Research On Rapid Detection Of Deoxynivalenol In Corn And Soybean Based On Fluorescent Lateral Flow Immunoassay

Deoxynivalenol（DON）,also known as vomit toxin,is a secondary metabolite produced by mycotoxic fungi,widely found in wheat,barley,oats,corn and other grains.DON has cytotoxic,immunotoxic and neurotoxic characteristics and cause vomiting in animals.In the last decade,there have been more and more reports about the adverse effects of DON on human.In this study,quantum dot nanobeads（QBs）lateral flow immunoassay（LFIA）based on single T line and double T（T₁,T₂）lines model were established for quantitative determination of DON in maize and soybean.The main research methods and conclusions are as follows:Firstly,the complete antigen was synthesized by carbondiimine method,and the serum titer of mice was monitored by enzyme-linked immunosorbent assay.Three mice with high positive rate and good inhibition rate were obtained and subjected to shock immunization,and their spleen cells were fused with myeloma cells.After cell fusion,screening and subcloning,there were only 9 cell culture wells with OD₄₅₀ value higher than 1.0,among which OD₄₅₀ value of 5C4 was 1.81 and OD₄₅₀ value of 4H4 was 1.36.The inhibition rate（200 ng/m L）was 54%and 62%,respectively.The cells with higher OD₄₅₀ value and better cell growth status were selected for subcloning by limited dilution method.After cell cloning,the cells were fully amplified.Indirect competitive enzyme-linked immunosorbent assay（IC-ELISA）was used to detect the culture supernatant of the clones,and no positive results were found.Secondly,a competitive LFIA was established based on anti-DON monoclonal antibody.The p H of antibody conjugation,the amount of EDC,the amount of mAb,the concentration of DON-BSA and the amount of probe were optimized.Under the optimal conditions,the half maximal inhibitory concentration(IC₅₀)of DON in corn and soybean were 340μg/kg and 351μg/kg,respectively.The standard recovery experiment showed that the established QBs-LFIA had a good recovery rate and a low coefficient of variation（CV）.In the corn sample,the intra assay recovery rate was98.56%-104.75%,and the CV was lower than 8.41%.The inter assay recovery rate was105.16%-109.32%,and CV was less than 7.57%.In the soybean samples,the intra assay recovery rate was 95.50%-105.53%and CV was lower than 7.42%,while the inter assay recovery rate was 101.18%-106.01%and CV was less than 7.95%.QBs-LFIA established in this chapter has a good prospect of industrial application.Thirdly,a double T（T₁,T₂）lines QB_S-LFIA was established to explore the effect of double T lines model on LFIA analytical performance.Under a certain total amount of total antigen,double T lines QB_S-LFIA was established according to the different ratios(C_T1:C_T2=4:6,C_T1:C_T2=5:5 and C_T1:C_T2=6:4).The results showed that when the distribution ratio of double T lines was C_T1:C_T2=6:4,the double T lines QBs-LFIA had better stability.Compared the analysis performance of traditional single T line with QBs-LFIA,it was found that the immune reaction time of double T lines model was shorter,and it could reach the stable state 2 min earlier,which saved the time for the field detection of large quantities of samples.In the accelerated preservation experiment,the double T lines model had higher stability（decrease range is less than6%）,which provided a scientific basis for the extension of shelf life.However,the recovery rate（104.50%-137.88%）and CV（less than 12.46%）of the double T lines model were both worse than the recovery rate（97.12%-107.50%）and CV（less than8.73%）of the traditional single T line model,which might affect the precision and accuracy of the product.The traditional single T line QBs-LFIA had higher accuracy and precision while the double T lines model QBs-LFIA had lower time cost and higher shelf-life stability,which provided a scientific basis for improving product performance.In summary,this study developed a QBs-LFIA based on anti-DON monoclonal antibody for the rapid and sensitive detection of DON in corn and soybean,providing a guarantee for the rapid detection of DON in grain.In addition,the influence of traditional single T line model and double T lines model on LFIA analysis performance were compared to provide scientific basis for commercialization of product.