| Diethylstilbestrol (DES) is a synthetic estrogen. It can produce the effects likenatural estrogens in the bodies of human beings and animals. DES was ruled not touse in food animals by any way and was not detected in animals and animal foodsbecause it can lead to cancer and deformation for humanity. The immunoassay ofresidual DES is one of the critical means in monitor of DES residues. The maincontents and experimental results of this study were as follows: The synthetic method of DES-HS was founded. In the condition of no light andat the room temperature, DES was mixed with definite quantity succinate anhydridein anhydrous pyridine, stirring 34h and placing in acid circumstance for one night,some light brown oil substances were extracted. The crude products?light browndepositions were obtained by filtering, after this crude products were purified andidentified by NMR, IR, high-resolution mass spectroscopy, the results indicated thatthe molecular mass of this brown substance is 368, the molecular type is C22H24O5, Ithas some critical groups such as phenol-hydroxy and groupcarboxy gruop. Which isthe derivative of DES-DES-HS. DES-HS was bonded to the carrier proteins (OVA, BSA) by the mixed acidanhydride method, artificial antigen of DES was obtained, which were examined byultraviolet ray scanning and SDS-PAGE, the results showed that the bond wassuccessful; and the labworks 4.0 Image Acquisition and Analysis software in UVPBioimaging system was used to assay and figure out that the bond rate of BSA andOVA with DES-HS were 1:30～32 and 1:25~28. Rabbits were immunized by the synthetic DES-HS-BSA, and after six timesstrengthening immunization, Agar double-diffusion experiment was used todetermined the specificity and titer of anti-DES antibody in immune serum. Themethod of indirect ELISA was also built which was used to determined the specificbond reaction of the anti-DES antibody in immune serum with the dissociate DES.The results showed that: in agar double-diffusion experiments, there were cleardeposit lines, and the deposit line between immune serum and BSA-HS-DES wasthicker than that of immune serum and DES-HS-OVA, the titer of anti-DES antibodyin immune serum was 22.5～22.6. After the optimal condition of the determination ofindirect ELISA was found out and the results manifested that: in the two group rabbits,the immune serums were respectively reacted with DES-HS-OVA and OVA, theOD490nm were 3.459±0.045,3.738±0.136 and 0.235±0.080,0.131±0.007; Thetiters of the two immune serums both were 1:6000. After confirmed the optimal diluteconcentration of which was 1:100, the indirect ELISA experiment indicated that thedissociate DES can specifically react with immune serum, and with the increase ofdissociate DES, the quantum of antibody in immune serum which can react withDES-HS-OVA was reduced continuously. All the results demonstrated that: afterrabbits were immunized by DES-HS-BSA which can stimulated the immune systemand produce anti-DES determinant antibody, which can not only recognizespecifically the DES determinant of artificial antigen such as DES-HS-BSA﹑DES-HS-OVA, but also can recognize and react specifically with dissociate DES. After Balb/c mice was immunized by DES-HS-BSA 4 times, which wasmeasured by indirect ELISA, the results showed that the titer of anti-DES antibody inimmunized mice all was in excess of 1:8×103 in 13 mice. The spleencytes of twoimmunized mice that the immunized effects was best were taken out,which was fusedwith sp2/0 cell in PEG. After filtration and 3 times cloning, two strains hybridomawhich grew stably and secreted anti-DES monoclonal antibody higher were obtainedand named respectively 3A8 and 4E6; the titers of anti-DES monoclonal antibody wasdetermined by indirect ELISA and the results showed that the titers in cell culturesupernatants and ascites of 4E6 were respective 1:6×104,1:1×107, and that of 3A8were 1:4×104,1:1×107. The anti-DES monoclonal...
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