己烯雌酚人工抗原的合成及多克隆抗体的制备

Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies

目的:合成己烯雌酚人工抗原,制备效价高、特异性好的己烯雌酚多克隆抗体。方法:以己烯雌酚(DES)、溴乙酸乙酯、牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)等作为合成的主要原料,采用亲核取代反应,合成己烯雌酚衍生物(DES-HS),用电喷雾电离质谱(ESI-MS)鉴定合成是否成功;然后,将DES-HS与BSA(OVA)偶联合成人工抗原,并用紫外扫描法以及高效液相色谱鉴定人工抗原是否制备成功。最后,用DES人工抗原动物免疫以获得DES多克隆抗体。结果:成功合成了偶联率为22∶1的己烯雌酚人工抗原。利用已合成的人工抗原制备出了效价为1∶25 600、IC50为10.81 ng/ml的己烯雌酚多克隆抗体。结论:建立了己烯雌酚人工抗原合成和鉴定以及制备己烯雌酚多克隆抗体的方法。

Objective： To synthesize artificial diethylstilbestrol （DES） antigen and to prepare DES polyclonal antibody with high titer and sensitivity. Methods： The derivative of DES （DES-HS） was synthesized from diethylstilbestrol, ethyl bromoacetate, bovine serum albumin （BSA） and chicken ovalbumin （OVA） with the nueleophilic substitution reaction; the compound was identified by eleetrospray ionization mass spectrometry（ESI-MS）. The DES-HS and the carrier proteins （BSA, OVA） were cross-linked to prepare the artificial antigen; the UV absorption spectrophotometry and high performance liquid chromatography （HPLC） were used to identify the prepared artificial antigen. The rabbits were immunized with the DES artificial antigen to prepare the DES polyclonal antibodies. Results： The DES-HS was synthesized. The DES artificial antigen was prepared successfully with a coupling rate of 22： 1. The DES polyclonal antibodies with a titer of 1 ： 25 600 and ICs0 of 10.81 ng/ml were prepared with DES artificial antigen. Conclusion： A set of methods to synthesize DES artificial antigen and to prepare the DES polyclonal antibodies has been developed successfully.