双抗原夹心ELISA法检测抗念珠菌烯醇化酶抗体  

Determination of antibodies against Candida albicans enolase by a double-antigen sandwich ELISA

作　　者：陈笑[1] 王颖[1] 黄梅[1] 胡毓安[1] 史利宁[1] 李芳秋[1] 

机构地区：[1]南京大学医学院附属金陵医院南京军区总医院,解放军临床检验医学研究所,南京210002

出　　处：《临床检验杂志》2016年第12期924-926,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81302536);南京军区医学科技创新重点课题(10Z027)

摘　　要：目的建立检测抗念珠菌烯醇化酶(Eno)特异性抗体的双抗原夹心ELISA法,并进行临床应用评价。方法用基因工程制备的重组Eno作为包被抗原和酶标抗原,通过棋盘滴定法对双抗原夹心ELISA法的反应条件进行优化,对方法的敏感性、特异性和精密度进行考察。用双抗原夹心ELISA法测定291例住院患者(侵袭性念珠菌病114例,念珠菌定植82例,细菌感染患者95例)以及200名健康人血清,并与本实验室前期自建的间接ELISA法检测抗Eno抗体的结果进行比较。结果双抗原夹心ELISA法工作条件为:Eno抗原包被浓度为0.5μg/m L,酶标抗原1∶4 000稀释;封闭液为含50 g/L脱脂奶粉的PBS-T,待测血清稀释度为1∶100。患者血清检测结果显示,批内变异系数(CV)分别为6.8%、7.4%和5.9%;不同批次重复测定15次,其批间CV分别为10.1%、9.6%和12.4%。重组抗原对血清中相应抗体的阻断率为92.2%。通过ROC曲线确定cut off值吸光度(A)为0.209。检测侵袭性念珠菌病(invasive candidiasis,IC)的敏感性和特异性分别为81.6%(93/114)和94.4%(356/377),敏感性高于检测抗Eno抗体的间接ELISA法(81.6%vs 76.1%)。结论建立了双抗原夹心ELISA法检测抗念珠菌Eno特异性抗体,可提高IC的诊断率,具有临床应用价值。Objective To establish a double-antigen sandwich enzyme-linked immunosorbent assay（ ELISA） for detecting the specific antibodies against Candida albicans enolase（ Eno） in human serum,and evaluate its clinical application. Methods The recombinant Eno prepared by the genetic engineering technology was used as coating and enzyme-labeled antigens. The reaction conditions of the established double-antigen sandwich ELISA were optimized by the chessboard titration,and its sensitivity,specificity and repeatability were evaluated. Then,the serum samples from 291 inpatients,including 114 patients with invasive candidiasis（ IC）,82 with Candida colonization and 95 with bacterial infection,and 200 healthy volunteers were collected and detected by the sandwich ELISA,and the obtained results were compared with those of indirect ELISA established by our laboratory previously. Results The working conditions of the established double-antigen sandwich ELISA were as follows： 0. 5 μg / m L of coating antigen Eno,1∶ 4 000 diluted of enzyme-labeled antigen,PBS-T containing 50 g / L of dried skimmed milk as the blocking buffer,and 1： 100 diluted of serum samples. The intraand inter-assay coefficients of variation of the established sandwich ELISA for the high,middle and low concentrations of serum samples were 6. 8%,7. 4%,5. 9% and 10. 1%,9. 6%,12. 4%,respectively. The blocking rate of anti-Eno antibodies in serum by the recombinant Eno was 92. 2%. The absorbance value of the cut-off determined by the Receiver Operating Characteristics（ ROC） curve was 0. 209. The sensitivity and specificity of the established sandwich ELISA for the diagnosis of IC were 81. 6%（ 93 /114） and94. 4%（ 356 /377）,respectively,and its sensitivity was higher than that of indirect ELISA（ 81. 6% vs 76. 1%）. Conclusion The established double-antigen sandwich ELISA for the detection of specific antibodies against Candida albicans Eno may be useful for the clinical diagnosis of IC.

关 键 词：侵袭性念珠菌病 血清学诊断 烯醇化酶抗体 双抗原夹心ELISA法 

分 类 号：R446.5[医药卫生—诊断学]