Antibodies | Systematic Review of Analytical Performance of Nanobodies in Bacterial and Toxin Detection

This study systematically evaluates the performance of nanobodies in detecting bacteria and toxins, revealing their advantages in high sensitivity, specificity, and stability, providing important reference for the development of rapid detection technologies.

 

Literature Overview

The article “Analytical Performance of Nanobody-Based Immunoassay and Immunosensing Platforms for Bacteria and Toxin Detection: A Systematic Review”, published in the journal “Antibodies”, reviews and summarizes the analytical performance of nanobody-based immunoassays and immunosensing platforms for detecting bacterial pathogens and toxins in food and clinical samples. The study includes 32 original experimental studies, systematically evaluating detection sensitivity, specificity, limit of quantification, practicality, and methodological quality. It finds that nanobody platforms exhibit significant advantages in sensitivity, stability, and engineering design, particularly achieving ultra-sensitive detection when combined with advanced signal amplification strategies. However, most studies rely on artificially spiked samples and lack large-scale validation with real-world samples, highlighting the need for enhanced standardization and practical application testing in the future. The paragraph is coherent and logical, ending with a Chinese period.

Background Knowledge

Bacterial pathogens and their toxins are major contributors to foodborne illnesses and antimicrobial resistance (AMR), causing approximately 600 million infections and millions of deaths globally each year. Although traditional detection methods such as culture, PCR, and conventional immunoassays are effective, they are time-consuming and technically complex, making them unsuitable for on-site rapid testing. Nanobodies, also known as single-domain antibodies (VHH), derived from the variable domain of heavy-chain-only antibodies in camelids, are ideal recognition elements due to their small molecular size, high stability, ease of genetic engineering, and prokaryotic expression. The absence of an Fc region minimizes non-specific binding, enhancing detection specificity. Additionally, their excellent thermal stability and resistance to denaturing environments make them suitable for complex sample analysis. In recent years, nanobodies have been widely used in constructing platforms such as ELISA, lateral flow immunoassays, electrochemical, and optical biosensors for detecting key pathogens including Salmonella, Escherichia coli O157:H7, Staphylococcal enterotoxins (SEs), and Clostridioides difficile toxins. Despite numerous reported applications, a systematic evaluation of their overall performance and practical applicability remains lacking. This study, based on the PRISMA guidelines, conducts a systematic review of existing literature to comprehensively assess the technical performance, advantages, and limitations of nanobody-based detection platforms, providing theoretical foundation and directional guidance for the development of future high-sensitivity, portable diagnostic tools.

 

 

Research Methods and Experiments

This study followed the PRISMA 2020 guidelines and was registered in PROSPERO (CRD420251088725). A systematic literature search was conducted in PubMed, Scopus, PMC, and ScienceDirect databases from inception to August 1, 2025. Inclusion criteria were original experimental studies using nanobodies (VHH) to detect bacteria or their toxins, providing quantitative performance metrics such as sensitivity, specificity, and limit of detection (LOD), and published in English or French. Studies related to viruses, fungi, parasites, or cancer were excluded. A total of 32 studies were included, from which information on target pathogens, detection platforms, nanobody sources, LOD, sensitivity, specificity, matrix recovery rates, and practicality was extracted. The adapted QUADAS-2 tool was used to assess risk of bias and applicability across four domains: patient selection, index test, reference standard, and flow and timing.

Key Conclusions and Perspectives

• Nanobody-based detection platforms demonstrate high sensitivity and specificity in bacterial and toxin detection, with some achieving LODs below 10³ CFU/mL or in the pg/mL range, significantly outperforming conventional methods
• Detection sensitivity can be further enhanced 10–100-fold through strategies such as phage-mediated signal amplification, chemiluminescence, and photothermal readout
• Nanobodies exhibit excellent thermal stability and engineering flexibility, enabling the construction of dimers, enzyme fusions, or nanoparticle probes to enhance detection signals and binding affinity
• Most studies use artificially spiked samples rather than naturally contaminated ones, potentially overestimating performance and limiting real-world applicability
• Over 30% of the studies did not employ a clear control method, affecting result comparability and bias assessment
• Applications of advanced sensing platforms such as electrochemical and multiplex detection remain limited; future work should focus on validating anti-interference capabilities and field applicability in complex matrices

Research Significance and Prospects

This study systematically reveals the immense potential of nanobodies in pathogen detection. Their high affinity, stability, and ease of modification make them core recognition elements for next-generation diagnostic reagents. Especially when combined with novel signal transduction mechanisms, they enable rapid, sensitive, and portable detection suitable for food safety, clinical diagnostics, and environmental monitoring.

However, current studies still face methodological limitations, such as insufficient sample representativeness, lack of standardized protocols, and large-scale validation. Future efforts should promote multi-center collaborative research, validate performance using real-world samples, and establish unified evaluation standards. Additionally, developing scalable, low-cost nanobody expression systems, combined with microfluidics and smartphone-based readouts, will facilitate true point-of-care testing (POCT), providing robust technological support for public health security.

 

 

Conclusion

This systematic review comprehensively evaluates the analytical performance of nanobody-based immunoassay and immunosensing platforms for bacterial and toxin detection. The findings indicate that due to their small molecular size, high stability, strong specificity, and ease of genetic engineering, nanobodies demonstrate superior sensitivity and practicality compared to conventional antibodies across various detection platforms, especially when integrated with signal amplification technologies enabling extremely low detection limits. The 32 included studies cover a range of technical approaches including ELISA, lateral flow assays, electrochemical, and optical sensing, targeting critical pathogens such as Salmonella, E. coli, Staphylococcal enterotoxins, and botulinum toxins, indicating broad application potential. However, most studies rely on artificially spiked samples without validation in real-world samples, and some lack control methods, affecting result reliability and generalizability. Future research should strengthen performance validation in real, complex matrices, promote the establishment of standardized protocols, and develop integrated, portable detection devices to enable the transition from laboratory to field applications, providing efficient tools for rapid monitoring of foodborne pathogens and public health interventions.

 

Aya Jalil, Nadia Touil, Omar Nyabi, Abdelaziz Benjouad, and Lamiae Belayachi. Analytical Performance of Nanobody-Based Immunoassay and Immunosensing Platforms for Bacteria and Toxin Detection: A Systematic Review. Antibodies.