文章摘要
陈 笑,李芳秋.双抗原夹心ELISA法检测烟曲霉硫氧还蛋白还原酶抗体[J].南京医科大学学报,2019,(7):1067~1070
双抗原夹心ELISA法检测烟曲霉硫氧还蛋白还原酶抗体
A double⁃antigen sandwich ELISA for detecting antibody to Aspergillus fumigatus thioredoxin reductase
投稿时间:2019-01-29  
DOI:10.7655/NYDXBNS20190727
中文关键词: 侵袭性念珠菌病  血清学诊断  硫氧还蛋白还原酶  抗体  双抗原夹心ELISA法
英文关键词: invasive aspergillosis  serodiagnosis  thioredoxin reductase  antibody  double⁃antigen sandwich ELISA
基金项目:国家自然科学基金资助项目(81302536);南京军区医学科技创新重点课题(10Z027);南京市卫生局基金(YKK18200)
作者单位
陈 笑 南京医科大学附属江宁医院检验科江苏 南京 211100 
李芳秋 东部战区总医院解放军临床检验医学研究所江苏 南京 210002 
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中文摘要:
      目的:建立检测烟曲霉硫氧还蛋白还原酶(thioredoxin reductase GliT,TR)特异性抗体的双抗原夹心ELISA法,同时对其临床应用进行评价。方法:利用基因工程技术制备重组TR作为包被抗原和酶标记抗原,采用棋盘滴定法优化双抗原夹心ELISA法的反应条件,并考察ELISA法的敏感性、特异性以及重复性。用双抗原夹心ELISA法测定273例住院患者(侵袭性曲霉病50例、侵袭性念珠菌病46例、念珠菌定植82例、细菌感染95例)以及200例健康人血清,并与实验室前期建立的间接ELISA法检测TR抗体的结果进行比较。结果:双抗原夹心ELISA法工作条件为TR抗原包被浓度为1.0 μg/mL,酶标抗原1∶500稀释;封闭液含50 g/L脱脂奶粉溶液的PBS?T,待测血清稀释度为1∶100。患者血清检测结果显示,高、中、低3份血清批内变异系数(CV)分别为11.2%、11.8%和12.3%;不同批次重复测定6次,其批间CV分别为10.9%、12.1%和13.5%。重组抗原对血清中相应抗体的阻断率为94.4%。通过ROC曲线确定吸光度的cut?off值为0.126。检测侵袭性曲霉病的敏感性和特异性分别为80.0%(40/50)和95.5%(404/423),敏感性高于检测TR抗体的间接ELISA法(83.3% vs. 72.2%)。结论:建立了双抗原夹心ELISA法检测烟曲霉TR特异性抗体,可提高侵袭性曲霉病的诊断率,具有临床应用价值。
英文摘要:
      Objective:This study aims to establish an immunological method for detecting specific antibody to Aspergillus fumigatus thioredoxin reductase(TR)in human sera,and evaluate its value in clinical prospect. Methods:The double?antigen sandwich ELISA for detecting TR antibody was applied with recombinant TR as coating and enzyme?labelled antigen. Reaction conditions were optimized by chessboard titration,and the sensitivity,specificity and repeatability for the ELISA were determined. Sera from 273 inpatients(50 cases with invasive aspergillosis,46 cases with invasive candidiasis,82 cases with Candida colonization and 95 cases with bacterial infection)and 200 healthy volunteers were collected and tested by the sandwich ELISA. The experiments results were compared with the previous results of indirect ELISA. Results:The TR was labeled with horseradish peroxidase(HRP)by the reformative sodium periodate method and the optimized condition were as follows:the coating concentration of TR was 1.0 μg/mL,the dilution of enzyme?labelled antigen was 1∶500,the blocking buffer was PBS?T containing 50 g/L dried skimmed milk,test serum was diluted to 1∶100. The intra? and inter?assay coefficients of variation for the developed ELISA were 11.2%,11.8%,12.3% and 10.9%,12.1%,13.5%,respectively. The blocking rate of sera with recombinant antigen was 94.4%. The absorbance value of 0.126 was chosen as cut?off value according to the ROC curve. The sensitivity of the sandwich ELISA for diagnosis of invasive aspergillosis was 80.0%(40/50),higher than that of indirect ELISA(83.3% vs. 72.2%) and the specificity was 95.5%(404/423) in this condition. Conclusion:A double?antigen sandwich ELISA for detecting the specific antibody to Aspergillus fumigatus TR was established successfully,which can be potentially applied in clinical diagnosis setting of invasive aspergillosis.
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