Studies On The Preparation And Confirmation Of Monoclonal Antibody Against Diethylstilbestrol And Trial Production Of Its IAC Column

As a non-steroidal synthetic estrogen,Diethylstilbestrol(DES) is widely used in clinical treatment and livestock production.Having many adverse effects on human body,DES is strictly prohibited from using in food animals producing in many countrys and regions.However,it is still used illegally for economic interests.In order to guarantee safety of food and export trade in animal and aquatic products,it is necessary to establish a rapid and sensitive detection technology.During DES residue analysis,the sample pre-treatment is the key.Compared with traditional sample pre-treatment methods,Immunoaffinity chromatography(IAC),having good application prospect,is characterized by simple operation,high selective purification and enrichment capability.In this paper,DES complete antigen and monoclonal antibodies were prepared and confirmed based on immunology,and then IAC column was studied preliminary, which lay a foundation for the sample pre-treatment technology.The main results were as follows:1.Diethylstilbestrol was derivatived into semi-succinate(DES-HS) by succinic anhydride method.The best preparation conditions were that molar ratio of DES to HS was 1:10,tempreture was 45℃,and reaction time was 48 hours.Complete antigen were prepared according to methods of mixed anhydride and EDC catalyze and were confirmed by UV,IR,SDS-PAGE,and MS.The number of DES-HS bound to BSA and was 26.2.Ballb/C male mice of 6～8 weeks were immunized with the complete antigen by routine method.Immunological methods,dose,carrier proteins and times having effects on immune were considered.The results showed that back subcutaneous many points injection was better than intraperitoneal injection,immunologica dose(100/50 and 200/100μg per one) had no significant effect on immune,as carrier protein KLH was better than BSA,and four immunity had a good immune response.3.A non-competitive indirect ELISA method were established.The optimal application conditions were as following:Coated antigen DES concentration was 1000 ng/mL.Blocking buffer was 5%of skimmed milk powder.HRP-IgG was diluted 3000 times.Coated antigen, primary antibody and HRP-IgG should be incubated 2 h under 37℃.Blocking buffer should be incubated overnight under 4℃.The substrate was incubated at 37℃for 15 min.4.Cell fusion was carded by using PEG method.Through three times subcloning, Monoclone antibody(McAb) hybridoma cell 18G5 was obtained,which was able to product IgG3 antibody subtypes.McAb against DES was obtained through mice cultivation and affinity purification.The concerntrion of the purified McAb is 1.015 mg/mL,molecular weight is approximately 150 KD,titer is 10000,the best working concentration is 1000-fold dilution and affinity constant is 1.48×10⁹.DES inhibition standard curve is y=-0.2145x+0.4713,R²=0.9965, and linear range is 0.1～20 ng/mL,IC₅₀ is 0.735 ng/mL.The cross reaction rate with Hexestrol and Dienestrol was 0.37 and 0.98.5.The recovery ratio of DES in IAC column is 62.2%,which was prepared based on Sepharose 4B and McAb.