3.3.2. Tetrodotoxin

Tetrodotoxin in the pufferfish skin and collagen sample was characterized based on a method described by Wang et al. [48] with slight modification. Smashed pufferfish skin and collagen sample were incubated in 0.5 mol/L acetic acid at 100 ◦C for 40 min and then centrifuged at 10,000× *g* for 15 min. The resulting supernatants were collected and analyzed directly with an enzyme-linked immunosorbent assay (ELISA) test kit (Beijing Zhongnuo Taian Technology Co. Ltd., Beijing, China) for tetrodotoxin. As described in the manual, samples and enzyme-labeled MAb-TTX were added to wells of a microplate in which standard tetrodotoxin had been immobilized as a competitive agent. The absorbance (OD value) was measured at 490 nm. The ratio of absorbance (Ai/Ao, where Ai is the absorbance of standard solution, and Ao is the absorbance of blank with no tetrodotoxin added)

was used as the ordinate. The logarithm of the corresponding standard solution concentration was plotted as the abscissa, a standard curve was prepared, and the tetrodotoxin content of the sample was calculated.
