Anti-Idiotypic Nanobody Alkaline Phosphatase Fusion Protein-Triggered On-Off-On Fluorescence Immunosensor for Aflatoxin in Cereals.

Nanobodies (Nbs) are widely used in immunoassays with the advantages of small size and high stability. Here, the nanobody employed as the surrogate of aflatoxin antigen and the recognition mechanism of antiaflatoxin mAb with nanobody was studied by molecular modeling, which verified the feasibility of Nbs as antigen substitutes. On this basis, a nanobody-alkaline phosphatase fusion protein (Nb-AP) was constructed, and a highly sensitive "on-off-on" fluorescent immunosensor (OFO-FL immunosensor) based on the calcein/Ce3+ system was developed for aflatoxin quantification. Briefly, calcein serves as a signal transducer, and its fluorescence can be quenched after it is bound with Ce3+. In the presence of Nb-AP, AP catalyzed p-nitrophenyl phosphate to generate orthophosphate, which competes in binding with Ce3+, leading to fluorescence recovery. The method has a linearity range of 0.005-100 ng/mL, and the IC50 of the OFO-FL immunosensor was 0.063 ng/mL, which was 18-fold lower than that of conventional enzyme-linked immunosorbent assay. The assay was successfully applied in food samples with a recovery of 88-121%.

"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.

Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products.

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.