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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 hyodeoxycholic acid (HDCA) artificial antigen, and to further prepare its specific antibody, a corresponding immunoassay method was established and laid the foundation for its application to the study of pharmacological effects. Methods HDCA artificial antigen (HDCA-bovine serum albumin) and coated antigen (HDCA-chicken ovalbumin) were synthesized by mixed acid anhydride method. The artificial antigen was identified by silica gel thin layer chromatography, BALB / c mice were immunized subcutaneously with Freund’s adjuvant emulsion 4 times after subcutaneous injection, the blood was collected from the caudal tips, and the antiserum was determined by enzyme-linked immunosorbent assay (ELISA) The potency and specificity. Results The results of TLC showed that the coupling of HDCA with bovine serum albumin (BSA) was successful. The titer of mouse antisera was more than 1: 8000. The competition inhibition curve showed that the antibodies in serum could specifically bind with HDCA . Antiserum and glycyrrhizic acid, baicalin, geniposide and other Chinese herbal compounds often do not cross-react with the application, but the cross-reactivity with cholic acid was 90%. Conclusion HDCA-BSA was successfully synthesized and the corresponding specific antibodies were induced in mice. Which laid the foundation for the further preparation of anti-HDCA monoclonal antibody and HDCA pharmacological targets.