盐酸可乐定人工抗原及其鼠源多抗的制备

Preparation of Clonidine Hydrochloride Artificial Antigen and Its Mouse Polyclonal Antibody

以盐酸可乐定(Clonidine hydrochloride,CLO)的衍生物盐酸阿可乐定(Apraclonidine hydrochloride,ACLO)为半抗原,采用重氮化法合成人工抗原,免疫小鼠制备高敏感性、特异性的CLO鼠源多抗并进行鉴定,为CLO单克隆抗体的制备奠定基础。将ACLO分别与牛血清白蛋白(Bovine serum albumin,BSA)和鸡卵清蛋白(Ovalbumin,OVA)偶联,合成免疫原CLO-BSA和包被原CLO-OVA,经紫外扫描和SDS-PAGE凝胶电泳鉴定偶联效果后,免疫BALB/c小鼠,并对获得的鼠源多抗采用间接ELISA和间接竞争ELISA鉴定其特性。结果显示,免疫的4只小鼠血清抗体效价均达到1∶25600以上,其中2号小鼠的抗体敏感性最高,半数抑制质量浓度(IC50)为7.026 ng/mL,与其他几种常见瘦肉精类药物的交叉反应率均小于0.1%,特异性良好。表明成功合成免疫原性良好的CLO人工抗原,并获得敏感性高、特异性强的CLO鼠源多抗,为其单克隆抗体的制备及免疫学快速检测方法的建立奠定良好基础。

In this study,apraclonidine hydrochloride(ACLO),a derivative of clonidine hydrochloride(CLO),was used as hapten to synthesize artificial antigen by diazotization method.Polyclonal antibody with high sensitivity and specificity was prepared from immunized mice and was identified,which will lay the foundation for the preparation of CLO monoclonal antibody.ACLO was conjugated with bovine serum albumin(BSA)and ovalbumin(OVA)to synthesize immune antigen CLO-BSA and coating antigen CLO-OVA.The conjugation effect was identified by UV scanning and SDS-PAGE.After that,BALB/c mice were immunized,and the reactivity of polyclonal antibody was identified by indirect ELISA and indirect competitive ELISA.The results indicated that the polyclonal antibody titer of four mice were more than 1∶25600.The antibody sensitivity of No.2 mouse was the best among the immunized mice,which half inhibitory concentration(IC50)was 7.026 ng/mL.The cross reactivity with other lean meat powder drugs were all less than 0.1%.In this study,CLO artificial antigen with good immunogenicity was successfully synthesized,and CLO mouse polyclonal antibody with high sensitivity and specificity was obtained,which laid a solid foundation for the preparation of monoclonal antibody and the establishment of rapid immunological detection method.