血清抗烟曲霉二肽基肽酶Ⅴ肽段IgG类抗体ELISA法的建立及初步应用  

Establishment and preliminary application of an ELISA method for detecting serum IgG antibody against Aspergillus fumigatus dipeptidyl peptidase Ⅴ fragment

作　　者：韩丹丹[1] 李芳秋[1] 史利宁[1] 张国勇[1] 李伟[1] 胡毓安[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》2014年第4期241-244,共4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：目的建立检测人血清中抗烟曲霉二肽基肽酶Ⅴ(dipeptidyl peptidasesⅤ,DPPⅤ)肽段IgG类抗体(抗DPPⅤ)的ELISA法,评估其对侵袭性曲霉病(IA)的早期诊断价值。方法用重组烟曲霉DPPⅤ肽段作为包被抗原,建立检测血清中抗DPPⅤ的ELISA法,检测105例IA患者、200例非IA患者及200例健康人血清中抗DPPⅤ水平,确定cut off值,考查方法的精密度和特异性。部分IA患者抗DPPⅤ结果与抗烟曲霉硫氧还蛋白还原酶(TR)抗体(抗TR)检测和曲霉半乳甘露聚糖(GM)检测结果对比,评价其临床应用价值。结果以建立的ELISA法检测吸光度(A)值为6.688、0.709的2份抗DPPⅤ抗体阳性血清,批内变异系数(CV)为4.1%和8.0%,批间CV为7.1%和10.7%。重组抗原对血清中相应抗体的阻断率>90%。以A值0.542为cut off值,对IA诊断的敏感性为66.7%,特异性为90.7%。非中性粒细胞缺乏的IA患者抗DPPⅤ阳性率(75.3%,55/73)高于中性粒细胞缺乏的IA患者(46.9%,15/32),差异有统计学意义(χ2=8.11,P<0.05);92例IA患者血清抗DPPⅤ、抗TR和GM测定总阳性率分别为63.0%、60.9%和73.9%,差异无统计学意义(P>0.05);中性粒细胞缺乏的IA患者GM阳性率(89.7%)显著高于抗DPPⅤ、抗TR的阳性率(41.4%和34.5%),χ2分别为14.96、18.75,P均<0.01。抗DPPⅤ与抗TR联合检测阳性率达到82.6%,抗DPPⅤ、抗TR和GM试验三者联合检测的阳性率可提高到96.7%。结论检测抗DPPⅤ抗体的ELISA法精密度和特异性较好,对侵袭性曲霉感染具有潜在的辅助诊断价值,联合多个指标检测可提高诊断的敏感性。Objective To establish an ELISA method for detecting human serum IgG antibody against Aspergillus fumigatus dipeptidyl peptidase Vfragment （anti-DPPV） and evaluate its early diagnostic value for invasive aspergillosis （IA）. Methods An indirect ELISA method to detect serum antibody against DPPV was established by using purified recombinant DPPV as coating antigen. Its cut-off value was determined based on the results of serum samples from 105 IA patients, 200 non-IA patients and 200 healthy donors. Meanwhile, its sensitivity, specificity and reproducibility were evaluated. In addition, the results of anti-DPPV, anti-TR （antibody against Aspergillus fumigatus thioredoxin reductase） and GM （Aspergillusfumigatus galactomannoglycan） test obtained from 92 IA patients were compared to evaluate the clinical application value of anti-DPPV. Results The intra-assay coefficients of variation of the established ELISA method for two serum, in which the A of antibody against DPPV are 1. 688 and 0. 709, were 4.1% and 8.0% respectively, and inter-assay coef- ficients of variation were 7.1% and 10.7%. The blocking rate of recombinant DPPV to serum anti-DPPV was more than 90%. Accord- ing to ROC curve, A450 nm 0. 542 was determined as the cutoff value. In this condition, the sensitivity and specificity of the method for the diagnosis of IA were 66.7% and 90.7%, respectively. The positive rate of anti-DPPV in non-neutropenic IA patients （75.3%, 50/73） was significantly higher than that in neutropenic IA patients （46.9%, 15/32） （X2 = 8.11, P 〈 0.05）. There was no significant differ- ence in 92 IA patients between the total positive rates of anti-DPPV （63.0%）, anti-TR （60.9%） and GM （73.9%） （P 〉 0.05 ）. However, the positive rate of GM （89.7%） in neutropenic IA patients was significantly higher than those of anti-DPPV （41.4%, X2 = 14.96, P 〈 0.01 ） and anti-TR （34.5%, X2 = 18.75, P 〈 0.01 ）. When combined anti-DPPV and anti-TR, the diagnostic sensitivity for IA patients i

关 键 词：侵袭性曲霉病 烟曲霉 二肽基肽酶 抗体 间接酶联免疫吸附试验 

分 类 号：R446.5[医药卫生—诊断学]