王咏枝 屈会化 邢洪霞 张越 成金俊 熊威 罗娟 赵琰 孔慧 王庆国

摘要：目的 合成大黃酸人工抗原，制备敏感性高、特异性强的大黄酸兔源多克隆抗血清。方法 采用碳二亚胺法制备大黄酸人工抗原，将大黄酸分别与牛血清白蛋白和卵清蛋白相偶联。用紫外扫描法鉴定大黄酸人工抗原是否偶联成功，免疫新西兰兔，制备多抗血清，通过间接竞争ELISA测定抗血清效价，通过间接竞争ELISA鉴定抗血清的敏感性和特异性，并分别用间接竞争ELISA和HPLC检测大黄中大黄酸的含量。结果 2只新西兰兔产生的多抗血清效价在1∶64 000以上，其中，2号兔多抗血清敏感性最好，半数抑制浓度为0.09 ng/mL，且具有良好的特异性。兔抗血清测得大黄中大黄酸含量为（0.026±0.001）%，HPLC测得大黄中大黄酸含量为（0.027±0.000）%。结论 本研究制备了敏感性高、特异性强的大黄酸兔源多克隆抗体血清，为建立大黄酸免疫亲和色谱分析技术和快速检测方法奠定了基础。

关键词：大黄酸；人工抗原；碳二亚胺法；兔源多抗血清；酶联免疫分析

DOI：10.3969/j.issn.1005-5304.2018.05.010

中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2018）05-0041-05

Preparation and Identification of Rhein Rabbit Polyclonal Antiserum

WANG Yong-zhi1， QU Hui-hua2， XING Hong-xia3， ZHANG Yue1， CHENG Jin-jun4，

XIONG Wei1， LUO Juan1， ZHAO Yan4， KONG Hui4， WANG Qing-guo4

1. School of Chinese Materia Medica， Beijing University of Chinese Medicine， Beijing 100029， China；

2. Beijing Institute of Traditional Chinese Medicine， Beijing University of Chinese Medicine， Beijing 100029， China； 3. Qufu Traditional Chinese Medicine Hospital， Qufu 273100， China；

4. School of Traditional Chinese Medicine， Beijing University of Chinese Medicine， Beijing 100029， China

Abstract： Objective To synthesize rhein （RHE） artificial antigens； To prepare high sensitivity and specificity polyclonal antiserum against RHE. Methods RHE was coupled respectively to bovine serum albumin （BSA） and ovalbumin （OVA） by using carbodimide two-step method. The RHE artificial antigen was identified by UV spectrophotometry， then it was injected to rabbits for antiserum preparation. Anti-serum titer was determined by indirect ELISA. Sensitivity and specificity were identified by indirect competitive ELISA. And the contents of RHE of Rhei Radix et Rhizoma were detected by indirect competitive ELISA and HPLC. Results Indirect ELISA showed a high titer above 1∶64 000. The sensibility of No.2 antiserum was better， which IC50 was 0.09 ng/mL and had good specificity. The content of RHE in Rhei Radix et Rhizoma was （0.026±0.001）% detected by rabbit polyclonal antiserum， and the content of RHE was （0.027±0.000）% detected by HPLC. Conclusion In this study， RHE artificial antigens is synthesized successfully. RHE rabbit polyclonal antiserum with high sensitivity and specificity is prepared， and these may provide basis for the immune affinity chromatography analysis technology and immunology fast detection methods.

Keywords： rhein； artificial antigen； carbodiimide two-step method； rabbit polyclonal antiserum； ELISA

基金项目：国家自然科学基金重点项目（81430102）

通讯作者：王庆国，E-mail：wangqg5858@sina.com

大黄酸（rhein，RHE）属单蒽核类1，8-二羟基蒽醌衍生物，是大黄、虎杖、何首乌等多种中药的主要有效成分[1]，也是大黄药材的质控指标之一。现代药理研究表明，大黄酸具有抗肿瘤[2]、抗菌[3]、抗炎[4]、降糖调脂[5]、保肝抗纤维化[6]等多种活性，在治疗糖尿病肾病及协同抗肿瘤方面表现尤其突出[7]。目前大黄酸常用检测方法为HPLC[8-9]，此方法依赖高精密仪器，分析成本高；……