Enhancing Nanobody Immunoassays through Ferritin Fusion: Construction of a Salmonella-Specific Fenobody for Improved Avidity and Sensitivity.

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.

Enhanced sandwich immunoassay based on bivalent nanobody as an efficient immobilization approach for foodborne pathogens detection.

BACKGROUND: Nanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation. RESULTS: Here, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM KD, as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 103 CFU mL-1 for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %. SIGNIFICANCE AND NOVELTY: In this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens.

Enhancing Oriented Immobilization Efficiency: A One-for-Two Organism-Bispecific Nanobody Scaffold for Highly Sensitive Detection of Foodborne Pathogens.

Nanobodies have gained widespread application in immunoassays. However, their small size presents a significant challenge in achieving effective immobilization and optimal sensitivity. Here, we present a novel "one-for-two"-oriented immobilization platform based on an organism-bispecific nanobody (O-BsNb) scaffold, enabling highly sensitive detection of two bacterial pathogens. Through genetic engineering, a bispecific nanobody (BsNb) was engineered, targeting Salmonella spp. and Vibrio parahaemolyticus. The O-BsNb scaffold allowed one nanobody to bind specifically to inactivated bacteria, forming an organism-oriented immobilization platform, while the other served as the capture antibody. Consequently, the O-BsNb bioscaffold-based ELISA (O-ELISA) for individual detection of S. enteritidis and V. parahaemolyticus was established. When compared to the sandwich ELISA utilizing passive immobilization of monovalent nanobodies, the O-ELISA exhibited a remarkable 13.4- and 13.7-fold improvement in LOD for S. enteritidis and V. parahaemolyticus, respectively, highlighting the enhanced immobilization efficacy of the O-ELISA. Furthermore, the feasibility and reproducibility of the assay in practical samples were meticulously evaluated, revealing exemplary performance in terms of recovery precision and assay stability. These findings demonstrate the significant potential of the O-ELISA platform for the sensitive detection of macromolecules, opening new avenues for efficient pathogen identification in foodborne safety and clinical diagnostics.

Phage-Displayed Nanobody as a Sensitive Nanoprobe to Enhance Chemiluminescent Immunoassay for Cronobacter sakazakii Detection in Dairy Products.

The exploitation of stable, high-affinity, and low-cost nanoprobes is essential to develop immunoassays for real-time monitoring of foodborne pathogens, so as to safeguard human health. The possible interaction of the Fc fragment of antibodies with spA protein on Staphylococcus aureus will result in unexpected interference. To address this consideration, we described herein for the first time the development of nanobodies that by definition are devoid of the Fc fraction. These nanobodies directed against Cronobacter sakazakii (C. sakazakii) were retrieved from a dedicated immune phage-displayed nanobody library. The binders showed superiority of low cost, strong stability, high binding affinity, and adequate load capacity. Thereafter, a phage-mediated sandwich enzyme-linked immunosorbent assay (ELISA) was constructed by using Cs-Nb2 as an antigen-capturing antibody and phage-displayed Cs-Nb1 as a detection probe. To further enhance the sensitivity, a chemiluminescent enzyme immunoassay (CISA) was established by replacing the substrate from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol, providing a limit of detection of 1.04 × 104 CFU/mL, with a recovery of 98.15-114.63% for the detection of C. sakazakii in dairy products. The proposed nanobody-based phage-mediated sandwich CLISA shows various advantages, including high sensitivity, cost effectiveness, enhanced loading capacity of the enzyme, and high resistance to the matrix effect, providing a strategy for the design of immunoassays toward foodborne pathogens.

Nanobody-based immunomagnetic separation platform for rapid isolation and detection of Salmonella enteritidis in food samples.

Rapid separation and identification of Salmonella enteritidis (S. enteritidis) in food is of great importance to prevent outbreaks of foodborne diseases. Herein, by using O and H antigens as targets, an epitope-based bio-panning strategy was applied to isolate specific nanobodies towards S. enteritidis. This method constitutes an efficient way to obtain specific antibody fragments and test pairwise nanobodies. On this basis, a double nanobody-based sandwich enzyme-linked immunosorbent assay (ELISA) coupled with immunomagnetic separation (IMS) was developed to rapid enrich and detect S. enteritidis in food. The detection limit of the IMS-ELISA was 3.2 × 103 CFU/mL. In addition, 1 CFU of S. enteritidis in food samples can be detected after 4-h cultivation, which was shortened by 2 h after IMS. The IMS-ELISA strategy could avoid matrix interference and shorten the enrichment culture time, which has great potential for application in monitoring bacterial contamination in food.

"Potential Scalpel": A Bioassisted Ultrafast Staining Lateral Flow Immunoassay from De Novo to Results.

It is of great importance to overcome potential incompatibility problems between dyestuffs and antibodies (mAbs) for extensive commercial application of a dyestuff-chemistry-based ultrafast colorimetric lateral flow immunoassay (cLFIA). Herein, inspired by traditional staining technologies, a basic dyestuff gallocyanine (GC)-assisted biogenic "potential scalpel"-based cLFIA (GC-ABPS-based cLFIA) by employing clenbuterol (CLE) as proof-of-concept was proposed to solve a high degree of incompatibility between the same potential dyestuffs and mAbs. Goat antimouse immunoglobulin (Ab2) could serve as the "potential scalpel" to form the positive potential value biomolecular network self-assemblers (BNSA) with anti-CLE mAbs (AbCLE) by noncovalent force. The cLFIA completed the entire detection process from de novo to detection results within 30 min thanks to the easy availability and ideal marking efficiency (≤1 min, saving 0.4-10 h) of GC. Encouragingly, the proposed ultrafast GC-ABPS-based cLFIA has also exhibited high sensitivity (0.411 ng mL-1) and low cost (300 times) compared with other cLFIAs. Also, the feasibility of the proposed cLFIA was demonstrated by detecting CLE in beef, pork ham, and skim milk. Finally, the proposed GC-ABPS-based cLFIA has broadened the application range of dyestuffs and provided an effective reference strategy for the application of dyestuffs in food safety monitoring.

A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen.

Rapid and sensitive detection of bacterial pathogens present in food and environmental samples is of crucial importance to ensure human health and safety. Here, we present a one-step label-free colorimetric strategy based on M13 bacteriophage-displayed nanobody (phage-Nb) derived from camelid heavy-chain antibodies specific to Vibrio parahaemolyticus (V. parahaemolyticus). The thiolation of phage-Nb (Phage-Nb-SH) on pVIII shell proteins induces the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between nanobody and bacteria prevents the aggregation of AuNPs, resulting in visible color change due to alteration of surface plasmon resonance properties. Based on this phenomenon, a simple and sensitive colorimetric immunosensor for V. parahaemolyticus was developed. The assay can be accomplished within 100 min, and exhibits a visual detection limit of 104 cfu/mL and a quantitative detection limit of 103 cfu/mL, with no cross-reactivity towards other bacterial species. This strategy takes full advantages of both the high specificity of phage-Nbs and the optical properties of AuNPs, enabling simple and rapid detection of bacterial pathogens.