狂犬病病毒抗原定量双抗体夹心ELISA检测方法的建立及应用 |
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引用本文: | 崔文广,杨屹,常军亮,井伟东,张秀霞,苗丽,丁丽丽,周长军,郭秀侠. 狂犬病病毒抗原定量双抗体夹心ELISA检测方法的建立及应用[J]. 中国生物制品学杂志, 2012, 25(12): 1684-1687 |
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作者姓名: | 崔文广 杨屹 常军亮 井伟东 张秀霞 苗丽 丁丽丽 周长军 郭秀侠 |
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作者单位: | 长春生物制品研究所有限责任公司疫苗研究室 长春 130062 |
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摘 要: | 目的建立狂犬病病毒(Rabies virμs,RV)抗原定量双抗体夹心ELISA检测方法,以应用于疫苗生产过程中RV含量的监测。方法将RV aGV株纯化抗原经腹腔注射免疫BALB/c小鼠,制备抗RV单克隆抗体,纯化后以HRP进行标记,建立双抗体夹心ELISA检测方法。以狂犬病疫苗(效力检定用)国家标准品为定量标准,建立剂量-反应曲线,确定该方法的灵敏度,并对该方法的专属性、精密性及适用性进行验证。结果制备了2株针对不同抗原位点的单克隆抗体1E9和2H1,间接ELISA法测定腹水效价为1∶105~1∶107,纯化后蛋白含量分别为10.285和7.64 mg/ml。建立的ELISA法对RV抗原的最低检出限为1.03 mIU/ml,标准曲线的最佳线性范围为1.03~66 mIU/ml,相关系数为0.991 9。该方法对检测过程中可能遇到的杂质和添加物(人血清白蛋白、牛血清、Vero细胞培养上清)的检测结果均为阴性;检测3个浓度(20、12.50和3.13 mIU/ml)狂犬病疫苗(效力检定用)国家标准品的试验内和试验间变异系数的平均值分别为7.78%和14.0%;用该方法检测不同毒株、不同细胞生产的狂犬病疫苗的RV抗原含量的平均值在0.67~4.86 IU/ml之间。结论成功建立了RV抗原定量双抗体夹心ELISA检测方法,可对来自不同毒株、不同细胞的狂犬病疫苗的RV抗原进行快速定量检测,为疫苗生产中的质量控制提供了简便的监测手段。
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关 键 词: | 狂犬病病毒 抗原 定量检测 酶联免疫吸附测定 |
Development and application of double antibody sandwich ELISA method for quantitative determination of rabies virus antigen |
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Abstract: | Objective To develop a double antibody sandwich ELISA method for quantitative determination of rabies virus(RV)antigen,and use for the monitoring of RV content during vaccine production.Methods Monoclonal antibody was prepared by immunizing BALB/c mice with purified antigen from RV aGV strain by intraperitoneal injection,then purified and labeled with HRP,based on which a double antibody sandwich ELISA method was developed.A dose-response curve was plotted using national reference for potency test on rabies vaccine as a quantitative standard,by which the sensitivity of the developed method was determined.The method was verified for specificity,precision and suitability.Results Two monoclonal antibodies against different antigenic epitopes,1E9 and 2H1,were prepared,of which the titers in ascites determined by indirect ELISA were 1 ∶ 105 ~ 1 ∶ 107,while the protein contents were 10.285 and 7.64 mg / ml after purification,respectively.The minimum detection limit of the developed ELISA method was 0.03 mIU / ml,while the linear range of standard curve was 1.03 ~ 66 mIU / ml,with a correlation coefficient of 0.991 9.All the detection results of foreign matters and additives by the method,such as human serum albumin,bovine serum and culture supernatant of Vero cells,were negative.National standard for potency test on rabies vaccine,at concentrations of 20,12.50 and 3.13 mIU / ml,were determined by the developed method,of which the mean coefficients of variation of results of intra-and interassays were 7.78% and 14.0%,respectively.The mean RV antigen contents in vaccines prepared with various strains and cells were 0.67 ~ 4.86 IU / ml.Conclusion A double antibody sandwich ELISA method for rapid quantitative determination of RV antigen in vaccines prepared with various strains and cells was developed,which provided a simple method for monitoring the quality control of vaccine during production. |
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Keywords: | Rabies virus Antigen Quantitative assay Enzyme-linked immunosorbent assay(ELISA) |
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