Direct competitive time-resolved fluorescence immunoassay for tetrodotoxin detection

Tetrodotoxin (TTX) is one of the most potent lethal non-protein neurotoxin. Outbreak of TTX food poisoning has been reported in various countries. It is important to develop the rapid method for detecting TTX in the food. In this study, the monoclonal antibody (McAb) against TTX was produced, and Eu³⁺-McAb was synthesized and purified by Sephadex G-25. The synthesized antigen was TTX linked to KLH or OVA by Mannich method. The time-resolved fluorescence immunoassay (TRFIA) was developed and optimized for TTX detection. The detection conditions of the dcTRFIA were confirmed: 4.0 μg/mL coating antigen, 5.0 μg/mL Eu³⁺-McAb, 45 min competitive time, at room temperature ( 20-25 °C ). The IC₅₀ value of the standard curve was 19.2 ng/mL. The detection limit was 1.2 ng/mL and the working ranges were 3.0-123.3 ng/mL. The one step dcTRFIA is sensitive and time-saving, hope this study will benefit the development of the TRFIA kit for detecting TTX.