Preparation Of Monoclonal Antibody And Establishment Of Immunoassay For The Analysis Of T-2 Toxin

T-2 toxin is a highly toxic single-terminal sporotrichum toxin produced by many fungi,which is widely found in cereals and feed.If people and animals eat cereals or feeds contaminated with T-2 toxin,it will pose a potential hazard to human and animal health.The immunoassay method has the characteristics of high sensitivity,specificity and rapid detection,and can be used for large-scale and rapid detection of T-2 toxin residues.In this paper,a hybridoma cell fusion technique was used to prepare monoclonal antibody against T-2 toxin.Through optimization of reaction conditions,an enzyme-linked immunosorbent assay,a chemiluminescence enzyme-linked immunosorbent assay and an immunoaffinity column HPLC were established.T-2 toxin was detected in grains such as wheat and corn samlpes.1.Preparation of monoclonal antibody against T-2 toxinThe Balb/c mice were immunized with artificial immune antigen T-2-BSA.After the SP2/0 myeloma cells were fused with mouse spleen cells.Two hybridoma cells3B2 and 8C3 secreting monoclonal antibody of T-2 toxin were successfully obtained,and 8C3 exhibited better competitive effect.Therefore,the 8C3 hybridoma cell was selected to inoculate mice to prepare T-2 toxin monoclonal antibody ascites.The monoclonal antibody ascites was purified by octanoic acid ammonium sulfate method and protein G column method,and the purity of the monoclonal antibody was 3.58mg/m L.2.Establishment of enzyme-linked immunosorbent assayThe enzyme-linked immunosorbent assay?ELISA?was established.by optimizing the concentration of antigen coating,dilution ratio of antibody,dilution ratio of secondary antibody,concentration of organic solvent,ionic strength,PH and other factors in the reaction system,we determined that the T-2 antigen coating concentration was 4 ug/m L,the antibody dilution ratio was 1:800,enzyme labeled anti-body dilution ratio was 1:2000,a series of standard solutions were prepared w ith5%methanol,T-2 antibody was diluted by PBS buffer with ionic strength of 5 m M and p H of 7.0.The IC₅₀ was 1.45 ng/m L,the minimum detection limit(IC₁₀)was 0.16ng/m L.The average recoveries of wheat and corn simples were 92.09%?114%,101.50%?107.70%,and the coefficients of variation were 3.50%?5.78%,2.82%?10.34%,respectively.3.Establishment of chemiluminescence enzyme-linked immunosorbent assayThe chemiluminescence enzyme-linked immunosorbent assay was established.through the optimization of the influence factors of reaction system,we determined that the T-2 antigen coating concentration was 1 ug/m L,the antibody dilution ratio was 1:400,enzyme labeled anti-body dilution ratio was 1:2000,a series of standard solutions were prepared with 20%methanol,T-2 antibody was diluted by PBS buffer with ionic strength of 15 m M and p H of 7.0.On the basis of optimization,the competition inhibition curve was established,the IC₅₀ was 0.94 ng/m L,and the minimum detection limit(IC₁₀)was 0.14 ng/m L.The average recoveries of wheat and corn simples were 83.16%?87.49%and 86.45%?107.02%,the coefficients of variation were 2.86%?7.56%and 2.19%?5.80%,respectively.4.Establishment and validation of the immunoaffinity column-HPLC methodThe validation of immunoaffinity column-HPLC method was established.Under the national standard parameters,the retention time of T-2 toxin target peak detected by fluorescence detector was 12.627 min.There was a good linear relationship in the concentration range of 10?500 ng/m L,the limit of minimum detection and the limit of quantitative were 2.61 ng/m L and 8.70 ng/m L.The average recoveries of wheat and corn simples were 102.07%?114.77%,94.17%?106.96%,the coefficients of variation were 3.29%?5.45%,4.48%?9.88%,respectively.At the same time,the correlation between ELISA,CLEIA and HPLC are studied,and the correlation coefficients were R₁²=0.99862 and R₂²=0.99731,respectively.The T-2 toxin content of 9 grain samples were detected by the above three methods.As a result,except for glutinous rice,buckwheat and soybeans,the T-2 toxin content in other samples was not detected by HPLC,but ELISA and C LEIA were detected,which indicated that ELISA and CLEIA were more sensitive and cost-effective than HPLC and could be used for rapid screening of T-2 toxin in actual samples selection and detection.