Development of a nanobody-based immunoassay for the detection of Escherichia coli O157:H7 in food samples.

Rapid and accurate detection of foodborne pathogens is crucial for food safety. E. coli O157:H7, a major foodborne pathogen, can cause severe illness even at low concentration, highlighting the necessity for effective detection methods. This study focuses on developing nanobodies specific to E. coli O157:H7, aiming to provide a more efficient, cost-effective, and stable analytical reagents which can be available internationally. An alpaca was immunized and a phage-displayed nanobody library was constructed. Following panning, nanobodies were expressed and characterized for their affinity and specificity. A nanobody-based immunoassay was developed, with a limit of detection of 8.7 × 10 cfu/mL. The matrix effect was evaluated, and the assay successfully detected E. coli O157:H7 in various food samples (with 5 cfu spiked), including ground beef, orange juice, and milk. The study developed a nanobody-based immunoassay for E. coli O157:H7 detection, underscoring the practical applicability of nanobodies for testing food safety monitoring.