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Aptamer-antibody sandwich lateral flow test for rapid visual detection of tetrodotoxin in pufferfish
Díaz-Avello, U. G., Skouridou, V., Shkembi, X., Reverté, J., Mandalakis, M., Peristeraki, P., Campàs, M., & O'Sullivan, C. K.
Sci Total Environ2025 May 25PubMed ID: 40245514Read Article
Applications: LFA Reactive species: Pufferfish
"Abstract: Tetrodotoxin (TTX) is a highly potent marine toxin which can cause severe poisoning following consumption of contaminated fish and seafood. Thus, a sensitive, reliable and simple test is required for rapid screening of samples and prevention of intoxication. Herein, we translated a previously reported microtiter plate hybrid aptamer-antibody assay into a rapid lateral flow assay (LFA) test. The test relies on an aptamer immobilized on the membrane and an antibody conjugated with gold nanoparticles to provide a visual result when TTX is present in the sample. The optimized test is simple (one-step), rapid (<20 min), highly sensitive (visual limit of detection of 0.3 ng/mL TTX in buffer corresponding to 0.78 mg TTX/kg tissue), specific, reproducible and with long storage life. It was validated by analyzing contaminated pufferfish tissue extracts and it successfully detected TTX below the current limits set by official bodies. The analysis performed with this device in combination with a simple LFA reader for quantification was in excellent agreement with other established methods, further demonstrating the value of this test as a simple, low-cost and reliable analytical tool to ensure food safety, protect human health, and broaden the knowledge on the correlation between biological parameters and environmental data." Article snippet: Mouse monoclonal α-TTX IgG antibody (CABT-L3089, CD Creative Diagnostics) was purchased from Deltaclon S.L. (Spain).
Figure 1. Sensitivity and reproducibility of the LFA tests.
Figure 2. Specificity of the LFA test.
Mechanistic insights into antibody recognition of tetrodotoxin analogues: Implications for neurotoxicological assessment
Jaume Reverté, Mounira Alkassar, Maria Rambla-Alegre, Andres Sanchez-Henao, Manolis Mandalakis, Panagiota Peristeraki, Francesc X Sureda, Jorge Diogène, Mònica Campàs
Chem Biol Interact2026 Jan 25PubMed ID: 41386469Read Article
Applications: Magnetic bead-based immunoassay Reactive species: Unspecified reactive species
"Abstract: Tetrodotoxins (TTXs) pose significant food safety risks due to their potent neurotoxicity. Growing concerns about the impact of these toxins on public health have driven the development of new detection methods, with immunoassays showing strong potential. However, limited knowledge of the cross-reactivity of anti-TTX antibodies with analogues may compromise the reliability of these assays in food safety applications. To address this, cross-reactivity factors (CRFs) for five TTX analogues (i.e., 11-norTTX-6(S)-ol, 11-deoxyTTX, 6,11-dideoxyTTX, 5,11-dideoxyTTX, and 5,6,11-trideoxyTTX) were assessed using a magnetic bead-based immunoassay. In parallel, the antibody's ability to neutralise the toxicity of TTX analogues was evaluated in Neuro-2a cells using automated patch clamp, a single-cell biosensing platform specifically designed for in vitro toxicity assessment and characterisation. Antibody cross-reactivity towards the tested analogues correlated with their relative toxicity, enabling a selective detection of the most hazardous compounds. These findings highlight the dual role of molecular structure in dictating both toxicological potency and immunological recognition, and support the use of immunoassays as effective tools for TTX monitoring in food safety applications." Article snippet: The anti-TTX monoclonal antibody (CABTL3089) was obtained from Creative Diagnostics (*).
Figure 1. Dose-response curves obtained from the analysis of TTX and TTX analogues with the MB-based colorimetric immunoassay
Background
Tetrodotoxin (TTX) is a potent neurotoxin that is found in various organisms, most notably in certain species of pufferfish (also known as blowfish), as well as other marine creatures such as certain species of octopus and newts. It is one of the most potent naturally occurring toxins known to humans. TTX exerts its toxic effects by selectively blocking voltage-gated sodium channels, which are essential for the generation and propagation of action potentials in nerve cells. By binding to specific sites on these channels, TTX prevents the influx of sodium ions, effectively halting the transmission of nerve impulses. This blockade leads to muscle paralysis and can result in respiratory failure and death if the toxin reaches a critical concentration. Due to its potent toxicity, TTX has gained significant attention in both scientific and medical fields. It is used as a valuable tool in neuroscience research to study the function and properties of sodium channels and their role in nerve signaling. TTX has contributed to the understanding of various neurological disorders and has been instrumental in elucidating the mechanisms underlying pain perception. The Anti-Tetrodotoxin Monoclonal Antibody is a specialized antibody designed for use in immunoassay technology. It serves as a powerful tool for the detection, analysis, and monitoring of TTX in a variety of samples, such as seafood, biological fluids, or environmental samples. Its specificity and high affinity enable accurate and reliable identification of TTX, contributing to research efforts, food safety measures, and environmental monitoring related to this potent neurotoxin.
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References
An Updated Review of Tetrodotoxin and Its Peculiarities
Marine Drugs
Authors: Katikou P, Gokbulut C, Kosker A R, et al.
Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide.
Development of ELISA and colloidal gold immunoassay for tetrodotoxin detetcion based on monoclonal antibody
A monoclonal hybridoma cell named 5B9 against tetrodotoxin (TTX) was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The 5B9 monoclonal antibody (McAb) with high affinity (about 2.55×109) is specific to TTX, and this McAb belongs to the immunoglobulin G (IgG) isotype. Finally, an enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay were established based on this McAb. The linear range of ELISA to detect TTX was 5–500 ng/mL, and the limit of detection (LOD) was 4.44 ng/mL. The average CV of intra- and inter-assay was less than 8%, with the samples recovery range of 70.93–99.99%. A competitive format colloidal gold strip was developed for detection of TTX in real samples, and the LOD for TTX is 20 ng/mL, and the assay time of the qualitative test can be finished in less than 10 min without any equipment. The result from test strip revealed that the test strip has a good agreement with those obtained from ELISA.
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