**Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)**

#### **Jérôme Viallon, Mireille Chinain and Hélène Taiana Darius \***

Institut Louis Malardé (ILM), Laboratory of Marine Biotoxins-UMR 241-EIO, 98713 Papeete-Tahiti, French Polynesia; jviallon@ilm.pf (J.V.); mchinain@ilm.pf (M.C.) **\***Correspondence:tdarius@ilm.pf

Received: 3 April 2020; Accepted: 24 April 2020; Published: 27 April 2020

**Abstract:** The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, ye<sup>t</sup> a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

**Keywords:** CBA-N2a; standardization; matrix effects; absorbance data; ciguatoxins; brevetoxins; saxitoxins; biological sample; seafood safety

**Key Contribution:** The present work is a revisit of several parameters of the CBA-N2a that improved assay implementation and reliable toxin detection. This study resulted in a practical guide accessible to both supervised beginners and experienced users, provided in the Supplementary Materials. This revisited CBA-N2a allowed for the improved detection of toxins acting on the voltage gated sodium channels (ciguatoxin, brevetoxin and saxitoxin) as evidenced by CTX estimation data obtained from fish samples of known toxic status.
