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

Abstract

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many food poisoning incidents and some fatalities. Although mainly associated with the consumption of pufferfish, in recent years, TTX has been found in shellfish, particularly in Europe. In this work, a magnetic bead (MB)-based colorimetric immunoassay was applied to the detection of TTX in Pacific oysters (Crassostrea gigas), razor clams (Solen marginatus) and mussels (Mytilus galloprovincialis). Effective LODs (eLODs) for TTX of 1 µg/kg in oysters and razor clams and 3.3 µg/kg in mussels, significantly below the EFSA guidance threshold (44 µg/kg), were obtained. The strategy was applied to the analysis of naturally-contaminated Pacific oysters (Crassostrea gigas) and mussels (Mytilus edulis) from the Netherlands, and TTX was detected in all samples. The approach, which takes less than 1.5 hours, proved to be useful as a rapid and simple method to detect TTX, support shellfish safety and protect consumers.

Hybrid Antibody–Aptamer Assay for Detection of Tetrodotoxin in Pufferfish

“…The TTX content in these tissues was 2.5–5-fold higher than the permissible levels in Japan (2 mg of TTXs/kg). For comparison, the samples were analyzed in parallel with a competitive magnetic bead-based ELISA (detailed in the SI), which was previously developed and exploited a different monoclonal antibody. , Some differences were observed, which may derive from the specificity of the assays toward the different TTX analogues. It is necessary to take into account that the cross-reactivity factors for the different TTX analogues may vary according to the biorecognition molecule (which in the case of the hybrid sandwich assay are both the antibody and the aptamer) and also the format of the assay.…”

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012

“…Analytical standards for TTX and its analogues are important because they are required for most analysis types. Certified reference materials for TTX are available from many sources: Funakoshi (purity = 96%, Tokyo, Japan), Nacalai tesque (purity ≥94%, Kyoto, Japan), Wako pure chemical industries (purity >99%, Osaka, Japan), Affixscientific Corporation (purity ≥98%, Fremont, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), Cifga (Lugo, Spain), ,,, Tocris Bioscience (purity ≥98.8%, Bristol, UK), ,, Biorbyt Ltd. (Cambridge, UK), Abcam plc (Cambridge, UK), Latoxan (Valence, France), , J&K Scientific Ltd. (Beijing, China), Must Bio-Tech Co., Ltd. (Chengdu, China), , among others. Sometimes, TTX standards contain its analogues, for example, a TTX standard solution from CIFGA (Lugo, Spain) contains TTX, 4,9-anhydroTTX, 11-deoxyTTX, 4-epiTTX, and trace levels of 5,6,11-trideoxyTTX and 11-norTTX-6-ol .…”

“…Remarkably, magnetic beads (MBs) have emerged as a kind of promising immobilization support for immunosensors . Compared with common immunoassays, immunoassays employing the MBs as TTX immobilization supports display numerous advantages, such as better assay kinetics, more efficient washing steps, lower matrix effects, and higher reproducibility. , In the work of Leonardo et al, TTX was conjugated to maleimide-activated MBs by forming cysteamine SAMs, realizing oriented and stable TTX immobilization, as demonstrated in Figure b. After optimization, the proposed electrochemical MB-based immunosensing tool provided a low LOD (1.2 ng/mL) and broad working range (1.2–52.7) with high reproducibility (RSD < 16%).…”

An Updated Review of Tetrodotoxin and Its Peculiarities

“…That is the case of the work described by Reverté and co-workers [308], where TTX was immobilized on gold electrode arrays and the subsequent biosensor, which attained an LOD of 2.6 ng/mL, was applied to the analysis of pufferfish. More recently, the use of magnetic beads (MBs) as TTX immobilization supports has been demonstrated to be a very good strategy to remove or at least decrease matrix effects in both immunoassays [309] and immunosensors [310]. The use of MBs in suspension certainly favors the antibody/antigen interaction and makes the washing steps more efficient, which result in lower LODs (0.5 and 1.2 ng/mL, respectively).…”