Development And Primary Application Of Indirect ELISA Detection Of Domoic Acid

Domoic acid(DA)is a marine bio-toxins toxin produced by pseudo-nitzschia diatoms.Seafood contaminated by DA may cause human diarrhea,vomiting and memory loss,and even coma and death when consumed.Therefore it is also called amnesic shellfish poison(ASP).Domoic acid is usually detected by mouse bioassay and HPLC method.However,it can not meet the demand for rapidly detection,affecting the edible safety of seafood seriously.Enzyme linked immunosorbent assay(ELISA)with high sensitivity,specificityare,easy operation and rapid detection is widely used in analysis of pollutants in the environment.For the establishment rapid detecting technology of domoic acid,Bovine albumin(BSA)was linked to DA using EDC method to prepare coating antigen DA-BSA successfully.The monoclonal antibodies was made.This study established indirect competitive ELISA(ic-ELISA)detection method of DA with the optimization of reaction conditions such as the dilution of the antigen,antibody and IgG-HRP,incubation conditions,blocking conditions,packet conditions and coloration conditions.Its specificity,OD variability,recovery and shelf life were also been studied.A comparison between ic-ELISA and HPLC method in detecting the content determination of DA in real samples was studied.The results were as follows:1.Bovine albumin(BSA)was linked to DA.Coating antigen DA-BSA was obtained Successfully.The protein concentration of DA-BSA was 1.0 mg·mL-1.2.Analyze the titer of monoclonal antibody by ic-ELISA.The titer reaches 1:64000 and the protein concentration of monoclonal antibody was 1.0 mg·mL-1.3.The results show that the optimal dilution of antigen,antibody and IgG-HRP were 1:12800,1:400 and 1:3000 respectively,choose 1%PVA-PBS as the best blocking solution,blocking at 37? for 120 min,packeting 12 h at 4?,then incubating 60 min at 37? after the monoclonal antibodies added,incubating 30 min at 43? after HRP-IgG added,the best coloration condition was at room temperature for 20 min.4.A standard curve of ic-ELISA kit was ranging from 1.60 ng-mL"1 to 200 ng-mL-1 and the limit of detection was 1.60 ng-mL'1.The linear regression equation was Y =-0.0844X + 0.9185 and the correlation coefficient R2 was 0.9943.5.There was no cross reaction with L-Glu and Kainic acid.The variability of within-assay and between-assay of OD value were both less the 10%and the recovery was 86.8%to 103.2%,illustrating that the method had good repeatability,the kit can be saved for two months.6.Five shellfish samples were detected by ic-ELISA,and the concentrations of DA were both less safety limits.7.The relativity between ic-ELISA and HPLC method was analyzed.The correlation coefficient was 0.817.There was a good correlation between the two methods.From above all,we can make a conclusion that the domoic acid ELISA kit was developed successfully which can met the demand for rapidly detection of seafood.