The Research Of Fast Determination Of Tetrodotoxin

Tetrodotoxin(TTX) is one of the best-known marine toxins that is predominantly isolated from puffer fish. Because of its high toxicity(LD5o 0.5mg/kg), the puffer fish is prohibited from eating and processing in China. In order to both bring the great accelerating role of puffer fish in economy into play and guarantee the safety of consumers, a reliable, advanced and especially fast determination method is in dire need to be a technological support. There are no such technologies existed inland. Endeavors in establishing such methods are done in this research and two fast determination methods are established.The first method is the utilization of immobilized enzyme of oxalate oxidase (OXO). After reacted with sodium hydroxide, TTX transfers into €9 base and oxalate. And I^Ch generated from oxalate by immobilized OXO is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by horseradish peroxidase (HRP) . Thus the content of TTX is determined. This method, with the minimum detection limit of 2.5umol/L, linear range from 4.5u,mol/L to 45umol/L and within and between assay coefficients of variation<10%, is immuno from the disturb of compounds in the fugu.The second method is the immunological method of Indirect competitive ELISA. Rabbit antiserum against tetrodotoxin is previously produced by the immunization of TTX coupled with keyhole limpet hemocyanin (KLH ) . Then the rabbit antiserum is purified by the method of caprylic acid-ammonium sulphate and used to the determination of TTX. The sensitivity and specificity of this technique are excellent. And the determination limit is low to 5ng with linear range from lOng/kg to 3QOng/kg and within and between assay coefficients of variation between 10% and 20%.These two methods are used to determine the content of TTX in some poisonous puffer fish livers. After contrasted with the result of mouse bioassay. the reliability and assurance of the fast determination methods are confirmed. These two methods arecomplementary to each, that the immobilized OXO is optimum to determine TTX low above Img/kg while indirect competitive ELISA is optimum to the content below Img/kg.