P12-21 Optimization and characterization of the Neuro-2a assay for the detection of the marine toxin tetrodotoxin

Tetrodotoxin (TTX) is a potent marine neurotoxin involved in human foodborne poisonings, characterized by severe neurological symptoms sometimes leading even to death. Originally identified as a natural contaminant of the Tetraodontidae pufferfish, TTX and its analogs have been detected also in other edible marine organisms, including bivalve mollusks from Pacific area and, in recent years, also in the Mediterranean Sea. The presence of these toxins in edible marine organisms in European countries has raised concerns about food safety due to the lack of EU regulations fixing a maximum TTX level in seafood and of adequate monitoring programs. For these purposes, rapid and sensitive methods for detecting these toxins are also urgently needed. Therefore, this study was aimed at optimizing the functional Neuro-2a assay for the detection of TTX in mollusks, an in vitro assay based on the toxin's mechanism of action (blockade of voltage-gated sodium channels, VGSC) and the use of the mouse neuroblastoma Neuro-2a cells. The assay allows TTX detection exploiting the toxin ability to inhibit the Na + -dependent cytotoxicity induced by cells co-exposure to veratridine, a VGSC activator, and ouabain, a Na + /K + ATPase inhibitor. Each step of the assay procedure was optimized to improve TTX detection and quantitation. The optimized assay was characterized according to Eurachem guidelines: the linearity range of the calibration curve (0.41-33 ng/mL), the limit of detection (LOD=0.132 ng/mL) and the limit of quantitation (LOQ=0.447 ng/mL) were defined with good intra- and inter-day (along a period of 6 months) assay repeatability. Assay cross-reactivity was evaluated considering marine toxins sharing the same mode-of-action (saxitoxin, 4,9-anhydrous-TTX) and other toxins possibly co-occurring in edible mollusks (palytoxin, azaspiracid-1, okadaic acid, dinophysiotoxin-1 and dinophysiotoxins-2). Lastly, the assay's suitability to quantify TTX in mussels was assessed evaluating matrix effect: different extracts from toxin-free mussels and extracts spiked with known amounts of TTX (0.41-33.3 ng/mL) were analyzed after 1:1, 1:10, 1:100, 1:1000 (v/v) dilutions to verify whether the mussel matrix could interfere with TTX quantitation. Results showed no interferences starting from a 1:100 dilution (e.g., 10 mg mussels’ meat equivalents/mL). The study is in progress to complete the characterization of the Neuro-2a assay performance for TTX quantitation in mussels by means of linearity range, LOD, LOQ and repeatability.