摘要
目的：建立双抗体夹心模式光激化学发光免疫（LiCA）技术定量检测血清促红细胞生成素（EPO）的方法,并对方法进行方法学评估。方法：采用棋盘格滴定法,用针对EPO抗原的一个单克隆抗体及两个多克隆抗体,分别包被发光微粒,及进行生物素化,两两配对筛选出最佳双抗体组合,进而确定最佳反应浓度,建立光激化学发光检测EPO的方法,建立标准曲线,评价该方法。研究中采用SPSS19.0软件和ELISA Calc进行统计分析。结果：筛选到一组最适抗体,分别与发光微粒和生物素进行连接,成功建立了EPO定量检测的双抗体夹心模式LiCA方法。该法检测的敏感性为0.09 ng/ml,在0.09~30 ng/ml范围内线性良好。批内变异系数和批间变异系数分别为2.93%和3.86%,EPO浓度高达30 ng/ml时无Hook效应。51例EPO增高及51例健康体检者EPO的测定结果与放射免疫法检测EPO比较具有良好的相关性（r=0.957）,建立的参考范围为0.76~6.80 ng/ml。结论：成功建立了双抗体夹心模式的光激化学发光定量检测EPO的方法。该检测方法均具有敏感性高、免洗板、线性范围宽、背景荧光低、快速简便等特点。在EPO检测中具有极好的应用前景,有助于EPO诊断试剂盒的研制。
Objective： To establish a double antibody sandwich mode quantitative light initiated chemiluminescence immunoassay（ LiCA） detect of serum erythropoietin（ EPO）,and assess this method. Methods： Using the checkerboard titration,with a monoclonal antibody and two polyclonal antibody to EPO coated with light emitting particles and been biotinylated separately. Filter out the best double-antibody combination pair,and then determine the optimum concentration of the reaction,to establish a light initiated chemiluminescence detection method of EPO with making a standard curve for evaluation. SPSS19. 0 software and ELISA Calc statistical analysis been used in the study. Results： Using the best double-antibody combination pair which been filtered out to conbine with the light-emitting particles and biotinylated,to established the double antibody sandwich mode LiCA of the EPO quantitative detection method successfully,with 0. 09 ng / ml sensitivity,good linear in 0. 09-30 ng / ml range. The coefficient of variation and the days of batch-to-batch variation coefficients were 2. 93% and 3. 86%,there is no Hook effect of EPO concentrations up to 30 ng / ml. There was a good correlation（ r = 0. 957） between this method and radioimmunoassay method when measured 51 cases of EPO increased and51 healthy subjects; establishing reference range at 0. 76-6. 80 ng / ml. Conclusion： Successfully established double antibody sandwich mode light chemiluminescence quantitative detection method of EPO. The assay has high sensitivity,disposable plates,wide linear range,low background fluorescence,quick and easy. EPO detection has excellent prospects,contribute to EPO diagnostic kits developement.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2013年第11期1202-1206,1215,共6页
Chinese Journal of Immunology
基金
天津市北辰区科学技术委员会项目基金(BCWS2012-07)资助
关键词
促红细胞生成素
光激化学发光
方法学评价
Erythropoietin
Light initiated chemiluminescence immunoassay
Methodological evaluation
