Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents. Methods The naive phage nanobody library was constructed and its diversity was analyzed. Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target, and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing. Results The capacity of the constructed naive phage nanobody library was 3.89×10⁹ CFU/mL, and the insertion of effective fragments was higher than 85%. Based on this library, seven anti-human CD22 nanobodies were screened, and the amino acid sequence comparison results showed that the overall similarity was 70.34%, and all of them were hydrophilic proteins. The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22, and the main forces were hydrophobic interaction and hydrogen bonding. Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22, successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.
Camelus/immunology* ; Single-Domain Antibodies/chemistry* ; Peptide Library ; Humans ; Animals ; Sialic Acid Binding Ig-like Lectin 2/genetics* ; Amino Acid Sequence ; Molecular Docking Simulation

Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain (RBD) of spike (S) protein to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus from attaching to angiotensin converting enzyme 2 (ACE2). This study reports a distinctive nanobody, designated as VHH21, that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes. Derived from camels immunized with multiple antigens, a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2, including SARS-CoV and BatRaTG13. Importantly, a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits. A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer. Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein, distinct from the ACE2 binding site on the RBD, which destabilizes the fusion process. This research highlights the potential of VHH21 as an abzyme-like nanobody (nanoabzyme) possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.
Single-Domain Antibodies/immunology* ; Spike Glycoprotein, Coronavirus/metabolism* ; SARS-CoV-2/immunology* ; Animals ; Humans ; Antibodies, Neutralizing/immunology* ; Camelus ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Angiotensin-Converting Enzyme 2

This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
Single-Domain Antibodies/biosynthesis* ; Peptide Library ; Complementarity Determining Regions/immunology* ; Animals

Hemoglobin, the principal protein in red blood cells, is crucial for oxygen transport in the bloodstream. The quantification of hemoglobin concentration is indispensable in medical diagnostics and health management, which encompass the diagnosis of anemia and the screening of various blood disorders. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. In this study, we immunized a Bactrian camel (Camelus bactrianus) with human hemoglobin and subsequently constructed a nanobody library. Utilizing a solid-phase screening method, we selected nanobodies and evaluated the binding activity of the screened nanobodies to hemoglobin. Initially, human hemoglobin was used to immunize a Bactrian camel. Following four immunization sessions, blood was withdrawn from the jugular vein, and a nanobody library with a capacity of 2.85×10⁸ colony forming units (CFU) was generated. Subsequently, ten hemoglobin-specific nanobody sequences were identified through three rounds of adsorption-elution-enrichment assays, and these nanobodies were subjected to eukaryotic expression. Finally, enzyme-linked immunosorbent assay and biolayer interferometry were employed to evaluate the stability, binding activity, and specificity of these nanobodies. The results demonstrated that the nanobodies maintained robust binding activity within the temperature range of 20-40 ℃ and exhibited the highest binding activity at pH 7.0. Furthermore, the nanobodies were capable of tolerating a 10% methanol solution. Notably, among the nanobodies tested, VHH-12 displayed the highest binding activity to hemoglobin, with a half maximal effective concentration (EC₅₀) of 10.63 nmol/L and a equilibrium dissociation constant (K_D) of 2.94×10^-7 mol/L. VHH-12 exhibited no cross-reactivity with a panel of eight proteins, such as ovalbumin and bovine serum albumin, while demonstrating partial cross-reactivity with hemoglobin derived from porcine, goat, rabbit, and bovine sources. In this study, a hemoglobin-specific high-affinity nanobody was successfully isolated, demonstrating potential applications in disease diagnosis and health monitoring.
Animals ; Camelus/immunology* ; Single-Domain Antibodies/immunology* ; Hemoglobins/immunology* ; Humans ; Peptide Library

Nanobodies (Nbs), the unique single-domain antibodies discovered in the species of Camelidae and sharks, are also known as the variable domain of the heavy chain of heavy-chain antibody (VHH). They offer strong antigen targeting and binding capabilities and overcome the drawbacks such as large size, low stability, high immunogenicity, and slow clearance of conventional antibodies. Nbs can be boosted by bioconjugation with toxins, enzymes, radioactive nucleotides, fluorophores, and other functional groups, demonstrating potential applications in the diagnosis and treatment of human and animal diseases. This article introduces the structures and characteristics of Nbs, the construction and screening of Nb libraries, and the strategies for affinity maturation and then reviews the current applications of Nbs in diagnosis and treatment, providing a reference for the development of diagnostic reagents and clinical therapies for infectious diseases.
Single-Domain Antibodies/chemistry* ; Animals ; Humans ; Camelidae/immunology*

Coronavirus disease (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with strong contagiousness, high susceptibility and long incubation period. cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain of the viral spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). Here, we briefly reviewed the mechanisms underlying the interaction between SARS-CoV-2 and ACE2, and summarized the latest research progress on SARS-CoV-2 neutralizing monoclonal antibodies and nanobodies, so as to better understand the development process and drug research direction of COVID-19. This review may facilitate understanding the development of neutralizing antibody drugs for emerging infectious diseases, especially for COVID-19.
Angiotensin-Converting Enzyme 2 ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19 ; Humans ; Peptidyl-Dipeptidase A/metabolism* ; Protein Binding ; SARS-CoV-2 ; Single-Domain Antibodies ; Spike Glycoprotein, Coronavirus/metabolism*

The aim of this study was to construct a recombinant adenovirus expressing extracellular domain gene of human epidermal growth factor receptor variant Ⅲ (EGFRvIII ECD), and to prepare single domain antibody targeting EGFRvIII ECD by immunizing camels and constructing phage display antibody library. Total RNA was extracted from human prostate cancer cell line PC-3 cells and reversely transcribed into cDNA. EGFRvIII ECD gene was amplified using cDNA as template, and ligated into pAdTrack-CMV plasmid vector and transformed into E. coli BJ5183 competent cells containing pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus expressing EGFRvIII ECD was obtained through transfecting the plasmid into HEK293A cells. The recombinant adenovirus was used to immunize Bactrian camel to construct EGFRvIII ECD specific single domain antibody library. The single domain antibody was obtained by screening the library with EGFRvIII protein and the antibody was expressed, purified and identified. The results showed that recombinant adenovirus expressing EGFRvIII ECD was obtained. The capacity of EGFRvIII specific phage single domain antibody library was 1.4×10⁹. After three rounds of enrichment and screening, thirty-one positive clones binding to EGFRvIII ECD were obtained by phage-ELISA, and the recombinant single domain antibody E14 with highest OD₄₅₀ value was expressed and purified. The recombinant E14 antibody can react with EGFRvIII ECD with high affinity in ELISA assessment. The results indicated that the EGFRvIII specific single domain antibody library with high capacity and diversity was constructed and the single domain antibody with binding activity to EGFRvIII was obtained by screening the library. This study may facilitate the diagnosis and treatment of EGFRvIII targeted malignant tumors in the future.
Adenoviridae/genetics* ; DNA, Complementary ; ErbB Receptors ; Escherichia coli/genetics* ; Genetic Vectors/genetics* ; Humans ; RNA ; Recombinant Proteins/metabolism* ; Single-Domain Antibodies

As a malleable and novel tool for antigen recognition and modulation, nanobodies have the advantages of small size, easiness of expression, screening and modification, as well as high affinity and stability. Nanobodies are capable of recognizing more cryptic antigenic epitopes that are difficult to be recognized by traditional antibodies, making them increasingly used in the diagnosis and treatment of various diseases and assays. Nanobodies are also playing an irreplaceable role in the basic research. This review summarized the recent development of nanobodies and their derivatives in the detection of small molecules, pathogenic microorganisms and diagnosis of diseases, as well as in the fields of targeted therapies, cellular and molecular imaging. Broad prospects of nanobodies in the field of protein conformation studies were also reviewed.
Single-Domain Antibodies

The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3'-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.
Animals ; Antibodies, Viral ; Coronavirus Infections/veterinary* ; Enzyme-Linked Immunosorbent Assay ; Porcine epidemic diarrhea virus/genetics* ; Reproducibility of Results ; Sensitivity and Specificity ; Single-Domain Antibodies ; Swine ; Swine Diseases

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.
Humans ; Immunoglobulin Heavy Chains ; chemistry ; Single-Domain Antibodies ; chemistry ; drug effects