目的 合成并鉴定猪去氧胆酸(hyodeoxycholic acid,HDCA)人工抗原,为进一步制备其特异性抗体,建立相应的免疫分析方法,并为其应用到药理药效的研究中奠定基础.方法 采用混合酸酐法合成HDCA人工抗原(HDCA-牛血清白蛋白),包被原(HDCA-鸡卵清白蛋白);应用硅胶薄层色谱法鉴定人工抗原、包被原合成是否成功;应用人工抗原与弗氏佐剂的乳化液背部皮下多点注射免疫BALB/c小鼠4次后,尾尖取血,通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)方法测定免疫小鼠抗血清的效价和特异性.结果 薄层色谱法检测结果表明,HDCA与牛血清白蛋白(BSA)偶联成功;小鼠抗血清效价大于1:8 000,竞争抑制曲线表明,血清中的抗体可与HDCA产生特异性结合.抗血清和甘草酸、黄芩苷、栀子苷等与其常配伍应用的中药化合物均无交叉反应,但与胆酸交叉反应率为90%.结论 成功合成了HDCA-BSA,并可诱发小鼠产生相应特异性抗体.为进一步制备抗HDCA单克隆抗体及HDCA药理作用靶点的研究奠定了基础.

Objective To synthesize and identify the hyodeoxycholic acid (HDCA) artificial antigen and to lay a foundation for the preparation of its specific monoclonal antibody, establishment of immunoassay method, and application in the pharmacological researches. Methods The HDCA artificial immunogen (HDCA-BSA) and coating antigen (HDCA-OVA) were synthesized by mixed anhydride method and then examined by TLC method. BALB/c mice were immunized with the emulgator containing the prepared HDCA-BSA conjugate and Freund's adjuvant by the back sc multipoint injection for three times following with a booster immunization by the prepared HDCA-BSA conjugate alone. Then the blood of mice was obtained from tail vein 24 h after the booster, and the sera were isolated for the titer and specificity test by ELISA. Results According to TLC, HDCA was successfully conjugated to BSA. The antibody against HDCA obtained from immunized-mice could bind to HDCA and the titer was over 1:8 000. As shown in the competitive inhibition curves, the antibodies in the serum were specifically bound to HDCA without cross-relativities with glycyrrhizic acid, baicalin, geniposide, etc, except cholic acid (90%). Conclusion The HDCA-BSA is successfully synthesized, which could be used for the preparation of anti-HDCA monoclonal antibody and the research on pharmacological targets of HDCA.

国家自然科学基金资助项目(81274043)