中国中药杂志

2015, v.40(08) 1463-1467

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大黄酸人工抗原合成及免疫原性鉴定
Preparation and identification of artificial antigen for rhein

张波;袁媛;黄璐琦;王保民;南铁贵;
ZHANG Bo;YUAN Yuan;HUANG Lu-qi;WANG Bao-min;NAN Tie-gui;College of Pharmacy,Shandong University of Traditional Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;College of Agronomy and Biotechnology,China Agricultural University;

摘要(Abstract):

大黄药理作用广泛,是临床常用中药材之一。大黄酸是其主要活性成分,大黄酸的含量是大黄质量控制的主要指标之一。目前对大黄酸的检测主要是通过仪器分析手段,如高效液相色谱法,但是仪器分析通常操作繁琐、耗时长、通量小且仪器设备昂贵。与之相比,酶联免疫检测法(ELISA)则具有灵敏度高、操作简单、设备仪器需求少、通量高等优点,是实现中药材快速检测的有效手段之一。该研究制备大黄酸(rhein)人工抗原及血清,为之后大黄酸单克隆抗体制备及建立酶联免疫吸附检测方法提供基础。通过碳二亚胺法活化大黄酸,分别与牛血清白蛋白(BSA)及鸡卵清白蛋白(OVA)偶联制备免疫抗原及包被抗原;通过紫外分光光度法检测人工抗原的半抗原与载体蛋白偶联比,分别为4.0:1(rhein-BSA)和2.6:1(rhein-OVA)。用免疫原免疫Bal b/c雌性小鼠,制备抗血清,通过酶联免疫吸附法(ELISA)检测抗血清效价及特异性。结果表明血清效价均高于8 000,特异性强,免疫原性良好。
The Rhei Radix et Rhizoma was one of the most widely used traditional Chinese medicine for its special biological activities.The content of rhein,one of its major compounds,was an important standard for the quantity control of Rhei Radix et Rhizoma.The major method used for the detection of rhein was instrumental analysis like HPLC,but it was complex,time-consuming and cannot detect large samples at the same time.The enzyme-linked immunosorbent assay(ELISA) was accurate,reliable,simple,low costs,and of a high-throughout.Recently,it was widely used for the determination of those small molecule compounds in some traditional Chinese medicinal plants.In this study,an artificial antigen were synthesized by the carbodiimide(CDI) method.Rhein-bovine(rhein-BSA) conjugate and rhein-ovalbumin(rhein-OVA) conjugate,were produced as the immunogen and coating antigen,respectively.The conjugate and the hapten number in the conjugate were determined by UV-Vis spectrophotometry(UV).The conjugation ratio of Rhein and BSA was about 4.0:1,rhein acid and OVA was 2.6:1,respectively.Rhein-BSA conjugate was used to immunize Bal b/c mice to produce antiserum.The antiserum titer of the Rhein were higher than 8 000 detected by ELISA.The successfully synthesized conjugate antigen rheinBSA implies its feasibility in the establishment of fast immunoassay for the rhein content determination.

关键词(KeyWords): 大黄酸;人工抗原;免疫原性;酶联免疫吸附检测法
rhein;artificial antigen;immune effect;ELISA

Abstract:

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基金项目(Foundation): 中医药行业科研专项(201407003)

作者(Author): 张波;袁媛;黄璐琦;王保民;南铁贵;
ZHANG Bo;YUAN Yuan;HUANG Lu-qi;WANG Bao-min;NAN Tie-gui;College of Pharmacy,Shandong University of Traditional Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;College of Agronomy and Biotechnology,China Agricultural University;

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