Preparation Of Monoclonal Antibody Against β-zearalenol And Preliminary Establishment Of ELISA Assay

β-Zearalenol(β-Zearalenol,β-ZOL)is one of the major derivatives of zearalenone(ZEA)metabolism in animals.β-ZOL like ZEA,have estrogen-like activity,the animals have a strong reproductive toxicity,but also has cytotoxicity,immunotoxicity and genotoxicity.β-ZOL is also one of the main residues of ZEA in animals,which directly affects the quality and the safety of animal-derived foods.Therefore,it is necessary to establish a rapid and accurate method for the detection of p-ZOL in order to detect the residues of p-ZOL in animal-derived foods.In this experiment,the small molecμLe β-ZOL was conjugated with the protein carrier by the active ester method to synthesize the complete antigen,after the purification,the BALB/c mice were immunized.The monoclonal antibody with high titer and specificity for β-ZOL was prepared by the monoclonal antibody preparation and enzyme-linked immunosorbent assay(ELISA).Based on this,an indirect competitive ELISA(CiELISA)detection method for p-ZOL was established.1.Synthesis and identification of artificial complete antigen of β-ZOL.β-ZOL belongs to the hapten.Its molecμLar structure does not contain carboxyl group and amino group.In this experiment,the β-ZOL molecμLar structure was modified by chemical reaction,the carboxyl group was introduced.The complete antigen was synthesized by the method of the active ester with the modified hapten P-ZOL combined with macromolecμLe carrier protein BSA and OVA.The compounds were identified by UV scanning,SDS-PAGE and immunological methods.The resμLts showed that β-ZOL was successfμLly coupled with the carrier protein.the concentration of synthetic antigen was detected by BCA protein concentration assay kit.As a resμLt,the concentrations of the synthesized β-ZOL-BSA and β-ZOL-OVA were 2.6 mg/mL and 3.2 mg/mL,respectively.2.Preparation of monoclonal antibody against P-ZOL.β-ZOL-BSA was used as the immunogen,β-ZOL-OVA as the coating original.The female BALB/c mice which were 6 to 8 weeks old were immunized according to routine immunization procedures.Severn days after the fifth immunization,the serum titer of mice was more than 1:10000.The third days after strengthen immunization,the spleen cells of the mouse with the highest immunogenicity were fused with SP2/0 myeloma cells.A strain of 1F5 was obtained which stable secreted the anti-β-ZOL antibody after screening by ELISA and limited dilution.The ascites was prepared by in vivo induction method in mice,the ascites of anti-β-ZOL antibody was prepared by using 1F5 hybridoma cell line,which was a total of 11mL,and the protein concentration of the ascites was 44.48 mg/mL.3.The establishment of CiELISA detection method.The establishment of indirect competitive ELISA method for detection residues of β-ZOL by 1F5 monoclonal antibody.The optimum dilution concentration of the coating was 1:1000,the working concentration of the antibody was 1:8000,the drug inhibition standard curve of β-ZOL showed that when the concentration is in 5～500ng/mL,the linear relationship of standard curve was good,the linear equation was y=0.4063x-0.2042(R2=0.9904),IC50 was 53.19 ng/mL,LOD was 6 ng/mL,LOQ was 26.39 ng/mL.The resμLts of monoclonal antibody specificity test showed that,the monoclonal antibody had 100%inhibition rate for β-ZOL,and it had no cross reaction with ZEA,p-estradiol,vomitoxin and fumonision B1.4.Recovery test.We conducted the addition and recovery test of P-ZOL in milk.The interference of sample matrix mainly disappeared when the milk sample was diluted for 8 times,in the range from 5 to 500ng/mL,the linear equation was y=0.3749x-0.1668,(R2= 0.9949),IC50 was 59.35 ng/mL,LOD was 6.57 ng/mL,LOQ was 57.47 ng/mL.the recovery rate of β-ZOL was between 91.24%and 110.07%,and the coefficient of variation was between 6.17%and 8.73%,when adding p-ZOL which the concentration were 10,100 and 400 ng/mL to milk.Conclusion:The monoclonal antibody of β-ZOL was prepared by monoclonal antibody preparation and ELISA.The CiELISA method was established for detecting P-ZOL.The specificity of the prepared monoclonal antibody was good,and the detection range was 5～500ng/mL,IC50 was 53.19 ng/mL.The resμLts show that the CiELISA method for detectingβ-ZOL is stable and reliable.