Preparation Of Monoclonal Antibody Against Diethylstilbestrol And Establishment And Application Of The ELISA Method For Detection

Diethylstibestrol (DES) is an artificial estrogen. It has a good effect in improving the metabolism of protein, the growth of the animal and decreasing the fat, so it was used in the feeding of the animal. The International Agency for Research on Cancer (IARC) has found that DES can lead to cancers, so it was ruled strictly not to be used in feeding animals by any way in many countries and regions. However, it is still used illegally for economic interests.Now, many physical and chemical methods to detect residue of DES have been established at home and abroad. Some methods are precise and sensitive, but not suitable for large batch samples’screening, because of their complication, long time consuming, big cost and high technical request. So it is necessary to establish a rapid and sensitive detection technology. Enzyme-linked immunosorbent assay (ELISA) is gradually being used to detect the residues of medicines, as a method which is sensitive, fast, powerful specificity and can screen a large number of samples at one time.In this paper, DES man-made antigen and anti-diethylstibestrol monoclonal antibody were composed. Finally anti-diethylstilbestrol monoclonal antibody was achieved and ELISA method to detect residue of diethylstilbestrol was established, using monoclonal antibody technique and ELISA technique.The haptens Diethylstilbestrol-mono-carboxylic propyl ether (DES-MCPE) which was synthesized and purified were covalengtly conjugated to carrier proteins BSA and HSA by the method of active ester or mixed anhydride to synthesis DES artificial antigens DES-MCPE-BSA and DES-MCPE-HSA respectively. The conjugates were demonstrated to be successful by UV-scaning and immunological methods.Balb/C female mice of6～8weeks 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 gradually strengthen the immune by back subcutaneous injection was better than the same dose of immune, the first immunization dose of100μg per one was better than50μg, as carrier protein BSA was better than HSA, and five immunity had a good immune response.One of the antigens named DES-MCPE-BSA synthesized by active ester method was used as an immune antigen while DES-MCPE-HSA composed via mixed anhydride was used for detection. The titer of blood serum collected after the fifth immunization reached1:256,000. Then a hybridoma cell line named2F6which could excrete anti-diethylstilbestrol monoclonal antibody steadily by hybridoma’s technique was received and then ascetic fluid was prepared. The protein concentration of ascetic fluid was37.4mg/mL, the titer of the McAb was1:512,000.The CiELISA’s method was established by the monoclonal antibody in the abdominal dropsy which was got by injecting hybridoma cells2F6into BALB/c mouse. The optimum conditions were established through the matrix test. The coating antigen was attenuated by1:32,000, the monoclonal antibody in the abdominal dropsy was attenuated by1:64,000. Under optimized conditions, the standard ELISA inhibition curve of DES to antibody-antigen reaction was developed and the regression equation was Y=-0.4657x+0.7373(R2=0.9925). The half-maximal inhibition (IC50) was4.16ng/mL. The cross reaction experiment showed that the very high specificity of the McAb for DES was expressed with endogenous estrogen and other compounds and drugs. The cross reaction rate with Hexestrol and Dienestrol was2.14%and1.46%.This method was initially applied in the addition and reclamation test of the liver tissue and muscle tissue of swine. The sample was to be determined after pre-processing. The intervention disappeared when the extraction of liver and muscle sample was diluted8times. The standard curve equation was Y=-0.3949x+0.7137(R2=0.9935) between0-50ng/mL. The recovery ratio were between89.7%～105.9%when adding1,5and10ng/mL DES to the liver tissue of swine. The intraassay coefficient of variation was between4.94%-8.24%, and the interassay coefficient of variation was between5.33%～6.85%.