Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels

Abstract

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.

A fast magnetic bead-based colorimetric immunoassay for the detection of tetrodotoxins in shellfish

“…Taking into account the LOD from the calibration curve (0.5 ng/mL) and the shellfish matrix concentrations allowed, effective LODs (eLODs) were calculated to be 1 µg/kg for oysters and razor clams and 3.3 µg/kg for mussels, well below the value proposed by EFSA (44 µg/kg). These eLODs are also lower than the ones found with our previous immunoassay using microtiter plates (20-50 µg/kg for oysters and 30 µg/kg for mussels) (Reverté et al, 2018). Working ranges upper limits were calculated to be 18.2 µg/kg for oysters and razor clams and 60.7 µg/kg for mussels.…”

“…The presence of TTX and its analogues in samples can be detected in a rapid, simple, specific, sensitive and reliable way using immunoassays. In our previous work, an immunoassay using microtiter plates for TTXs was developed and applied to the analysis of shellfish (Reverté et al, 2018), but that immunoassay experienced strong shellfish matrix effects (antibody binding percentage values lower than 80% in the absence of TTX). These undesirable effects were solved with a solid-phase extraction (SPE) clean up followed by solvent evaporation prior to the immunoassay.…”

First Detection of Tetrodotoxin in Bivalves and Gastropods from the French Mainland Coasts

“…In addition to the Iberian locations, two gastropod samples from the Moroccan coast [2] revealed the presence of TTXs (maximum value: 20 µg TTX eq/kg). In the Netherlands, the maximum level observed in 2017 was 51 µg TTX eq/kg in an oyster sample [29,30]. This maximum value is below the highest reported values, observed in 2015 and 2016, of 124 and 253 µg TTX eq/kg respectively in two oyster samples.…”

Public Health Risks Associated with Tetrodotoxin and Its Analogues in European Waters: Recent Advances after The EFSA Scientific Opinion

“…Antibody-based methods, such as competitive inhibition enzymatic immunoassay (ELISA) or surface plasmon resonance biosensor (SPR), are potentially useful for qualitative identification, but not ideal for routine screening at present, due to fact that there is no cross reactivity for all the known TTX analogues. Recent developments in TTXs detection in this type of methods comprise a nanoarray planar waveguide biosensor, initially tested on pufferfish samples [32] and the modification of a self-assembled monolayer-based Immunoassay (mELISA) previously developed for TTX detection in pufferfish [43], which has now been adapted to the analysis of oyster and mussel samples [30]. As regards the biosensor method, TTX detection was possible at levels of 0.4–3.29 μg/g puffer fish tissue in 10 min; repeatability and reproducibility were assessed at 0.4 and 0.8 μg/g, showing relative standard deviation (RSD) values below 15% and toxin recoveries within 85–115%.…”

“…The mELISA showed high selectivity for TTX as no cross-reactivity of the antibody with co-occurring PSP toxins was observed. Analysis of naturally-contaminated samples, in comparison with LC-MS/MS quantifications, indicated that the mELISA method could be a promising tool for the rapid (< 2 h on the same day) detection of TTX in oyster and mussel samples, with potential application in routine monitoring programs [30].…”