Against Interleukin-6 Receptor Decelerate Experimental Arthritis Progression Via Modulating JNK Expression.

The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signalling that cause collateral damage to protective signalling cascades. The single domain chain firstly discovered in Camelidae displays fully functional ability in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. In this study, we established a simple prokaryotic expression system for a dual target-directed single domain-based fusion protein against the interleukin-6 receptor and human serum, albumin, the recombinant anti-IL-6R fusion protein (VHH-0031). VHH-0031 exhibited potent anti-inflammatory effects produced by LPS on cell RAW264.7, where the major cytokines and NO production were downregulated after 24 h incubation with VHH-0031 in a dose-dependent manner. In vivo, VHH-0031 presented significant effects on the degree reduction of joint swelling in the adjuvant-induced arthritis (AIA) rat, having a healthier appearance compared with the dexamethasone. The expression level of JNK protein in the VHH-0031 group was significantly decreased, demonstrating that VHH-0031 provides a low-cost and desirable effect in the treatment of more widely patients.

Potential approaches to combat COVID-19: a mini-review.

The outbreak of a novel coronavirus namely SARS-CoV-2, which first emerged from Wuhan, China, has wreaked havoc not only in China but the whole world that now has been engulfed in its wrath. In a short lapse of time, this virus was successful in spreading at a blistering pace throughout the globe, hence raising the flag of pandemic status. The mounting number of deaths with each elapsing day has summoned researchers from all around the world to play their part in driving this SARS-CoV-2 pandemic to an end. As of now, multiple research teams are immersed in either scrutinizing various antiviral drugs for their efficacy or developing different types of vaccines that will be capable of providing long-term immunity against this deadly virus. The mini-review sheds light on the possible approaches that can be undertaken to curb the COVID-19 spread. Possible strategies comprise viral vector-based, nucleic acid-based, protein-based, inactivated and weakened virus vaccines; COVID-19 vaccine being developed by deploying Hyleukin-7 technology; plant-based chimeric protein and subunit vaccines; humanized nano-bodies and human antibodies; intravenous immunoglobulin (IVIG) infusion therapy; inhibitors for ACE-2, Angiotensin 1 receptor (AT1R), complement system, viral proteins, host cell protease and endocytosis; shield immunity; IL-6R, NKG2A and hACE2-SARS-CoV-2-RBD interaction blocking monoclonal antibodies; SARS-CoV RdRp-based drugs, traditional Chinese medicine, repositioned and anti-viral drugs. These vaccines and drugs are currently being screened in the clinical trials as several of them have manifested positive results, hence increasing the probability of becoming one of the potential treatments for this disease.

Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom.

Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.

Simultaneous Unlocking Optoelectronic and Interfacial Properties of C60 for Ultrasensitive Immunosensing by Coupling to Metal-Organic Framework.

Due to exceptional electron-accepting ability, light-absorption, and a delocalized conjugated structure, buckminsterfullerene (C60) has attracted fascinating interest in the field of organic solar cells. However, poor delocalization and accumulation of electrons for pristine C60 in physiological aqueous solution and difficulties in conjugation with biomolecules limit its extended photovoltaic applications in bioassay. Herein, we reported the noncovalent coupling of C60 to an electronically complementary porphyrin-derived metal-organic framework (PCN-224) with carboxyl-group terminals. Such assembly not only offered a friendly interface for bioconjugation but also resulted in a long-range ordering C60@PCN-224 donor-acceptor system that demonstrated an unprecedented photocurrent enhancement up to 10 times with respect to each component. As an example, by further cooperating with Nanobodies, the as-prepared C60@PCN-224 was applied to a photoelectrochemical (PEC) immunosensor for S100 calcium-binding protein B with by far the most promising detection activities. This work may open a new venue to unlock the great potential of C60 in PEC biosensing with excellent performances.

Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach.

The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane ß-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last ß-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.

A novel nanobody and mimotope based immunoassay for rapid analysis of aflatoxin B1.

Mimotopes could replace mycotoxins and their conjugates to develop immunoassay methods. The mimotopes obtained by phage display technology were mainly using monoclonal antibodies or polyclonal antibodies as targets. However, the mimotope of recombinant antibody has not been selected and applied to immunoassay for mycotoxin. The purpose of this study was to prove that an immunoassay for mycotoxin could be developed based on both recombinant antibody and its mimotope. Using aflatoxin B1 (AFB1) as a model system, mimotopes of an aflatoxin nanobody Nb28 were screened by phage display. A rapid magnetic beads-based directed competitive ELISA (MB-dcELISA) was developed utilizing Nb28 and its mimotope ME17. The 50% inhibitory concentration and the detection limit of the MB-dcELISA were 0.75 and 0.13 ng/mL, respectively, with a linear range of 0.24-2.21 ng/mL. Further validation study indicated good recovery (84.2-116.2%) with low coefficient of variable (2.2%-15.9%) in spiked corn, rice, peanut, feedstuff, corn germ oil and peanut oil samples. The developed immunoassay based on nanobody and mimotope provides a new strategy for the monitoring of AFB1 and other toxic small molecular weight compounds.