中国中药杂志

2015, v.40(22) 4359-4363

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木犀草苷人工抗原合成及免疫原性鉴定
Preparation and immunogenicity identification of artificial antigen for luteoloside

张波;詹志来;康利平;袁媛;黄璐琦;丁凤伟;南铁贵;
ZHANG Bo;ZHAN Zhi-lai;KANG Li-ping;YUAN Yuan;HUANG Lu-qi;DING Feng-wei;NAN Tie-gui;College of Pharmacy,Shan Dong 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;Shandong C.P.Freda Pharm Co.,Ltd.;

摘要(Abstract):

金银花是常用大宗中药材之一,市场流通量大,但存在较多质量问题,建立一种金银花质量快速检测方法具有重要意义。木犀草苷是其质量控制指标之一,目前木犀草苷的含量检测主要以仪器检测为主,无法满足简单、快速、高通量的需求。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,是实现中药材有效成分快速检测的有效手段之一。该研究以木犀草素-7-O-β-D-葡萄糖醛酸苷为原料,通过活泼酯法,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;紫外分光光度法检测半抗原与载体蛋白的偶联比,分别为3.7∶1(LG-BSA)和1.0∶1(LG-OVA);免疫原(LG-BSA)免疫Bal b/c雌性小鼠,制备抗血清,ELISA检测抗血清效价并建立血清特异性标线。结果显示血清效价>2 000,以抗血清建立特异性标线,IC_(50)=298.7μg·L~(-1),标线范围18.4~4 852.4μg·L~(-1),表明所制备的完全抗原免疫原性良好。
Lonicerae Japonicae Flos was one of the most widely used traditional Chinese medicine for its special biological activities. The content of luteoloside,one of its major compounds,was an important standard for the quantity control of Lonicerae Japonicae Flos. The major method used for the detection of luteoloside was instrumental analysis. Compared with the ELISA method,instrumental analysis was time-consuming,complex pretreatment and low-throughout. Thus,it was significantly important to develop an enzymelinked immunosorbent assay( ELISA) for luteoloside analysis. Here,the conjugates of luteoloside-bovine( LG-BSA) and luteolosideovalbumin( LG-OVA) were produced as the immunogen and coating antigen by the carbodiimide( CDI) method,respectively. The conjugation ratio of carrier protein and the hapten in the conjugate were determined by UV-Vis spectrophotometry( UV). LG-BSA conjugate was used to immunize Bal b / c mice to produce antiserum. The titer and specificity of antiserum were detected by ELISA. The conjugation ratio of hapten and carries protein were 3. 7 ∶ 1( LG-BSA) and 1. 0 ∶ 1( LG-OVA). The antiserum titer was higher than2 000 with the linear range of 18. 4-4 852. 4 μg·L~(-1),R~2= 0. 988 4 and IC50= 298. 7 μg·L~(-1). The result showed that the conjugate antigen LG-BSA was synthesized successfully and the mice can produce specific antiserum injected with artificial antigen.

关键词(KeyWords): 木犀草苷;人工抗原;免疫原性;酶联免疫吸附检测法
luteoloside;artificial antigen;immunogenicity;ELISA

Abstract:

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基金项目(Foundation): 国家自然科学基金项目(81373959);; 国家中医药行业科研专项(201407003)

作者(Author): 张波;詹志来;康利平;袁媛;黄璐琦;丁凤伟;南铁贵;
ZHANG Bo;ZHAN Zhi-lai;KANG Li-ping;YUAN Yuan;HUANG Lu-qi;DING Feng-wei;NAN Tie-gui;College of Pharmacy,Shan Dong 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;Shandong C.P.Freda Pharm Co.,Ltd.;

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