Preparation Of Monoclonal Antibody Against Tenuazonic Acid And Research Of Immunoassay

Tenuazonic acid（TeA）has many side effects such as carcinogenesis,which toxicity is the first toxin of Alternaria mycotoxin.Recently,it has been detected in various agricultural products and foods such as grains,fruits and vegetables,seriously endangering people’s health.Therefore,it is important to strengthen the detection method of TeA in food.Instrumental analysis method has the advantages of high sensitivity and good accuracy,but it can not meet the requirements of fast,simple and low cost for modern food safety.Immunoassay has become a research hots pot in the field of TeA detection because of its sensitivity,rapidity and high throughput,which can meet the needs of rapid food safety screening.This thesis takes TeA as the research object,aiming to prepare TeA monoclonal antibody and establish two immunoassay methods for the detection of TeA.The main research contents are as follows:1.Synthesis and identification of TeA hapten and artificial antigenIn this study,five kinds of TeA haptens were summarized,and the single point energy,optimal spatial conformation and molecular orbital of each hapten were calculated by molecular simulation technology.TeA,hapten 1 and hapten 2 were selected and synthesized,among which hapten 1 and hapten 2 were prepared by TeA molecular derivatization.Four kinds of immunogen and four kinds of coated antigen were prepared by coupling with carrier proteins（bovine serum albumin and ooalbumin）by different methods.The artificial antigens were identified by UV and SDS-PAGE.2.TeA monoclonal antibody was prepared and ic-ELISA assay was establishedAfter cell fusion,hybridoma cell screening and subclonal screening,two hybridoma cell lines 3C7 and4F7 with stable antibody secretion were obtained.4F7 was selected to prepare ascites and purify monoclonal antibody of TeA.An ic-ELISA was established based on monoclonal antibody for TeA.The optimal reaction conditions for ic-ELISA were as follows:concentration of TeA-OVA-CDI was 0.36μg/m L,concentration of monoclonal antibody was 0.45μg/m L,diluent of TeA stock solution was 0.01 M PBS（p H7.4,0.2%Na Cl）.The reaction time of competitive response was 40 min,The secondary reaction with the enzyme-labeled conjugate was allowed durinng 30 min,and the color developing reaction at 15 min.Under the conditions above,The IC₅₀value for TeA was estimated to be 20.50 ng/m L with the linear detection range(IC₂₀-IC₈₀)of 3.30 to 165.00 ng/m L,and the limit detection of the assay was 1.20 ng/m L.There was no crossing reaction with AOH,AME,AFB1 and OTA.The recoveries of tomato,grape,wolfberry and jujube were 88.78%to 117.53%.3.Establishment of a colloidal gold immunochromatographic strip for TeA detectionColloidal gold nanoparticles were prepared by sodium citrate reduction method and gold-labeled antibody probes were prepared.The experimental conditions of colloidal gold immunochromatographic strips were optimized.The optimal conditions were as follows:The p H of the gold-labeled probe was 2μL0.1 M K₂CO₃per m L colloidal gold,the antibody addition was 6μg antibody per m L colloidal gold,the dilution ratio of the gold-labeled probe was 3×and the concentration of T-line coating antigen was 0.25mg/m L.The performance of the strip detection method was evaluated under optimal conditions,and the standard curve obtained by Imag J analysis was y=5665.06-9.89x（R²=0.98）.The linear detection range(IC₂₀～IC₈₀)was 105.18 to 455.90 ng/m L,the limit of detection(IC₁₀)was 46.73 ng/m L,and the test strip had good specificity and no cross-reaction with AOH,AME,AFB1 and OTA.The recoveries of tomato,grape,wolfberry and jujube were 90.60%to 100.23%.