A Study Of The Artificial Antigen Synthesis And Indirect Competitive ELISA For Deoxynivalenol

Deoxynivalenol(DON),the secondary metabolite of Fusatium fungi,is most member of the trichothecene family of mycotoxins.DON appears predominantly in wheat,corn,rye, rice and barley all over the world.The eatent of infection is dependent on weather conditions and storage conditions of cereal crops.Although DON is,aming the least toxic of the trichothecenes,it is the most frequently detected one throughout the word and its occurrence is considered to be an indicator of possible presence of other more toxic trichothecenes.In addition,DON is a very stable compound,during both storage and the processing/cooking of food,and it needs quite harsh conditions to get substantial breakdown.Consumption of contaminated feeds by livestock has been associated with a variety of adverse health effects including feed refusal,reduced weight gain,diarrhea and emesis.IARC had classified DON in group 3 "not classifiable as to its carcinogenicity to humand".In the present study,we had produced deoxynivalenol antibody and had established DON's immunoassay method.We prepared immunogen of DON by two methods.Then dectected anti-sera titer from mice immunized with different immunogen.We optimized and established ELISA process.The main results were as following:1.Synthesis immunogen with two methods—One was that DON obtained a carboxyl after deriving,and then complete antigen of DON was synthesized by succinic anhydride; The other was that DON was activated by 1,1-Diclohexylcarbodiimide.The products were detected by UV spectrum,SDS-PAGE and ELISA kit,and the concentration of DON in DON-BSAwas 0.797μg/μL,the DON-OVA was 0.518μg/μL.2.Synthetical antigen was mixed with the same quantity adjuvant and immunized mice.After each immunization,the mice were blooded from tail.Separated sera,detected sera antibody titer by ELISA to monitor the variation of antibody.At last we conclude that the best antibody titer could be get after the sixth immunization.3.The optimal working concentration of the coated antigen and the Balb/c mice sera dilutions in the indirect ELISA were 3μg/mL and 1:200,respectively.4.Optimized indirect ELISA process.The best reaction-conditions were that plate coated at 4℃for 12 h,anti-sera incubated 60 min,IgG-HRP incubated 60min,substrate action time were 10min.5.The experimentation showed:Samples were extracted with acetronile-water(84:16, V/V);recovery rate were 86.4～95.6%;cutoff value was 2.4;reproducibility and specificity were good.6.The method of the indirect competitive ELISA for DON was established. Competitive inhibitory indirect ELISA standard curve equation was:Y=-37.945X+134.45. The detection limit was 1μg/mL.Samples with DON levels from 1 to 1000μg/mL could be quantitated.7.Compared the ELISA with ELISA kit by detection of DON in samples,the result Show that no significant difference.