Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay

Abstract

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus

“…These two compounds have been already identified in puffer fish and newt from Japan and Solomon Islands (Kudo et al, 2012;Puilingi et al, 2015). 6,6, previously determined (Reverté et al, 2015) to the individual LC-MS/MS and LC-HRMS quantifications. Cross-reactivity factors (in percentage) are 29.9 for 5-deoxyTTX and 11-deoxyTTX, 2.9 for 11-norTTX-6(R)-ol and 11-norTTX-6(S)-ol, <10 -2 for 4,9-anhydroTTX, and <10 -4 for 5,6,11-trideoxyTTX; CRF for 4-epiTTX was assumed to be 1; CRFs for 5,11-dideoxyTTX/6,11-dideoxy and the two new 5,6,11-trideoxyTTX analogues were not available and thus these analogues were not included in the sum of TTXs.…”

“…All puffer fish were dissected and gonads (only in females), liver, skin and muscle were retrieved. Extraction of TTXs was performed as previously described (Reverté et al, 2015). Briefly, a double extraction of each organ with 0.1% acetic acid was performed.…”

“…LC-MS/MS analysis was performed by a TSQ Quantum system (Thermo Fisher Scientific, Bremen, Germany) as previously described (Reverté et al, 2015). Briefly, analytical separation was performed on a HILIC XBridge Amide column; a binary gradient elution was programmed with water (mobile phase A) and acetonitrile/water The recently developed mELISA, based on the immobilization of TTX through dithiolcarboxylate self-assembled monolayers (Reverté et al, 2015) was used to determine the TTX equivalent contents in the L. sceleratus tissues.…”

“…LC-MS/MS analysis was performed by a TSQ Quantum system (Thermo Fisher Scientific, Bremen, Germany) as previously described (Reverté et al, 2015). Briefly, analytical separation was performed on a HILIC XBridge Amide column; a binary gradient elution was programmed with water (mobile phase A) and acetonitrile/water The recently developed mELISA, based on the immobilization of TTX through dithiolcarboxylate self-assembled monolayers (Reverté et al, 2015) was used to determine the TTX equivalent contents in the L. sceleratus tissues. Briefly, carboxylate-dithiol was self-assembled on maleimide-activated plates; carboxylic groups of dithiols were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) for subsequent reaction with ethylenediamine; remaining carboxylic groups were deactivated with ethanolamine; TTX was immobilized through formaldehyde crosslinking (amino-amino reaction); competition between free (standard or extract) and immobilised TTX for the anti-TTX monoclonal antibody took place; a blocking step with BSA was performed; immunoglobulin G-horseradish peroxidase (IgG-HRP) was added;…”

Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics

“…For small analytes (molecular weight <1000 Da), an inhibition assay is often used, where small analytes are premixed with antibodies and unbound antibody sites are captured by the small analytes immobilized on a sensor surface [43]. This approach has been used widely with SPR biosensors for the detection of aquatic toxins ([6,7,12,13,14,26,32,40,42]). Variations in SPR technology is available with different capabilities in analysis due to the utilisation of single or multiple flow channels (Fig.…”