Preparation Of Deoxynivalenol Monoclonal Antibody And Establishment Of Chemiluminescence Enzyme Immunoassay Method

Deoxynivalenol(DON)is mainly produced by wheat Fusarium graminearum,is microorganisms toxin with high toxicity to human and animals.The establishment of rapid detection methods,has important significance for ensuring food safety.The paper studied the DON monoclonal antibody and chemiluminescent enzyme immunoassay method.DON hapten was synthesized.Ovalbumin(OVA)and DON hapten were conjugated to prepare coating antigen by the activated ester method.The commercial DON immunizing antigen which conjugated with the bovine serum albumin(BSA)was used to immunize BALB/c mice.Detecting serum,selecting best-immunized mouse,its spleen cells and myeloma cells Sp2/0 were used to do fusing.After several rounds of screening and subcloning,as well as hybridoma cell culture supernatant was detected,eventually we got the hybridoma cell strain 5E8 which secreting anti-DON monoclonal antibody.The antibody’s subtype was IgG1,the titer was 8×105.There are no significant cross-reactivity between DON and other toxins.By selecting the chemiluminescent substrate buffer and the kind of enhancer,optimizing the concentration of enhancer,luminol and oxidant,determining the retention time of luminescent sensitizing solution,finally,the chemiluminescent substrate solution was manufactured.Based on the solution,we optimized the concentration of coating angen,antibody,ionic,acetonitrile and goat anti-mouse IgG,and selected closure,optimized pH value.Ultimately,the determination conditions were:coating antigen was diluted 8000 times,the closure was 3%skim milk,the working solution containing 10%acetonitrele,pH 7.4,0.4M Na+ was phosphate buffer,goat anti-mouse IgG was diluted 5000 folds.In optimal conditions,the standatd curve of competitive indirect chemiluminescent enzyme immunoassay for deoxynivalenol was established.Its detection limit IC10 was 0.5602μg/L,the sensitivity IC50 was 18.62μg/L.Wheat,oats,and corn were added DON standard,and detected,the average recovery was 89.47%-114.38%,while the coefficient of variation was 5.71%-11.42%.The established method was evaluated by gas chromatography,analysis results with high degree of similarity,the correlation coefficient R2 was greater than 0.99.DON enzyme-labeled antigen was prepared,the concentration of antibody and enzyme-labeled antigen was optimized,acetonitrile concentration and competition reaction time was selected.Based on the chemiluminescent substrate solution and the enzyme-labeled antigen,the optimal conditions were:coating antibody was diluted 10000-fold,enzyme-labeled antigen was diluted 4000-fold,the system’s competitive reaction time was 30min,the content of the working solution in acetonitrile was 5%.In optimal conditions,the standatd curve of competitive direct chemiluminescent enzyme immunoassay for deoxynivalenol was established.Its detection limit IC10 was 0.6931μg/L,the sensitivity IC50 was 30.46μg/L.Wheat,oats,and corn were added DON standard,and detected,the average recovery was 88.57%-116.38%,while the coefficient of variation was 7.31%-10.21%.The established method was evaluated by gas chromatography,analysis results with high degree of similarity,the correlation coefficient R2 was greater than 0.99.Several pesticides were used to control the wheat scab.After collecting wheat samples,the established competitive indirect and direct chemiluminescent enzyme immunoassay was used to test the deoxynivalenol(DON).Considering the drift interference and matrix effects on the determination,the test results of competitive indirect and direct methods was comparatively analyzed,and the test results of commercially available vomitoxin(DON)ELISA kit were correlated analyzed.The results showed that two chemiluminescent enzyme immunoassay methods can accurately determine the content of DON,and their results have good correlation with commercially available ELISA kit,the correlation coefficient R2 was greater than 0.99.The results showing that the established chemiluminescent enzyme immunoassay was reliable,accurate,analytical methods have good prospects in the field detection of DON in wheat samples.