检测抗烟曲霉分泌蛋白抗体的ELISA法及其诊断价值

An ELISA-based method for detection of antibody against Aspergillus fumigatus secretary proteins and its value in diagnosis of invasive aspergillosis

目的建立检测人血清中抗烟曲霉特异性抗体的ELISA法,评估该法在侵袭性烟曲霉病(IA)诊断中的价值。方法用沙氏液体培养基震荡培养烟曲霉,TCA/丙酮沉淀法分离菌体蛋白和培养上清中的分泌蛋白。用烟曲霉播散感染的家兔血清及确诊IA患者血清,分别与菌体蛋白和分泌蛋白进行免疫印迹分析,选择反应强的分泌蛋白作为包被抗原,建立测定抗烟曲霉特异抗体的ELISA,并对方法的精密度和特异性进行考察、确定cut off值。对实验动物模型血清以及5例确诊、24例临床诊断为IA的患者血清样本进行测定,并与血清半乳甘露聚糖(GM)测定结果进行比较。结果免疫印迹结果显示培养获得的烟曲霉分泌蛋白与感染动物和病人血清均呈现强反应,以其作为包被抗原建立的ELISA法精密度良好,批内CV=12.0%,批间CV=8.1%;阻断试验证实此法对烟曲霉抗体具特异性。依据200份健康人血清测定结果,取吸光度(A)值1.03作为cut-off值。健康人抗体阳性率为2.0%(4/200),IA患者阳性率41.4%(12/29),二者差异显著(P<0.01)。2例临床诊断病人血清GM试验阴性,但抗体阳性,占8.3%(2/24)。实验感染的家兔均在感染后1周内测出抗体,且抗体滴度快速上升。结论建立了检测抗烟曲霉分泌蛋白抗体的ELISA法,在IA早期诊断中具有潜在应用价值。

Objective To establish an ELISA-based method for detecting specific antibody against AspergiUus fumigatus, and evaluate its usefulness in diagnosis of invasive Aspergillosis（IA）. Methods Aspergillus fumigatus was cultured in Sabouraud＇s media, at 37 ℃ with shaking. Mycelia proteins and secreted proteins from culture supernatant were separated with TCA/acetone precipitation. Serums from rabbits with disseminated infection and patients with final diagnosed invasive Aspergillosis were used to select strong reactive antigens from mycelia proteins and secreted proteins in Western blot. An ELISA for detecting specific antibodies against Aspergillus fumigatus were established with secreted proteins as coating antigens which exhibited strong reaction in Western Blot. The specificity, repeatability was investigated and cut-off value was determined. The serums from animal models and 5 patients with final diagnosed, 24 patients clinical diagnosed invasive Aspergillosis were detected and the results compared with galactomannan （GM） test. Results Aspergillus fumigatus secretary proteins separated from Sabouraud＇s culture were selected as ELISA coating antigen because their strong reaction with serums of animal patient infected with Aspergillusfumigatus. The precision of ELISA was good with within-run CV 12.0% and between-run CV 8.1% respectively. The specificity of ELISA was confirmed by blocking assay. The cut-off value of 1.03 has been derived on basis of detecting 200 healthy sera. The positive rates of antibody were 2.0% （4/200） and 41.4% （ 12/29）, respectively. Two clinical diagnosed cases with GM negative but antibody positive （8.3%, 2/24）. Conclusions An ELISA-based method was developed for antibody against AspergiUusfumigatus secretary proteins and showed potential useful value in early diagnose of invasive Aspergillosis considering its well specificity and repeatability.