基于免疫指纹图谱法筛查双黄连注射剂的致敏成分
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摘要
该文采用免疫指纹图谱法,以酶联免疫吸附法(ELISA)结合HPLC/MS法筛查双黄连注射剂(SHLI)的致敏性成分,制备SHLI的鼠抗兔血清免疫球蛋白E抗体(抗Ig E抗体),通过ELISA包埋抗Ig E抗体可成功吸附SHLI及其不同给药途径的含药血浆样品中的致敏原,提示SHLI可引起大鼠产生Ⅰ型超敏反应。建立抗Ig E抗体吸附前后各样品的HPLC指纹图谱和质谱图,由HPLC指纹图谱的相似度和MS图谱归纳结果可知,不同给药方式可改变SHLI的致敏性,SHLI及其含药血浆中可被特异性抗Ig E抗体吸附的成分有22种,多为酸类、酯类及含氮化合物,基于超分子理论推断这些酸类、酯类及含氮化合物来源于SHLI或机体,可能形成超分子半抗原,致使其口服不过敏而作为注射剂使用时具有免疫毒性引发过敏反应。比较传统的中药注射剂致敏原筛查方法,免疫指纹图谱法不仅限于单成分致敏原筛查,该方法纳入成分更全面,灵敏度高,操作方便,可为今后中药注射剂过敏反应的研究方法提供参考。
In this paper,immune fingerprint was used to screen the allergenic components of Shuanghuanglian Injection(SHLI) by enzyme-linked immuno sorbent assay(ELISA) combined with HPLC/MS method. ELISA-embedded anti-IgE antibody could successfully adsorb allergens in SHLI and its plasma samples containing drugs through different routes of administration,suggesting that SHLI can induce type I hypersensitivity in rats. HPLC fingerprints and MS map of SHLI and drug-containing plasma samples from different routes of administration before and after anti-IgE antibody adsorption were established. According to the similarity evaluation of HPLC fingerprints and analysis results MS map,the sensitization of traditional Chinese medicine injections can be changed by different administration methods. There were 22 kinds of components that can be adsorbed by specific anti-Ig E antibodies in Shuanghuanglian Injection and its drug-containing plasma,most of them were acids and nitrogen compounds. Based on supramolecular theory,it was inferred that these compounds came from SHLI or body,and may form supramolecular hapten,which results in immunotoxicity and allergic reaction when being used as injection instead of oral liquid. Immune fingerprint is not only used to screen out single component allergen,but also more comprehensive,sensitive and easy to operate. It can provide reference for the future research methods of allergic reaction of traditional Chinese medicine injections.
In this paper,immune fingerprint was used to screen the allergenic components of Shuanghuanglian Injection(SHLI) by enzyme-linked immuno sorbent assay(ELISA) combined with HPLC/MS method. ELISA-embedded anti-IgE antibody could successfully adsorb allergens in SHLI and its plasma samples containing drugs through different routes of administration,suggesting that SHLI can induce type I hypersensitivity in rats. HPLC fingerprints and MS map of SHLI and drug-containing plasma samples from different routes of administration before and after anti-IgE antibody adsorption were established. According to the similarity evaluation of HPLC fingerprints and analysis results MS map,the sensitization of traditional Chinese medicine injections can be changed by different administration methods. There were 22 kinds of components that can be adsorbed by specific anti-Ig E antibodies in Shuanghuanglian Injection and its drug-containing plasma,most of them were acids and nitrogen compounds. Based on supramolecular theory,it was inferred that these compounds came from SHLI or body,and may form supramolecular hapten,which results in immunotoxicity and allergic reaction when being used as injection instead of oral liquid. Immune fingerprint is not only used to screen out single component allergen,but also more comprehensive,sensitive and easy to operate. It can provide reference for the future research methods of allergic reaction of traditional Chinese medicine injections.
引文
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[2]杨帅才.中药注射剂过敏反应的原因分析[J].中医中药健康必读杂志,2011(2):153.
[3]鞠艳芳,刘蓉,杨金菊,等.氨甲酰磷酸合成酶单克隆抗体的制备、鉴定及应用[J].细胞与分子免疫学杂志,2008,24(5):479.
[4]曾姣丽,贺福元,唐昱,等.双黄连注射剂中黄芩苷致敏原性的研究[J].中国实验方剂学杂志,2012,18(1):102.
[5]陈锋,贺福元,罗杰英,等.鱼腥草注射液和新鱼腥草素钠注射液小鼠免疫毒性的评价[C].运城:全国中药创新与研究论坛,2009.
[6]卫生部.药品标准(中药成方制剂第11册)[S].2010.
[7]张彦昭,文占权,刘传绪,等.2013—2016年国家药品不良反应监测年度报告统计分析[J].药物流行病学杂志,2017,26(11):763.
[8]翁维良,林洪生.4种中药注射剂皮肤过敏试验方法与意义的探讨[J].中国中药杂志,2007,32(24):2649.
[9]陈伟,李存玉,彭国平,等.超滤工艺对双黄连注射剂致敏性的影响[J].医药导报,2012(3):292.
[10]贺福元,邓凯文,周逸群,等.中药注射剂(类)致敏反应的超分子作用分析与研究方法探讨[J].湖南中医药大学学报,2013,33(9):3.
[11]中药、天然药物免疫毒住(过敏性、光过敏反应)研究的技术指导原则课题研究组.中药、天然药物免疫毒性(过敏性、光变态反应)研究技术指导原则[S]. 2005.
[12] Lin K H,Cheng S Y. An efficient method to purify active eukaryotic proteins from the indusion bodies in Ewherichia coli[J].Biotechniques,1991,11(6):149.
[13] Chanan-Khan A,Szebeni J,Savay S,et al. Complement activation following first exposure to pegylated liposomal doxorubicin(Doxil):possible role in hypersensitivity reactions[J]. Ann Oncol,2003,14:1430.
[14] Bodo M,Szebeni J,Baranyi J,et al. Cerebrovascular involvement in liposome induced cardiopulmonary distress in pigs[J].J Liposome Res,2005,15:3.
[15]乔海灵,赵永星,马统勋.青霉素类抗生素致敏反应机制及诊断的新近研究进展[J].国外医药·抗生素分册,2002,23(5):197.
[16] Sueki H. Drug allergy[J].Nippon Rinsho,2009,67(11):2139.
[17]贺福元,邓凯文,曾姣丽,等.中药注射剂致敏成分群筛查方法研究的现状与免疫综合法[J].中国中药杂志,2012,37(19):2836.