The Common Representation And Hazard Identification Of Masked T-2Toxin In Litopenaeus Vannamei

The experiment was performed with the infected shrimp, but there is no T-2toxinaccumulation phenomenon was observed. The mice were treated different tissuehomogenate of the infected shrimp by intragastric administration. Detect the immunefunction indexes, serum biochemical parameters and genotoxicity indicators of mice.According to general probabilistic to analysis and identify the damage effect on the micethat was cause by shrimp. The results showed that T-2toxin infected shrimp has significantharms on immune functions and genetic characteristics of mice (high probability P<0.05),but not significant impact on serum biochemical indexes of mice (low probability P<0.05).These indicated that T-2toxin infected shrimp has immune toxicity and genetic toxicity,but it caused no significant damages to the liver and kidneys of mice. Although no T-2toxin accumulation phenomenon of the infected shrimp was observed, mice appearimmune and genetic function accumulation toxicity phenomenon after gavage the infectedshrimp. It showed that the infected shrimp may contain masked T-2toxin, and it can doharms on mice through the food chain of shrimp.Organic solvent extracted the shrimp of20d different T-2oral dose accumulationinfected then treated the extracted residua and organic solvent by Trifluoroacetic acid,LC-MS/MS detective T-2toxin of the dissociation sample, the dissociation of T-2toxinfrom sample confirmed masked T-2toxin exist in shrimp.determined masked T-2toxin isconcentrated in the hepatopancreas, blood, muscles and head of shrimp. Dissociation rateof masked T-2toxin was positively correlated with shrimp infected dosage.Hepatopancreas as the major part that masked T-2toxin exists. T-2toxin increment in thesupernatant of one shrimp hepatopancreas dissociation sample is about23.6ng and theresidual increment is about11.0ng. T-2toxin increase in the residue samples indicate thatorganic solvent extract masked toxin is incomplete.Base on NMR and mass spectrometry the synthesis of target T-2toxin nucleushapten3-Ac-NEOS was demonstrated and using erythrocyte enzymatic hydrolysis methodto acquire Hapten3-Ac-NEOS is feasible and high purity. After we optimized itspreparation and purified by column chromatography, the yield of hapten was60%or so. Inthe steam bath conditions,3-Ac-NEOS and HS synthesized to be3-Ac-NEOS-HS with connecting arm, then we prepared T-2toxin nucleus complete antigen bycarbodiimide method with conjugating the hapten with carrier protein, and used UVscanning and SDS-PAGE gel electrophoresis, finally we determined the conjugatedsuccess of the hapten3-Ac-NEOS with bovine serum albumin and ovalbumin. Thecoupling ratio between3-Ac-NEO and BSA was8.76:1and the coupling ratio between3-Ac-NEOS and OVA was7.24:1, both were in the range of (3~45):1and hadimmunogenic. The coupling rate of immunogenic was8.76:1that was in the range of(8~25):1, which indicated to produce efficient antiserum.Using the T-2toxin completely the parent nucleus antigen which was preparedimmune the New Zealand rabbits, Indirect competitive enzyme-linked immunosorbentassay (ic-ELISA) to detection.After the fifth booster immunization, the antiserum titer was1:64000and it can be used for antibody purification.The indirect competitive ELISA wasestablished based on antibodies purification for analysis the sensitivity and specificity ofantibodies, Results show that parent nucleus antibody of T-2toxin relative to theSingle-family trichothecene A with mother nucleus structure specificity is poor, butrelative to the Single-family trichothecene B toxins DON without mother nuclear structurespecificity is strong. Indicating T-2toxin nucleus antibodies specifically recognize thestructure of the nucleus of A single-family trichothecene toxins. Using indirect competitiveELISA the detection limit for T-2toxin was49ng/mL and IC50was0.228μg/mL.Using the coating antigen3-Ac-NEOS-HS-OVA that conjugate with carboxyl beadsto prepared identifiable T-2toxin mother nucleus antibody immunized beads (T2-IMB),that is the immune beads indirect competitive ELISA (T2-IMB-ELISA). T2-IMB-ELISAfor T-2toxin mother nucleus detection limit was equivalent10.7ng/mL of T-2toxin, andsignificantly lower than the detection limits49ng/mL. of conventional indirect competitiveELISA method. Labeling of T-2toxin detection of the average recovery was92.37%andthe variation coefficient are between3.26~6.43, shows that T2-IMB-ELISA has highrecovery rate and detection accuracy. T2-IMB-ELISA has been successfully applied to thedetection of masked T-2toxin in the shrimp, the results showed that detective the maskedT-2toxin in the one hepatopancreas of the highest dose group is about0.057nmol, if allmasked toxin was dissociation increments of the T-2toxin is26.7ng, higher than thegroup which under condition of acid hydrolysis increment is23.6ng. Indicating that acidhydrolysis is not complete,there is partial loss. T2-IMB-ELISA is suitable for detection ofmasked T-2toxin in vivo of shrimp.