Phage display was an efficient technique to produce selective antibodies for tumor-related antigens, viruses, bacteria and other targets. Since being made by McCafferty et al. over 30 years, phage antibody display has become a widely used method for Nb screening (Liu et al., 2021). The construction and validation of camelid-derived nanobody phage display libraries is a standard approach for isolating Nbs against various antigens. Phage display is a method of clonal protein display that inserts the coding DNA for a foreign peptide/protein into the coding gene of phage coat protein gene, thus the exogenous protein is expressed displayed on the phage surface when the phage particle is assembled, and the expressed displayed peptides/proteins can maintain original spatial configuration and biological activity, which can specifically recognize and bind target molecules (Salvador et al., 2019). This approach provides a quick screening process on the large library with much higher efficiency and costs less compared with the conventional hybridomas technique. It displays various scFv libraries on the surface of filamentous phage, selectively binding to the antigen. The in vitro evolution is achieved through multiple rounds of biopanning that to enrich the high affinity clones to the corresponding target antigen. Filamentous phage display, lytic phage display and T4 phage display systems are the commonly used phage display systems. Phage display has a broad range of applications, although there are also a number of constraints with regard to its practicality (Jin et al., 2023). Some of them are listed as follows:

Bacterial surface display is a technique that utilizes genetic engineering to present functional peptide or protein fragments (target proteins) on the cell surface by fusing them with bacterial outer membrane proteins (carrier proteins). This method relies on membrane-anchoring motifs, which facilitate the translocation of heterologous peptides across the cell membrane to the extracellular surface. Bacterial display offers several advantages, including high transformation efficiency, rapid growth rates, and genetic stability (Duan et al., 2021). In addition, this technique can be combined with flow cytometry for the screening of antibodies specific to target antigens. Fusion of the exogenous protein with the carrier protein can be achieved in three main formats: C-terminal fusion (with the N-terminus anchored in the outer membrane), N-terminal fusion (with the C-terminus anchored), and sandwich fusion (insertion of the foreign protein at a permissive internal site within a carrier protein that contains both a signal peptide and outer membrane anchor sequence). In traditional bacterial surface display systems, the exogenous protein is typically fused to bacterial outer membrane proteins, lipoproteins, or structural subunits of surface appendages (such as pili and flagella). More recently, alternative anchoring scaffolds such as ice nucleation proteins, autotransporters, and S-layer proteins have also been utilized. Early studies primarily focused on Gram-negative bacteria due to their well-characterized genetic backgrounds, which facilitate precise control over protein display. However, from an application standpoint, Gram-positive bacteria offer several advantages: they can tolerate the insertion of larger exogenous proteins, have only a single cytoplasmic membrane to traverse, and possess a thick but accessible cell wall, making them more amenable to surface display (Jovcevska and Muyldermans, 2020).

When combined with fluorescence-activated cell sorting (FACS) or flow cytometric analysis, bacterial surface display becomes a powerful high-throughput screening method. The main advantages of this approach include:

Compared to phage display, bacterial surface display offers unique advantages in vaccine development, enabling faster and more efficient screening. However, it also presents certain limitations, such as size constraints of displayed proteins, mislocalization, inclusion body formation, and outer membrane instability. Therefore, ongoing efforts are required to develop novel display systems and further optimize the technology for broader applications.

Yeast surface display is a eukaryotic protein expression system that has developed rapidly in recent years. The basic principle involves fusing a gene encoding a foreign target protein with the C-terminal coding sequence of a yeast-encoded agglutinin (which contains a GPI anchor signal sequence), and inserting the fusion gene downstream of a signal peptide in a plasmid vector. Upon induction, the fusion protein is directed for extracellular secretion via the signal peptide, and the C-terminal GPI-anchored agglutinin sequence facilitates anchoring of the protein to the yeast cell wall, thereby displaying the protein on the yeast cell surface. This system was developed following the success of phage display technologies. Yeast cells are relatively large, which makes them well-suited for screening and sorting by flow cytometry. Currently, the most commonly used yeast surface display systems are: (1) target protein–α-agglutinin fusions, and (2) α-agglutinin–target protein fusions. In the former, the target protein is fused to the N-terminus of the C-terminal portion of α-agglutinin, and the complex is displayed on the yeast cell surface. The agglutinin is covalently linked to the glucan network of the cell wall through its C-terminal 320 amino acid residues, which are rich in serine/threonine (Ser/Thr) residues. These Ser/Thr-rich regions are extensively O-glycosylated, forming a rod-like structure that acts as a spatial scaffold for display. To date, multiple heterologous proteins have been successfully displayed using the C-terminal domain of α-agglutinin, with the first reported example being α-galactosidase (Muyldermans, 2013).

Currently, yeast surface display has been widely applied in protein–protein interaction studies, directed evolution, and novel vaccine development. In terms of directed evolution, this system has been successfully used to evolve proteins for improved affinity and stability, with one of the earliest applications being antibody affinity maturation (McMahon et al., 2018). In vaccine development, yeast cells can serve as live oral vaccines. Proteins displayed on the yeast surface are readily accessible to antibodies and thus easily recognized by the immune system. Even small peptides can become immunogenic when displayed on the cell surface, making yeast a promising platform for expressing heterologous antigenic proteins for vaccine development. Yeast surface display offers several advantages. It mimics the natural process of antibody selection that occurs in the human immune system and enables the identification of polyclonal populations with different binding specificities in a single experiment (Kajiwara et al., 2020). Compared to phage and bacterial display systems, yeast enables post-translational modifications and correct folding of complex eukaryotic proteins. Moreover, glycosylation improves antibody solubility, and co-expression of chaperone proteins facilitates proper folding in the endoplasmic reticulum. Although the library capacity is relatively limited (~10⁷–10⁹ clones), the diversity is high, and unique high-affinity antibodies can be readily isolated with simple screening steps.

PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a significant swine pathogen. As of now, it is lack of broadly applicable anti-PRRSV strategies. Recently, Deng et al. identified several high affinity anti-CD163 Nbs. Of interest, Nb2 exhibited substantial inhibitory effects on diverse PRRSV lineages and suppressed virus-related NF-κB signaling by interfering with viral attachment and reducing CD163 transcription. In particular, the SRCR5 domain of CD163, a critical region implicated in PRRSV infection, was recognized by these Nbs, thereby facilitating the establishment of broad-spectrum approaches against PRRSV. Similarly, Yang et al. developed Nb-peptide conjugates (NPCs) by fusing PRRSV-specific, non-neutralizing Nbs with peptides derived from CD163 that recognize the receptor-binding domain (RBD) of PRRSV proteins. These NPCs showed potent inhibitory activities against diverse lineages of PRRSV (Yang H. et al., 2024). In addition, Zhou et al. screen a nanobody with the neutralizing activities for PRRSV. By integrating the adhesion protein and Nb, the probiotics enhanced its adhesion to IPEC-J2 cells and thus was utilized to prevent PRRSV fecal transmission (Zhou et al., 2024).

Notably, Nbs were also employed to develop enzyme-linked immunosorbent assay for PRRSV, indicating high sensitivity and accuracy (Duan et al., 2021; Sun et al., 2024). Importantly, the Nb could suppress PRRSV replication by inhibiting self-interaction of the viral nucleocapsid protein (Duan et al., 2024).

African swine fever virus (ASFV) is a virulent swine pathogen that causes huge losses to the swine industry. ASFV is large in size, thus vaccines of great efficacy are in urgent needed; however, Nbs targeting ASFV proteins are developed by many research groups of monitoring. Nbs of CD2v, p54, and p72 have been used to develop ELISA kit to monitor ASFV infection (Zhang et al., 2022; Zhao H. et al., 2022; Zhao J. et al., 2022; Zhu et al., 2024). Interestingly, Nbs are also helpful to identify epitopes of host immnuno-recognization. Zhao et al. screened one Nb via utilizing ASFV P54. The epitope recognized by this Nb also was reactive to the inactivated serum of ASFV naturally infected pigs (Zhao et al., 2023). Similarly, Wei et al. identified an epitope by utilizing ASFV K205R under the Nb verification (Wei et al., 2025). Notably, the structural proteins of ASFV, including p30, p54, and p72, were utilized to generate Nb-based TRIM-aways. These constructs exhibited significant potency in degrading viral structural proteins, thereby underscoring potential pathways for the development of novel antiviral strategies against ASF (Yang F. et al., 2024).

Highly pathogenic avian influenza viruses present significant threats to the poultry industry, public health, and have the potential to induce global pandemic risks. Nbs have extensive applications in the detection, differential diagnosis and prevention of avian influenza viruses. For example, Xu et al. developed a Nb of broadly neutralizing versatile clades of IAV H5 subtype by binding to HA1 (Xu et al., 2025). Chen et al. isolated a Nb with cross-group neutralization across various IAV subtypes. Critically, the epitope recognized by this Nb could elicits cross-reactive antibodies and provides partial protection by lethal viral challenges (Chen et al., 2025). In addition, Ji et al. established a Nb-based competitive ELISA (cELISA) in monitoring anti-IAV antibodies (Ji et al., 2022). Interestingly, a Nb-based reporter system was used for living cell sensing of IAV infection, providing a robust and advanced tool for the analysis of IAV infections.

Notably, besides interfering with viral infection, the Nb was also utilized as the cellular delivery vehicle. For instance, the FMDV particles were delivered by Nbs into dendritic cells to enhance host immune responses (Cheng et al., 2023).

Staphylococcus aureus (S. aureus) is capable of producing a variety of toxins, including staphylococcal enterotoxin type B and C (SEB/C), which play a critical pathogenic role in host infections and foodborne intoxications. As described above, Nbs were also employed to detect SEB/C (Graef et al., 2011; Ming et al., 2023) and clumping factor A (ClfA) (Mei et al., 2024). Nbs were also proved to neutralize SEB and thus hold promises for combating SEB-relative diseases. Detailed information demonstrated Nbs bound to SEB at the T-cell receptor interface, blocking the SEB toxicity (Zong et al., 2024). Zhang et al. develop a Nb (plus RNAbody)-based sandwich ELISA system, which could readily detect S. aureus α-hemolysin in milk and pork (Zhang et al., 2024).

Bacillus anthracis (B. anthracis) is a zoonotic spore-forming pathogen, can lead to anthrax, which is a highly resilient and deadly for animals and humans. It can secret toxins into the bloodstream once the infection occurred. Cecil et al. characterized a group of Nbs against the anthrax toxins via binding to the B. anthracis edema factor and lethal factor, which thus prevented entry of the toxin into the cells. Interestingly, Nbs with neutralizing function protected mice against the lethal anthrax spore infection, indicating a potent blocking role in in vivo tests (Vrentas et al., 2016).

Nbs inhibited the self-assembly of the S-layer proteins Sap of B. anthracis attenuated its growth and pathology in vivo. Subcutaneous delivery of these inhibitory Nbs cleared B. anthracis infection and prevented mice lethality, highlighting a therapeutic intervention of B. anthracis infection (Fioravanti et al., 2019). Recent work established a system of molecular dynamics simulations of these inhibitory Nbs, paving a road for Nb therapeutics (Cecil et al., 2024).

In addition, Nbs targeting the protective antigen of anthrax toxin were also developed. Nbs fused with the β-gal targeted the Bacillus collagen-like protein of anthracis (BclA) enabled sensitive detection of B. anthracis.Mycobacterium tuberculosis (M. tuberculosis), the etiological agent responsible for tuberculosis, exhibits high pathogenicity and results in severe morbidity and mortality.

Recently, Fay et al. developed a system by using the anti-ALFA Nb fused with a fluorescent protein to recapitulate the protein localization by fluorescent microscopy in living cells, serving as a versatile platform for the discovery of protein biology in mycobacteria (Fay et al., 2025). It was found that the Nb specific for ESAT-6 could inhibit M. tuberculosis growth in macrophages (Bates et al., 2024). Nb was also utilized to stabilize the M. tuberculosis to dissect the cryo-EM structure of its adenylyl cyclase transmembrane region (Mehta et al., 2022). Nbs were also applied to detection of Escherichia coli (E. coli) in food samples (He et al., 2025). They were also generated to bind to the extracellular fimbriae of enterotoxigenic E. coli (ETEC)/Shiga toxin producing E. coli (STEC) and thus blocked their adhesion in the gastrointestinal tract (Moonens et al., 2014).

It should be noted that E. coli and Lactobacillus could be utilized as delivery vectors to display the Nbs. Many researches applied these systems to produce and purify Nbs. For example, the E. coli could be utilized as a tool to screen Nbs capable of binding to and neutralizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and bovine viral diarrhea virus (BVDV) (Liu et al., 2024; Ma et al., 2024; Zhao et al., 2025).

Oral administration of Nbs was utilized to prevent GI E.coli infection. Lactococcus lactis was used to deliver Nbs to inhibit norovirus infection (Yuki et al., 2022).

In addition to their applications in viruses and bacteria research, Nbs were also used to inhibit parasite infection. For example, the Nbs could suppress the trypanosomatid pyruvate kinases activity by an allosteric mechanism (Pinto Torres et al., 2025). Trypanosoma brucei is a parasitic protozoan causing African trypanosomiasis, or sleeping sickness, transmitted by infected tsetse flies to animals and humans. Neutralization of Trypanosoma brucei Q586B2 Nbs hampered myeloid-derived IL-10 production and lowered parasitemia (Stijlemans et al., 2024). Genetically engineered Sodalis glossinidius expressing Nbs significantly compromised Trypanosoma brucei development in the tsetse fly midgut (De Vooght et al., 2022). Interestingly, Broster Reix et al. demonstrated Nbs also were able to suppress Trypanosoma brucei propagation. They screened one Nb targeting the T. brucei cytoskeletal protein Tb BILBO1, which could killed parasites producing phenotypes similar to the RNA knockdown, suggesting Nbs should be used to prevent T. brucei infection (Broster Reix et al., 2021). Notably, because Nbs do not contain the Fc fragments, they should hold less background interference in the ELISA assay. Another commonly causing agent of African trypanosomiasis is Trypanosoma congolense, which continues to impose a heav burden in livestock of Sub-Saharan Africa. Nbs were employed to establish the ELISA assay and improved Nb-based lateral flow assay to monitor Trypanosoma congolense infection, which showed increased sensitivity in detection of cattle samples (Odongo et al., 2016; Pinto Torres et al., 2018). Similarly, Abeijon et al. established an ELISA assay using Nbs specific for visceral leishmaniasis-an important zoonotic parasite, which was beneficial to increase the kit sensitivity (Abeijon et al., 2018).

Toxoplasma gondii (T. gondii) is a critical zoonotic pathogen which can infect a broad range of hosts, including cats, dogs and humans. Pathogenic mechanism study could help uncover the cellular processes of T. gondii within the hosts. By employing the actin Nbs, Periz et al. revealed how the dynamic F-actin networks connected Toxoplasma progeny and spread in the replicative vacuole (Periz et al., 2017), which should provide hints for novel strategies to decline intracellular T. gondii infection.