检测3种侵袭性真菌感染相关抗体的蛋白质芯片法

Development of a protein microarray method for detecting three kinds of antibodies associated with invasive fungal infections

目的用念珠菌烯醇化酶(Eno)、果糖二磷酸醛缩酶(Fba1)及烟曲霉硫氧还蛋白还原酶(TR)3种基因重组蛋白质,建立检测这3种蛋白质特异性抗体的蛋白质芯片法,并进行临床应用评价。方法用重组Eno、Fba1和TR作为固相抗原构建蛋白质芯片。对芯片制备及血清中相应抗体测定的反应条件进行优化。对方法的重复性、稳定性及特异性进行考察,并测定临床确诊的侵袭性念珠菌病(IC)、侵袭性曲霉病(IA)患者血清中的相应抗体。结果蛋白质芯片以硝酸纤维素膜为固相载体,以Tris-HCl为载样液,重组Eno、Fba1、TR最适点阵浓度分别为0.5 mg/mL、0.04 mg/mL和1.0 mg/mL,用含50 g/L脱脂奶粉的PBS溶液封闭,待测血清稀释度为1∶10。抗Eno抗体和抗Fba1抗体诊断IC的敏感性和特异性分别为67.6%(71/105)和96.5%(299/310),64.8%(68/105)和90.3%(280/310);联合检测抗Eno抗体和抗Fba1抗体,可将敏感性提高至81.0%。抗TR抗体诊断IA的敏感性为71.4%(30/42),特异性为98.1%(366/373)。结论建立了同时检测两种真菌的3种不同抗体的蛋白质芯片方法,敏感性尚可,可用于高危人群的筛查。

Objective To develop a protein microarray assay for detecting antibodies associated with invasive fungal infections using recombinant proteins, including enolase （Eno） and fructose-bisphospbate aldolase （Fbal） of Candida, and thioredoxin reductase （TR） of Aspergillusfumigatus, and evaluate its clinical application. Methods Purified recombinant Eno, Fbal and TR proteins were used as the immobilized antigens to construct a protein microarray. Then, the conditions for the microarray reacted with the antibodies in serum were optimized, and the sensitivity, specificity, reproducibility and stability of the assay evaluated. Further, the antibodies to Eno, Fbal and TR proteins in serum from the patients with clinically diagnosed invasive candidosis （IC） and invasive aspergillosis （IA） were detected. Results Nitrocellulose filter was used as solid carrier of the protein microarray, and Tris-HC1 as loading buffer. The loading concentrations of Eno, Fbal and TR proteins were 0.5 mg/mL, O. 04 mg/mL and 1.0 mg/mL, respectively. PBS with 50 g/L skimmed milk was used as blocking buffer. The dilution of the patients＇ serum was 1： 10. The sensitivity and specificity of anti-Eno antibody for diagnosing IC were 67.6% （71/105） and 96.5% （299/310）, respectively, while those of anti-Fbal antibody were 64.8% （68/105） and 90.3% （280/310） , respectively. When the anti-Eno and anti-Fbal antibodies were combined, the detection sensitivity increased to 81%. In addition, the sensitivity and specificity of anti-TR antibody for diagnosing IA were 71.4% （30/42） and 98.1% （366/373）, respectively. Conclusion A protein microarray assay with satisfactory sensitivity and specificity was suc- cessfully developed, which could be used to detect anti^Eno, anti-Fbal and anti-TR antibodies from IA and IC patients simuhaneously.