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Tetrodotoxin

The Severity of Food Contamination Caused by Tetrodotoxin

Tetrodotoxin (TTX) is an alkaloid contained in pufferfish and other organisms. Pufferfish has a long history of eating in Asian countries such as China and Japan. However, TTX is one of the most toxic neurotoxins found in nature. Its toxicity is more than 1250 times higher than sodium cyanide, and 0.5 mg can cause death. TTX food poisoning is often caused by improper processing. TTX and analogues not only exist in various hogfish but also widely distributed in various higher and lower organisms. The accumulation and distribution of TTX in the body vary with different seasons and locations. The puffer is very toxic during the reproductive season, and among different parts, the ovaries and liver contain the most toxins. Different types of pufferfish have different distributions of toxins in their bodies. Among them, there are 34 species of pufferfish with TTX in the liver, 32 species with TTX in the ovary, etc.

The Necessity of Monitoring Tetrodotoxin Contamination with ELISA

The Necessity of Monitoring Tetrodotoxin Contamination with ELISA Fig. 1 The chemical structures of Tetrodotoxin

TTX is an amino perhydroquinazoline compound, white crystals, odourless and tasteless, slightly soluble in water, insoluble in organic solvents. TTX has a local stimulating effect on the intestinal tract. After absorption, it quickly acts on the nerve endings and nerve centre, blocking the conduction of nerves and muscles, causing nerve paralysis and death. The specific mechanism of action is to block the voltage-dependent sodium channel by binding to the sodium ion channel receptor, thereby blocking the action potential, leading to the obstruction of related physiological activities. The inhibition of pufferfish toxin on respiration and cardiovascular is the result of the joint action of central and peripheral nerves. 1972 to 1993, 1258 people were poisoned by Japanese pufferfish alone, resulting in 279 deaths. The toxicity of TTX has aroused widespread concern in society, and all localities have strict regulations on the detection content of TTX. Because the ELISA test procedure is simple, fast, and sensitive, it has broad application prospects in the quantitative detection of TTX and the prevention of pufferfish poisoning.

ELISA Types

Indirect competitive ELISA

The Advantages of ELISA Testing

Can detect TTX specifically
Save testing time and not subject to site restrictions
Provide a reliable method for detecting TTX in food

ELISA Procedure for Tetrodotoxin Testing

The microtiter plates were coated with 100 μL conjugate diluted in PBS (2 μg/mL), and incubated 1 h at room temperature (RT).

After washing three times with 250 μL of PBS-T, 200 μL/well of blocking solution (1% PBS-BSA) was added, and incubated for 1 h at RT.

After washing, 50 μL of standards or samples are added to wells. Then 50 μL of anti-TTX antibodies diluted to the optimal concentration are added to wells, incubated for 1 h at RT.

After washing, 1000 μL of anti-mouse IgG + IgM antibodies are added to all wells except the positive and blank controls, incubated 1 h at RT.

After washing five times with PBST, 100 μL/well of goat anti-rabbit IgG conjugated to HPR was added, incubated for 1 h at RT.

After washing, 50 μL of secondary antibodies are added to the positive control wells, then 200 μL of a 1 mg/mL pNPP solution is added to all of the wells. The plate is protected from light and incubated for 10 min at RT.

The absorbance was read at 405 nm by a microplate reader. Readings are taken every 5 min after the initial reading.

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References

Noguchi, T.; et al. TTX accumulation in pufferfish. Comp Biochem Physiol Part D Genomics Proteomics. 2006, 1(1): 145-152.
Stokes, A.N.; et al. An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification. Biol Proced Online. 2012, 14: 3.

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