文章摘要
刘丽萍,孙丽洲,卞智萍,顾春荣,杨 笛,张寄南.定量检测ProMBP双抗体夹心ELISA方法的建立[J].南京医科大学学报,2008,28(7):836~840
定量检测ProMBP双抗体夹心ELISA方法的建立
Establishment of a sandwich ELISA for quantitative measurement of ProMBP
投稿时间:2007-11-20  
DOI:10.7655
中文关键词: 定量检测  嗜酸性主要碱性蛋白前体  ELISA双抗体夹心
英文关键词: quantitative measurement  ProMBP  sandwich ELISA
基金项目:江苏省医学重点学科-重大疾病分子诊断和生物治疗的高技术平台(XK200705);江苏省医学重点实验室-临床生物学诊断和治疗实验室(SK200205)
作者单位
刘丽萍 南京医科大学第一附属医院妇产科,江苏 南京 210029 
孙丽洲 南京医科大学第一附属医院妇产科,江苏 南京 210029 
卞智萍 南京医科大学第一附属医院妇产科,江苏 南京 210029 
顾春荣 南京医科大学第一附属医院妇产科,江苏 南京 210029 
杨 笛 南京医科大学第一附属医院心血管病研究所,江苏 南京 210029 
张寄南 南京医科大学第一附属医院心血管病研究所,江苏 南京 210029 
摘要点击次数: 1398
全文下载次数: 119
中文摘要:
      目的:建立定量检测ProMBP的双抗体夹心ELISA方法?方法:采用Protein A亲和层析法纯化自制的抗ProMBP单克隆抗体(mAb),并行SDS-PAGE 和Western blot对纯化抗体特性进行鉴定;利用简易过碘酸钠法对抗ProMBP mAb进行标记辣根过氧化物(HRP)后行抗体配对实验;通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度;以纯化的妊娠相关血浆蛋白A(PAPP-A)/ProMBP抗原为标准品建立标准曲线;以重复性?灵敏性和回收性实验评价ELISA方法,初步对人血清中的ProMBP水平进行检测?结果:最佳配对组合为抗ProMBP mAb 4C6E5B9和HRP标记的抗ProMBP mAb 9G4A6G10,最适工作浓度分别为2.5 μg/ml和1 ∶ 3 000,该方法的批内?批间变异系数分别为4.6%~6.2%和4.6%~10.5%,灵敏度达2.0 ng/ml,回收率为92%~113%?正常非妊娠血清?9~11周妊娠血清?15~17周妊娠血清ProMBP水平分别为(26.37 ± 2.90)ng/ml?(357.71 ± 33.60)ng/ml和(1088.77 ± 58.13)ng/ml?结论:建立了一种可用于检测ProMBP的双抗体夹心ELISA方法?
英文摘要:
      Objective:To establish a sandwich ELISA for quantitative measurement of ProMBP. Methods:Anti-ProMBP monoclonal antibodies(mAbs) prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western blotting to test their characteristics. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method, and antibody pairs were performed using anti-ProMBP mAb as coating antibody and HRP labeled anti-ProMBP mAb as labeled antibody,which optimal concentrations were defined by titration. Standard curve was performed using purified PAPP-A/ProMBP tetramer and was judged by sensitivity,reproducibility and recovery rate. The ProMBP levels of sera were measured with this assay. Results:The optimal paired antibodies were anti-ProMBP mAb 4C6E5B9 and HRP labeled anti-ProMBP mAb 9G4A6G10 which optimal concentrations were 2.5 μg/ml and 1:3 000,respectively. The sensitivity of this assay was 2.0 ng/ml. The coefficients of variation were 4.6% to 6.2% within assay and 4.6% to 10.5% between assays. The recovery rate was 92% to 113%.The ProMBP levels of serum were(26.37 ± 2.90)ng/ml,(357.71 ± 33.60)ng/ml and(1088.77 ± 58.13)ng/ml, which were collected from normal non-pregnant women,9 to 11 weeks gestation and 15 to 23 weeks gestation respectively. Conclusion:A sandwich ELISA assay for detecting ProMBP was obtained successfully.
查看全文   查看/发表评论  下载PDF阅读器
关闭