Hybrid Antibody–Aptamer Assay for Detection of Tetrodotoxin in Pufferfish

Abstract

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody−aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

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 .…”

“…Aptamers are artificial single-stranded DNA (ssDNA) and ribonucleic acid (RNA) that can interact with target molecules by strong affinity and specificity. As biorecognition molecules, they are considered preferable alternatives to antibodies, since they have significant advantages over antibodies, including in vitro selection, facile preparation, high specificity, reproducible chemical synthesis, minor batch-to-batch variation, easy modification, and long-term storage stability …”

“…As biorecognition molecules, they are considered preferable alternatives to antibodies, since they have significant advantages over antibodies, including in vitro selection, facile preparation, high specificity, reproducible chemical synthesis, minor batch-to-batch variation, easy modification, and longterm storage stability. 80 Aptamers are usually obtained through systematic evolution of ligands by exponential enrichment (SELEX). Notably, developing aptamers for small molecular targets is a difficult task, which may be attributed to the difficulty of immobilizing small molecules on solid surfaces.…”

“…They are easy to operate and applicable to most laboratories. Selecting a proper extractant is important, and the most common one is acetic acid (AA). ,, To further clean up the sample mixtures, trichloroacetic acid or acetonitrile is often used for protein precipitation, while hexane or dichloromethane is used for fat removal. In recent years, ultrasonic-assisted extraction (UAE) and accelerated solvent extraction (ASE) have been more applied to sample preparation, since they overcome the disadvantages of traditional extraction techniques, such as low extraction rates and large solvent consumption. ,, …”

An Updated Review of Tetrodotoxin and Its Peculiarities

“…Recently, a new aptamer has been produced using a capture-SELEX process, in which the oligonucleotide library was immobilized on magnetic beads, and exploited in a hybrid antibody-aptamer assay [321]. This is one of the few examples of sandwich assay for small molecules.…”