红景天苷单克隆抗体的制备及间接竞争酶联免疫方法的建立
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摘要
【目的】制备红景天苷单克隆抗体,建立红景天苷间接竞争酶联免疫方法,实现经济、高效、简便快速批量检测红景天药材及其制品中的红景天苷含量,为其商品化检测试剂盒开发奠定基础。【方法】用已经成功偶联的红景天苷-BSA作为免疫原,免疫6～8周龄Balb/c小鼠,ELISA检测血清效价,取效价最高的小鼠脾细胞与骨髓瘤细胞进行融合,经有限稀释法筛选、克隆后获得能够稳定产生红景天苷单克隆抗体的细胞株,体内诱生腹水制备大量红景天苷单克隆抗体。利用该抗体,建立红景天苷间接竞争酶联免疫分析方法并进行方法学确认,包括日内精密度、日间精密度、回收率等指标。【结果】建立的间接竞争酶联免疫吸附分析方法的线性范围为4.07～598.45ng·mL~(-1),检测限IC10为1.77ng·mL~(-1),半数抑制率IC50为49.33ng·mL~(-1),日内精密度和日间精密度均小于4.5%,加标样品的平均回收率为94.3%,红景天苷含量检测结果与高效液相色谱法一致。【结论】本研究成功制备了红景天苷单克隆抗体,建立了间接竞争酶联免疫分析方法,能够用于为红景天苷含量的快速检测提供依据。
【Objective】To prepare monoclonal antibody of salidroside for the development of an indirect competitive enzyme linked immunosorbent assay(ic-ELISA)methodology to accurately and rapidly detect salidroside in medicinal materials.【Method】Balb/c mice of 6-8 weeks old were immunized with the prepared salidroside-BSA.Specificity of the anti-serum was determined by ELISA.Spleen cells from the mice with positive result were fused with SP2/0 myeloma cells.Monoclonal hybridoma cells were screened by indirect ELISA with a limited dilution.Ascitic antibodies were induced and prepared from the positive cell line.Specific antibody against salidroside was used to establish ic-ELISA detection method that was verified by its specificity,precision and recovery rate on a known standard.【Result】The sensitivity of ic-ELISA was 49.33 ng·mL~(-1) with a liner range of 4.07-598.45 ng·mL~(-1).The detection limit of the method was 1.77 ng·mL~(-1).The newly developed method detected salidroside in spiked samples with a recovery rate of 94.3% within the designed range.The relative standard deviation of the measurements was less than 4.5% on both intra-and inter-day assays.The determination by ic-ELISA agreed with that obtained by HPLC.【Conclusion】The newly established ic-ELISA method based on the salidroside monoclonal antibody was considered appropriate for the detection.
【Objective】To prepare monoclonal antibody of salidroside for the development of an indirect competitive enzyme linked immunosorbent assay(ic-ELISA)methodology to accurately and rapidly detect salidroside in medicinal materials.【Method】Balb/c mice of 6-8 weeks old were immunized with the prepared salidroside-BSA.Specificity of the anti-serum was determined by ELISA.Spleen cells from the mice with positive result were fused with SP2/0 myeloma cells.Monoclonal hybridoma cells were screened by indirect ELISA with a limited dilution.Ascitic antibodies were induced and prepared from the positive cell line.Specific antibody against salidroside was used to establish ic-ELISA detection method that was verified by its specificity,precision and recovery rate on a known standard.【Result】The sensitivity of ic-ELISA was 49.33 ng·mL~(-1) with a liner range of 4.07-598.45 ng·mL~(-1).The detection limit of the method was 1.77 ng·mL~(-1).The newly developed method detected salidroside in spiked samples with a recovery rate of 94.3% within the designed range.The relative standard deviation of the measurements was less than 4.5% on both intra-and inter-day assays.The determination by ic-ELISA agreed with that obtained by HPLC.【Conclusion】The newly established ic-ELISA method based on the salidroside monoclonal antibody was considered appropriate for the detection.
引文
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[4]王芸,李正,段亚飞,等.红景天提取物对凡纳滨对虾抗氧化系统及抗低盐度胁迫的影响[J].南方水产科学,2018,14(1):9-19.WANG Y,LI Z,DUAN Y F,et al.Effect of dietary Rhodiola rosea extract on antioxidant system and anti-low salinity stress of Litopenaeus vannamei[J].South China Fisheries Science,2018,14(1):9-19.(in Chinese)
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[8]林建阳,续晋洁,林秋婕,等.LC-MS/MS法测定红景天苷在大鼠的血药浓度[J].中国临床药理学杂志,2013,29(11):862-864.LIN J Y,XU J J,LIN Q J,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].The Chinese Journal of Clinical Pharmacology,2013,29(11):862-864.(in Chinese)
[9]温时媛,周江韬,陈燕燕,等.红景天药材的H-NMR指纹图谱研究[J].中国中药杂志,2015,40(13):2629-2633.WEN S Y,ZHOU J T,CHEN Y Y,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].China Journal of Chinese Materia Medica,2015,40(13):2629-2633.(in Chinese)
[10]金贞姬,杨长青.高效液相色谱法测定高山红景天超微粉中红景天苷和苷元的含量[J].中国医院药学杂志,2012,32(4):313-314.JIN Z J,YANG C Q.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Chinese Journal of Hospital Pharmacy,2012,32(4):313-314.(in Chinese)
[11]王伟,胡立新,伏光华.红景天苷人工抗原的合成与鉴定[J].福建农业学报,2018,38(5):540-543.WANG W,HU L X,FU G H,et al.Synthesis and identification of artificial antigen of Salidroside[J].Fujian Journal of Agricultural Sciences,2018,38(5):540-543.(in Chinese)
[12]于生兰,欧阳臻,徐加兵,等.黄芪甲苷单克隆抗体的制备及鉴定[J].天然产物研究与开发,2013,25:1568-1571.YU S L,OUYANG Z,XU J B,et al.Prepration and Identification of Monoclonal Antibody against Astragaloside IV[J].Nat Prod Res Dev,2013,25:1568-1571.(in Chinese)
[13]张越,屈会化,吴婷婷,等.甘草酸单克隆抗体的制备与鉴定[J].药物分析杂志,2013,33(5):770-774.ZHANG Y,QU H H,WU T T,et al.Preparation and identification of monoclonal antibodies against glycyrrhizic acid[J].Chin J Pharm Anal,2013,33(5):770-774.(in Chinese)
[14]秦高风.柴胡皂苷D人工抗原的合成及单克隆抗体的制备[D].北京:北京中医药大学,2017.QIN G F.The physio-chemical mechanism and differential expressions of proteins for the browning of fresh-cut lotus root[D].Beijing:Beijing University of Chinese medicine,2017.(in Chinese)
[15]赛佳洋,屈会化,冯会宾,等.人参皂苷Re人工抗原的合成及单克隆细胞株的筛选[J].中国药学杂志,2014,49(13):1104-1108.SAI J Y,QU H H,FENG H B,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Chinese Pharmaceutical Journal,2014,49(13):1104-1108.(in Chinese)
[16]张波.基于单克隆抗体的金银花指标性成分快速检测技术研究[D].济南:山东中医药大学,2016.ZHANG B.Rapid determination of the index component of flos lonicerae japonicae based on monoclonal antibody technology[D].Jinan:Shandong University of Traditional Chinese Medicine,2016.(in Chinese)
[17]苏歆,屈会化,赵琰,等.基于黄芩苷单克隆抗体的ELISA快速检测方法的建立[J].药物分析杂志,2013,33(6):946-949.SU X,QU H H,ZHAO Y,et al.Establishment of quick enzyme-linked immunosorbent assay with monoclonal antibody against baicalin[J].Chin J Pharm Anal,2013,33(6):946-949.(in Chinese)
[18]张越.甘草酸、柚皮苷单克隆抗体的制备及其酶联免疫分析方法的建立[D].北京:北京中医药大学,2013.ZHANG Y.Preparation of naringin and glycyrrhizic monoclonal antibodies and establish study of enzyme linked immunosorbent assays[D].Beijing:Beijing University of Chinese medicine,2013.(in Chinese)
[19]冯盛岚.一种新柚皮苷单克隆抗体的制备与应用[D].北京:北京中医药大学,2015.FENG S L.Preparation and application study of naringin monoclonal antibodies[D].Beijing:Beijing University of Chinese medicine,2015.(in Chinese)
[2]魏日胞,王永新,杨悦,等.复方红景天胶囊对小鼠免疫调节作用的实验研究[J].中国实验血液学杂志,2012,20(40):187-191.WEI R B,WANG Y X,YANG Y,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Journal of experimental hematology,2012,20(40):187-191.(in Chinese)
[3]WU T,ZHOU H,JIN Z,et al.Cardioprotection of salidroside from ischemia/reperfusion injury by increasing N-acetylgluco-samine linkage to cellular proteins[J].Eur JPharmacol,2009,613(1-3):93-99.
[4]王芸,李正,段亚飞,等.红景天提取物对凡纳滨对虾抗氧化系统及抗低盐度胁迫的影响[J].南方水产科学,2018,14(1):9-19.WANG Y,LI Z,DUAN Y F,et al.Effect of dietary Rhodiola rosea extract on antioxidant system and anti-low salinity stress of Litopenaeus vannamei[J].South China Fisheries Science,2018,14(1):9-19.(in Chinese)
[5]张琛,杨长庚,文华,等.饲料中添加红景天对吉富罗非鱼耐低温特性的影响[J].淡水渔业,2016,46(5):75-80.ZHANG C,YANG C G,WEN H,et al.Effects of Rhodiola sachaliensis on improving low-temperature tolerance characteristics of Tilapia(Oreochromis niloticus)[J].Freshwater Fisheries,2016,46(5):75-80.(in Chinese)
[6]李龙,王宏辉,刘锁珠,等.红景天对高原缺氧条件下肉鸡免疫功能的影响[J].中国家禽,2013,35(2):19-21.LI L,WANG H H,LIU S Z,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].China Poultry,2013,35(2):19-21.(in Chinese)
[7]王洋,张璞,于涛,等.高效液相色谱法测定红景天甙含量方法的研究[J].植物研究,2001,21(1):113-115.WANG Y,ZHANG P,YU T,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Bulletin of Botanical Research,2001,21(1):113-115.(in Chinese)
[8]林建阳,续晋洁,林秋婕,等.LC-MS/MS法测定红景天苷在大鼠的血药浓度[J].中国临床药理学杂志,2013,29(11):862-864.LIN J Y,XU J J,LIN Q J,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].The Chinese Journal of Clinical Pharmacology,2013,29(11):862-864.(in Chinese)
[9]温时媛,周江韬,陈燕燕,等.红景天药材的H-NMR指纹图谱研究[J].中国中药杂志,2015,40(13):2629-2633.WEN S Y,ZHOU J T,CHEN Y Y,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].China Journal of Chinese Materia Medica,2015,40(13):2629-2633.(in Chinese)
[10]金贞姬,杨长青.高效液相色谱法测定高山红景天超微粉中红景天苷和苷元的含量[J].中国医院药学杂志,2012,32(4):313-314.JIN Z J,YANG C Q.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Chinese Journal of Hospital Pharmacy,2012,32(4):313-314.(in Chinese)
[11]王伟,胡立新,伏光华.红景天苷人工抗原的合成与鉴定[J].福建农业学报,2018,38(5):540-543.WANG W,HU L X,FU G H,et al.Synthesis and identification of artificial antigen of Salidroside[J].Fujian Journal of Agricultural Sciences,2018,38(5):540-543.(in Chinese)
[12]于生兰,欧阳臻,徐加兵,等.黄芪甲苷单克隆抗体的制备及鉴定[J].天然产物研究与开发,2013,25:1568-1571.YU S L,OUYANG Z,XU J B,et al.Prepration and Identification of Monoclonal Antibody against Astragaloside IV[J].Nat Prod Res Dev,2013,25:1568-1571.(in Chinese)
[13]张越,屈会化,吴婷婷,等.甘草酸单克隆抗体的制备与鉴定[J].药物分析杂志,2013,33(5):770-774.ZHANG Y,QU H H,WU T T,et al.Preparation and identification of monoclonal antibodies against glycyrrhizic acid[J].Chin J Pharm Anal,2013,33(5):770-774.(in Chinese)
[14]秦高风.柴胡皂苷D人工抗原的合成及单克隆抗体的制备[D].北京:北京中医药大学,2017.QIN G F.The physio-chemical mechanism and differential expressions of proteins for the browning of fresh-cut lotus root[D].Beijing:Beijing University of Chinese medicine,2017.(in Chinese)
[15]赛佳洋,屈会化,冯会宾,等.人参皂苷Re人工抗原的合成及单克隆细胞株的筛选[J].中国药学杂志,2014,49(13):1104-1108.SAI J Y,QU H H,FENG H B,et al.Extraction and determination of total phenol in luffa(Luffa cylindrical Roem.)[J].Chinese Pharmaceutical Journal,2014,49(13):1104-1108.(in Chinese)
[16]张波.基于单克隆抗体的金银花指标性成分快速检测技术研究[D].济南:山东中医药大学,2016.ZHANG B.Rapid determination of the index component of flos lonicerae japonicae based on monoclonal antibody technology[D].Jinan:Shandong University of Traditional Chinese Medicine,2016.(in Chinese)
[17]苏歆,屈会化,赵琰,等.基于黄芩苷单克隆抗体的ELISA快速检测方法的建立[J].药物分析杂志,2013,33(6):946-949.SU X,QU H H,ZHAO Y,et al.Establishment of quick enzyme-linked immunosorbent assay with monoclonal antibody against baicalin[J].Chin J Pharm Anal,2013,33(6):946-949.(in Chinese)
[18]张越.甘草酸、柚皮苷单克隆抗体的制备及其酶联免疫分析方法的建立[D].北京:北京中医药大学,2013.ZHANG Y.Preparation of naringin and glycyrrhizic monoclonal antibodies and establish study of enzyme linked immunosorbent assays[D].Beijing:Beijing University of Chinese medicine,2013.(in Chinese)
[19]冯盛岚.一种新柚皮苷单克隆抗体的制备与应用[D].北京:北京中医药大学,2015.FENG S L.Preparation and application study of naringin monoclonal antibodies[D].Beijing:Beijing University of Chinese medicine,2015.(in Chinese)