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.

Figure 1

Selection of TTX-binding aptamers. (A) Structures of the target TTX (upper panel) and the counter-selection molecules (lower panel). (B) Evolution of the selections using Dynabeads and SiMAG SA-MB. DNA eluting in the presence of buffer alone or TTX under the specific conditions from the selected rounds was detected after PCR amplification.

Figure 2

Hybrid antibody–aptamer assay for the detection of TTX. (A) TTX calibration curve with the monoclonal IgG antibody-D3 aptamer pair. (B) Specificity of the assay.