Preparation Of Zearalenone And Its Derivative Monoclonal Antibodies And Establishment Of Immunoassay Test

Zearalenone?ZEN?is mainly produced by Fusarium graminearum,a mycotoxin widelypresenting in cereals and food crops.There are many kinds of its derivatives,and there are five common ones.ZEN can cause pollution problems to food and has estrogen effects on animals,bringing a series of economic losses to the breeding industry.Therefore,it is necessary to establish a fast,simple and specific detection method.In this study,artificial antigens of ZEN were prepared.After immunizing animals,ZEN mAb was selected by cell fusion technology,and a ZEN immunoassay test method was established.Provided technical support for food safety testing and residue detection of ZEN in grain in China.The main research contents and conclusions are as follows:1.Chemically modify the ZEN molecule,introduce a group that can be coupled to thecarrier protein?-COOH?to synthesize the ester ZEN-CMO,and use the mixed anhydride method?MA?to combine the hapten ZEN-CMO with the carrier protein bovine serum albumin?BSA?)Coupling,synthesis of antigen ZEN-BSA;modification of ZEN molecule through oxime reaction between carboxymethylhydroxylamine hemihydrochloride and ZEN,synthesis of ester ZEN-CMO,using carbodiimide method?EDC?and carrier protein chicken Serum albumin?OVA?was coupled to synthesize coated antigen.The physical and chemical properties of artificial antigen ZEN-BSA were tested and identified by ultraviolet?UV?,infrared?IR?and protein gel electrophoresis?SDS-PAGE?.The test results showed that the artificial antigen was successfully synthesized;Female Balb/C mice were immunized to prepare polyclonal antibodies?pAbs?.The titers of immunized mice pAb reached 1?2.56×10⁵,and the indirect ELISA detected half the inhibitory concentration IC₅₀ value was 38.9 ng/mL.?-ZER and?-ZOL have a cross reaction rate of 100%,?-ZER has a cross reaction rate of 30%,ZAN has a cross reaction rate of 20%,and?-ZOL has a cross reaction rate of 10%.There was no cross-reactivity with AFB1,ABG1,ABG2,DON,T-2,FB1 and other mycotoxins.2.Immunize female Balb/C mice with synthetic artificial antigen ZEN-BSA,and useindirect ELISA and indirect competition ELISA to select cells for fusion.The cell fusion technique was used to fuse the spleen cells of mouse 3 with SP2/0 cells under the action of PEG-1500,screen positive hybridoma cell lines to prepare ZEN mAb,and analyze their immune characteristics.The results showed that after cell fusion,three hybridoma cell lines were selected and named as 1B12,3A5,4G10.Among them,the 3A5 strain was the best,and the karyotype identification of the three hybridoma cell strains is performed.The average number of chromosomes of the three hybridoma cell strains obtained was 98.7.The antibody prepared by in vitro culture method and in vivo induced ascites method has high titer.After 9 subcultures,the antibody secretion is stable,the cell culture supernatant titer was 1?128,the ascites antibody titer was 1?5.12×10⁵,IC₅₀ was 7.93 ng/mL.ZEN can be recognized 100%,and the cross-reaction rates with?-ZER,?-ZOL,?-ZER,?-ZOL,and ZAN were 100%,100%,22.91%,16.28%,and 9.42%,respectively.The affinity constant of the ZEN mAb secreted by the hybridoma cells of 3A5 strain was 6.79×10⁹ L/mol,indicating that the hybridoma cell strain has a high affinity.A ZEN mAb with high titer,good sensitivity and strong specificity was prepared,which laid the foundation for the establishment of ZEN immunoassay test method.3.The ZEN ELISA kit was prepared using ZEN monoclonal antibody 3A5.After theoptimization of the kit,the linear regression equation curve was good.The regression equation was y=-41.014x+111.14,R² was 0.9809,IC₅₀ was 30.9 ng/mL,detection The range was 5.75¹65.96 ng/mL.With good accuracy and precision,it can be stored at room temperature for one year or more.The colloidal gold was prepared by using trisodium citrate reduction method with chloroauric acid as the material,and the antibody was labeled by the Mey's series stabilization method to establish the detection method of ZEN colloidal gold immunoassay paper.After scanning by ultraviolet?UV?and transmission electron microscopy,the test results showed that the particle size of colloidal gold was 25±1.0 nm,indicating that the colloidal gold was successfully prepared;the optimal labeling concentration of ZEN mAb was determined to be 4.5?g/mL;ZEN immunocolloid gold test strip?ZEN-Strip?,ZEN-Strip has fast?5-10 min?,sensitive?3.91 ng/mL?,specific?only reacts with ZEN,no CR with other biological toxins?,broad spectrum?It can simultaneously detect its derivatives?-ZER,?-ZER,?-ZOL,?-ZOL,ZAN?,simple?no need for other instruments and reagents?and other advantages,has good market application value and development prospects.