Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels

Tetrodotoxin (TTX) is a potent marine neurotoxin responsible for severe seafood poisoning in humans, characterized by neurological symptoms that may be fatal. Originally identified as a natural contaminant of pufferfish (Tetraodontidae family), over the last few years TTX and its analogs have also been detected in other edible marine organisms, including mollusks, gastropods and crustaceans. Consequently, there is a need for rapid, sensitive, and reliable methods for TTX detection in seafood. In this study, a functional assay based on the use of mouse neuroblastoma Neuro-2a cells has been optimized and characterized for TTX detection in mussels. The assay is based on the toxin's ability to block voltage-gated sodium channels, thereby counteracting the sodium-dependent cytotoxicity induced by veratridine and ouabain. The linear range of the TTX standard curve fell between 0.44 and 33 ng/mL, with limits of TTX detection (LOD) and quantitation (LOQ) of 0.132 ng/mL and 0.439 ng/mL, respectively, and good intra- and inter-day repeatability (RSDr= 15 and 11%, respectively). The assay also detected saxitoxin, which shares the same mechanism of action as TTX, but was less sensitive towards 4,9-anhydro-TTX. The minimum mussel extract dilution of 1:100 did not result in matrix-related interference, allowing accurate TTX quantitation, with a LOQ of 0.54 µg TTX equivalents/kg mussel meat. Given its sensitivity, the optimized Neuro-2a assay represents a promising tool for toxicity-based TTX quantitation in mussels before their consumption.