烟曲霉硫氧还蛋白还原酶双抗体夹心ELISA法的建立及初步应用  

Development of sandwich ELISA for detecting thioredoxin reductase Gli T of Aspergillus fumigatus and its preliminary application

作　　者：史利宁[1] 陈勇[1] 胡毓安[1] 盛福梅 李晓军[1] 李芳秋[1] 

机构地区：[1]南京军区南京总医院临床中心实验科,南京210002

出　　处：《临床检验杂志》2015年第9期645-647,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金青年科学基金(81302536)

摘　　要：目的建立检测烟曲霉硫氧还蛋白还原酶Gli T(thioredoxin reductase Gli T,TR)的双抗体夹心ELISA法,并用于几种真菌培养上清液中TR蛋白的检测。方法纯化鼠抗人TR单克隆抗体并进行辣根过氧化物酶(HRP)标记。以纯化羊抗TR多克隆抗体为包被抗体,HRP标记鼠抗人TR单克隆抗体为检测抗体,建立双抗体夹心ELISA法。用方阵滴定法确定包被抗体和检测抗体的最适浓度;对方法的精密度、特异性和最低检测限进行评价;比较分析其检测3种常见曲霉(烟曲霉、黄曲霉、黑曲霉)培养上清中天然TR蛋白的能力,并与3种常见假丝酵母菌(白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌)作比较。结果方阵滴定的结果显示最适包被抗体浓度为1μg/m L,最适检测抗体的稀释度为1∶3 000。抗原最低检测限达2.25ng/m L。重组TR浓度为5 ng/m L和20 ng/m L时,批内和批间变异系数分别为8.57%、12.69%和6.73%、10.45%。阻断实验显示该法有较好的特异性,阻断率达84.7%。该法可以检出烟曲霉24 h培养上清中的天然TR,TR含量与烟曲霉菌丝含量呈正相关。本法与另2种曲霉和3种假丝酵母菌培养上清无交叉反应。结论成功建立并优化了检测烟曲霉TR抗原的ELISA法。该法具有良好的精密度及特异性,对烟曲霉感染有潜在的诊断价值。Objective To establish a sandwich ELISA for detecting thioredoxin reductase（ TR） Gli T of Aspergillus fumigatus,and try to detect Gli T in the supernatant of different fungi cultures. Methods HRP-conjugated rat anti-TR monoclonal antibody and anti-TR goat polyclonal antibody was used as the detector antibody and coating antibody respectively. Preliminary checkerboard titration experiments were performed to determine the optimal concentration of the coating antibodies and the detecting antibodies. The performance parameters of the sandwich ELISA including precision,specificity and limit of detection were evaluated. This assay was applied to determine TR levels in the supernatant of Aspergillus fumigatus,Aspergillus flavus,Aspergillus niger,Candida albicans,Candida tropicalis and Candida glabrata cultures. Results The optimal concentration of coating antibody was 1 μg / m L and the dilution of the detecting antibody was 1∶ 3 000. The limit of detection was 2. 25 ng / m L. The intra and inter-coefficient of variation was 8. 57%,12. 69%and 6. 73%,10. 45%,at the TR concentration of 5 ng / m L and 20 ng / m L,respectively. The sandwich ELISA could specifically detected TR in Aspergillus fumigatus cultrue after incubation at 37 ℃ for 24 h,and no cross-reactivity to Aspergillus flavus,Aspergillus niger,Candida albicans,Candida tropicalis or Candida glabrata was found. The level of TR in supernatant of fungi cultures increased with growth of hyphae. Conclusion A specific and sensitive sandwich ELISA for detection of Aspergillus fumigatus antigen TR was developed. This method might be valuable in diagnosis of invasive aspergillosis.

关 键 词：烟曲霉 硫氧还蛋白还原酶 ELISA 侵袭性曲霉感染 

分 类 号：R446.5[医药卫生—诊断学]