Development and Validation of a Monoclonal Antibody-Based Lateral Flow Immunochromatographic Assay Using Gold Nanoparticles for Rapid Detection of Vibrio vulnificus in Seafood
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Abstract
Vibrio vulnificus is a highly virulent marine bacterium associated with seafood-borne infections and represents a significant public health concern. Conventional detection methods are often labor-intensive and time-consuming, highlighting the need for a simple, rapid, and reliable detection approach. In this study, we developed a monoclonal antibody-based gold nanoparticle lateral flow immunochromatographic assay (LFIA) for the rapid visual detection of V. vulnificus. Two hybridoma-derived monoclonal antibodies, VV-213 and VV-301, were generated and characterized and exhibited different immunoreactivity patterns. These antibodies were integrated into a sandwich-format LFIA, wherein VV-213 was employed as the gold-conjugated detection antibody, while VV-301 was immobilized on the test line as the capture antibody. The developed assay demonstrated 100% specificity across 20 V. vulnificus isolates, with no observed cross-reactivity against other tested Vibrio species or non-Vibrio Gram-negative bacteria. When evaluated directly in application buffer, the analytical detection limit was determined to be 6.25 × 106 CFU/mL. The feasibility of the developed LFIA was evaluated in artificially contaminated oyster homogenates, and the assay achieved a detection limit of 10 CFU/mL following 6 h of pre-enrichment, which was comparable to PCR under similar conditions. Notably, the LFIA yielded visually interpretable results within 15 min, without requiring specialized instrumentation, thereby enhancing its field applicability. Furthermore, the assay remained functional after accelerated aging, indicating an estimated shelf life of at least 2 years under ambient conditions. Overall, these findings suggest that the developed LFIA represents a simple and practical tool for the rapid screening of V. vulnificus in seafood samples and may support routine seafood safety surveillance.
期刊介绍：
Food Analytical Methods publishes original articles, review articles, and notes on novel and/or state-of-the-art analytical methods or issues to be solved, as well as significant improvements or interesting applications to existing methods. These include analytical technology and methodology for food microbial contaminants, food chemistry and toxicology, food quality, food authenticity and food traceability. The journal covers fundamental and specific aspects of the development, optimization, and practical implementation in routine laboratories, and validation of food analytical methods for the monitoring of food safety and quality.
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