基于黄芪甲苷单克隆抗体的ELISA检测方法的建立
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摘要
目的建立黄芪甲苷的酶联免疫分析(enzyme-linked immunosorbent assay,ELISA)检测方法。方法以制备的黄芪甲苷单克隆抗体为基础,选择其最佳工作浓度,建立黄芪甲苷间接竞争ELISA法,并用该方法检测黄芪饮片中的黄芪甲苷含量。结果该方法线性范围为39～1 830 ng·mL~(-1),组内和组间差异均≤10%,平均加样回收率为102.7%(n=6)。采用该方法检测黄芪饮片中黄芪甲苷的含量,所得结果与HPLC法基本一致。结论建立了黄芪甲苷的ELISA检测方法,可为黄芪饮片及其制品的质量控制提供方法补充。
OBJECTIVE To establish an enzyme-linked immunosorbent assay(ELISA) method for the determination of Astragaloside Ⅳ(AST). METHODS Based on the preparation of monoclonal antibody against AST, an indirect competitive enzyme immunoassay method which could detect the content of AST in Radix Astragali was established by selecting the optimum working concentration. RESULTS The calibration curves were linear within the range of 39-1 830 ng·mL~(-1). The average recovery(n=6) was 102.7%, and the relative standard deviations(RSD) of measurements in the intra-assay and in the inter-assay were ≤10%. The analysis result of ELISA method was consistent with that of HPLC test in AST determination of Radix Astragali. CONCLUSION ELISA method for the determination of AST is well established and can be applied to the quality control for Radix Astragali and its products.
OBJECTIVE To establish an enzyme-linked immunosorbent assay(ELISA) method for the determination of Astragaloside Ⅳ(AST). METHODS Based on the preparation of monoclonal antibody against AST, an indirect competitive enzyme immunoassay method which could detect the content of AST in Radix Astragali was established by selecting the optimum working concentration. RESULTS The calibration curves were linear within the range of 39-1 830 ng·mL~(-1). The average recovery(n=6) was 102.7%, and the relative standard deviations(RSD) of measurements in the intra-assay and in the inter-assay were ≤10%. The analysis result of ELISA method was consistent with that of HPLC test in AST determination of Radix Astragali. CONCLUSION ELISA method for the determination of AST is well established and can be applied to the quality control for Radix Astragali and its products.
引文
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[14]YU S L,OUYANG Z,XU J B,et al.Prepration and identification of monoclonal antibody against astragaloside IV[J].Nat Pro Res Develop(天然产物研究与开发),2013,25(11):1568-1571.
[15]ZHANG J H,ZHOU J,WU Z L,et al.Determination of astragaloside IV in radix astragali and Jinqi jiangtang tablet by HPLC-ELSD[J].J Tianjin Med Univ(天津医科大学学报),2010,11(1):26-29.
[16]POWERS J L,RIPPE K D,IMARHIA K,et al.A direct competitive enzyme-linked immunosorbent assay(ELISA)as a quantitative[J].J Chem Edu,2012,89(12):1587-1590.
[17]SAI J Y.Prepration of monoclonal antibody against GRe and establishment of enzyme-linked immunosorbent assay[D].Beijing:University of Beijing Chinese Medicine,2014.
[18]WANG J,LIU J H,GUO X,et al.A sensitive indirect competitive enzyme-linked immunosorbent assay for the detection of sarsasapogenin in rat plasma[J].Acta Pharmacol Sinica,2010,31(2):984-989.
[2]中国药典.一部[S].2015:附录283-284.
[3]WANG Y X,ZENG D F,ZHU L,et al.Determination of astragaloside IV and total saponins in Huangqi granules[J].West China J Pharm Sci(华西药学杂志),2011,26(4):375-377.
[4]DU Z H.Determination of astragalosideⅣin Shengqijiangtang granules by HPLC-ELSD[J].Qilu Pharm Affa(齐鲁药事),2010,29(2):86-87.
[5]WANG J F,ZHANG Q,MU X.Determination of astragaloside IV in Yupingfeng oral solution by HPLC-ELSDmethod[J].Agric Basic Sci Technol,2015,16(2):197-199.
[6]TENG H Y,YU Y Y.Application of immunoassay in chemical composition of traditional Chinese medicine[J].Chin J New Drugs(中国新药杂志),2012,21(21):2511-2515.
[7]QU H H,QU B P,WANG,X Q,et al.Rapid,sensitive separation of the three main isoflavones in soybean using immunoaffinity chromatography[J].J Sep Sci,2016,39(6):1195-1201.
[8]XU F M.Prepration and application of monoclonal antibody against Baicalin[D].Xiamen:University of Xiamen,2009.
[9]SU X.Prepration of monoclonal antibody against astragaloside IV and establishment of enzyme-linked immunosorbent assay[D].Beijing:University of Beijing Chinese Medicine,2013.
[10]LIU Y.Synthesis of artificial antigens for Saikosapoins-a and prepration of monoclonal antibody against it[D].Beijing:University of Beijing Chinese Medicine,2013.
[11]SHAO Y Y.Prepration and immunoassay of monoclonal antibody against Bioactive paeoniflorin of Chinese materia medica[D].Xiamen:University of Xiamen,2008.
[12]余伯阳,刘吉华.中药有效成分皂苷类化合物快速微量免疫分析方法研究[C].药用植物化学与中药有效成分分析研讨会论文集(上),2008:10.
[13]YU S L,OUYANG Z,XU J B,et al.Synthesis and evaluation of artificial antigens for astragaloside IV[J].Rev Bras Farmacogn,2014,25(24):282-287.
[14]YU S L,OUYANG Z,XU J B,et al.Prepration and identification of monoclonal antibody against astragaloside IV[J].Nat Pro Res Develop(天然产物研究与开发),2013,25(11):1568-1571.
[15]ZHANG J H,ZHOU J,WU Z L,et al.Determination of astragaloside IV in radix astragali and Jinqi jiangtang tablet by HPLC-ELSD[J].J Tianjin Med Univ(天津医科大学学报),2010,11(1):26-29.
[16]POWERS J L,RIPPE K D,IMARHIA K,et al.A direct competitive enzyme-linked immunosorbent assay(ELISA)as a quantitative[J].J Chem Edu,2012,89(12):1587-1590.
[17]SAI J Y.Prepration of monoclonal antibody against GRe and establishment of enzyme-linked immunosorbent assay[D].Beijing:University of Beijing Chinese Medicine,2014.
[18]WANG J,LIU J H,GUO X,et al.A sensitive indirect competitive enzyme-linked immunosorbent assay for the detection of sarsasapogenin in rat plasma[J].Acta Pharmacol Sinica,2010,31(2):984-989.