栀子苷单克隆抗体的制备与鉴定
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摘要
目的:制备特异性的栀子苷(Geniposide,GEN)单克隆抗体。方法:采用高碘酸钠氧化法合成栀子苷免疫抗原,通过对BALB/c小鼠进行免疫;对血清进行检测阳性后,取上述免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0-Ag14按10∶1的比例融合;用间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;用阳性细胞株诱导制备腹水抗体;用辛酸硫酸铵法纯化抗体,并进行交叉反应检测。选择血清效价高且敏感性好的小鼠,采用杂交瘤细胞技术进行细胞融合,筛选分泌栀子苷单克隆抗体的杂交瘤细胞株;采用体内诱生腹水法制备栀子苷单克隆抗体,并通过正辛酸纯化的方法纯化腹水中抗体蛋白,采用SDS-PAGE法对抗体蛋白的纯化纯度进行检测。结果:获得了能分泌抗栀子苷单克隆抗体稳定的杂交瘤细胞株。SDS-PAGE结果说明经过纯化腹水中大量的杂蛋白能够被除去,获得的栀子单克隆抗体纯度较高,纯化后腹水中抗体效价与纯化前比均为60 000∶1左右。结论:成功获得了能分泌栀子苷单克隆抗体稳定的杂交瘤细胞株,特异性、灵敏度都比较高,为样品中的栀子苷快速微量检测及免疫亲和色谱柱的制备奠定了基础。
To prepare a specific monoclonal antibody against geniposide( GEN). Methods: BALB/c mice were immunized with the GEN-BSA conjugate which was synthesized by applying the method of sodium periodate oxidation. When the serum detection was positive,spleen cells isolated from the mice were mixed together with myeloma cells of SP2/0-Ag14 at a ratio of 10∶ 1. Monoclonal hybridoma cells were screened by indirect ELISA and limited dilution. The ascites antibodies were induced and prepared by the positive cell line,and then were purified by bitter-ammonium sulfate law and tested by SDS-PAGE. Results: The monoclonal antibody hybridoma cell line steadily secreting geniposide antibodies was obtained,the results of SDS-PAGE indicated that a great number of mongrel proteins in ascites were eliminated by purification and the higher purity of geniposide monoclonal antibody was achieved. The results manifested that the hybridoma cell lines secreting geniposide monoclonal antibody were successfully screened by cell fusion. The ratio of the ascites antibody titers after and before purification were about 60 000 ∶ 1. Conclusion: The anti-GEN antibody-secreting hybridomas are obtained with high sensitivity and specificity,which lays a foundation for the trace detection and immunoaffinity column preparation of geniposide in samples.
To prepare a specific monoclonal antibody against geniposide( GEN). Methods: BALB/c mice were immunized with the GEN-BSA conjugate which was synthesized by applying the method of sodium periodate oxidation. When the serum detection was positive,spleen cells isolated from the mice were mixed together with myeloma cells of SP2/0-Ag14 at a ratio of 10∶ 1. Monoclonal hybridoma cells were screened by indirect ELISA and limited dilution. The ascites antibodies were induced and prepared by the positive cell line,and then were purified by bitter-ammonium sulfate law and tested by SDS-PAGE. Results: The monoclonal antibody hybridoma cell line steadily secreting geniposide antibodies was obtained,the results of SDS-PAGE indicated that a great number of mongrel proteins in ascites were eliminated by purification and the higher purity of geniposide monoclonal antibody was achieved. The results manifested that the hybridoma cell lines secreting geniposide monoclonal antibody were successfully screened by cell fusion. The ratio of the ascites antibody titers after and before purification were about 60 000 ∶ 1. Conclusion: The anti-GEN antibody-secreting hybridomas are obtained with high sensitivity and specificity,which lays a foundation for the trace detection and immunoaffinity column preparation of geniposide in samples.
引文
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[7] ZHAO H,OSAMU M,HIROYUKI T,et al. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines[J]. Biolog Pharm BuLl,2003,26(6):862-866.
[8] TIAN M,HIROYUKI T,SHANG M,et al,Production,characterization of a monoclonal antibody against aristolochic acid-Ⅱand development of its assay system[J],Am J Chin Med,2008,36(2):425-436.
[9]屈会化,张桂亮,赵琰等.栀子苷人工抗原的合成与鉴定[J].北京中医药大学学报,2013,36(6):387-392.
[10]蔡碧双.中药活性成分甘草酸的单克隆抗体制备及其免疫测定法的建立[D].厦门:厦门大学,2009:25-30.
[11]王梓淞.基于单克隆抗体特异性敲除技术的栀子苷与栀子改善HUVEC细胞氧化应激损伤作用的相关性研究[D].北京:北京中医药大学,2017:60-65.
[12]刘儒彪,胡骁飞,张改平,等.沙拉沙星单克隆抗体制备及鉴定[J].华北农学报,2016,31(2):87-91.
[2] CHENG X X,XIANG Y. Quantitative method research of glycyrrhizic acid in licorice and its preparations[J]. Lishizhen Med Mater Med Res,2000,11(4):380-381.
[3] SHOYAMA Y. New strategy for drug discovery using monoclonal antibody[J]. J Curr Drug Discov Technol,2011,8(1):1-2.
[4] Shang M Y,TIAN M,TANAKA H,et al. Quality control of traditional Chinese medicine by monoclonal antibody method[J]. J Curr Drug Discov Technol,2011,8(1):60-65.
[5] ZHAO Y,QU H H,ZHANG G L,et al. Study of the metabolic characteristics of geniposide in the different encephalic region of the SD rats[J].Neurochem Res,2012,37(10):2272-2283.
[6] YIN J,YIN L H,JIN S H. Synthesis and identification of baicalin complete antigen andpreparation of polyclonal antibody against baicalin[J].Chin J Pharm Anal,2012,32(5):738-743.
[7] ZHAO H,OSAMU M,HIROYUKI T,et al. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines[J]. Biolog Pharm BuLl,2003,26(6):862-866.
[8] TIAN M,HIROYUKI T,SHANG M,et al,Production,characterization of a monoclonal antibody against aristolochic acid-Ⅱand development of its assay system[J],Am J Chin Med,2008,36(2):425-436.
[9]屈会化,张桂亮,赵琰等.栀子苷人工抗原的合成与鉴定[J].北京中医药大学学报,2013,36(6):387-392.
[10]蔡碧双.中药活性成分甘草酸的单克隆抗体制备及其免疫测定法的建立[D].厦门:厦门大学,2009:25-30.
[11]王梓淞.基于单克隆抗体特异性敲除技术的栀子苷与栀子改善HUVEC细胞氧化应激损伤作用的相关性研究[D].北京:北京中医药大学,2017:60-65.
[12]刘儒彪,胡骁飞,张改平,等.沙拉沙星单克隆抗体制备及鉴定[J].华北农学报,2016,31(2):87-91.
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