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

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet 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 incubatio...

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Main Authors: Jérôme Viallon, Mireille Chinain, Hélène Taiana Darius
Format: Article
Language:English
Published: MDPI AG 2020-04-01
Series:Toxins
Subjects:
Online Access:https://www.mdpi.com/2072-6651/12/5/281
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author Jérôme Viallon
Mireille Chinain
Hélène Taiana Darius
author_facet Jérôme Viallon
Mireille Chinain
Hélène Taiana Darius
author_sort Jérôme Viallon
collection DOAJ
description The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet 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 EC<sub>50</sub> 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.
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spelling doaj.art-20f4c2dcadea4727874373bb56d3f3412023-11-19T22:50:22ZengMDPI AGToxins2072-66512020-04-0112528110.3390/toxins12050281Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)Jérôme Viallon0Mireille Chinain1Hélène Taiana Darius2Institut Louis Malardé (ILM), Laboratory of Marine Biotoxins-UMR 241-EIO, 98713 Papeete-Tahiti, French PolynesiaInstitut Louis Malardé (ILM), Laboratory of Marine Biotoxins-UMR 241-EIO, 98713 Papeete-Tahiti, French PolynesiaInstitut Louis Malardé (ILM), Laboratory of Marine Biotoxins-UMR 241-EIO, 98713 Papeete-Tahiti, French PolynesiaThe neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet 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 EC<sub>50</sub> 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.https://www.mdpi.com/2072-6651/12/5/281CBA-N2astandardizationmatrix effectsabsorbance dataciguatoxinsbrevetoxins
spellingShingle Jérôme Viallon
Mireille Chinain
Hélène Taiana Darius
Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
Toxins
CBA-N2a
standardization
matrix effects
absorbance data
ciguatoxins
brevetoxins
title Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
title_full Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
title_fullStr Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
title_full_unstemmed Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
title_short Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)
title_sort revisiting the neuroblastoma cell based assay cba n2a for the improved detection of marine toxins active on voltage gated sodium channels vgscs
topic CBA-N2a
standardization
matrix effects
absorbance data
ciguatoxins
brevetoxins
url https://www.mdpi.com/2072-6651/12/5/281
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