双抗原夹心ELISA法检测烟曲霉Afmp1cr/Afmp2cr抗体  

Double- antigen sandwich ELISA for detecting Aspergillus fumigatus anti- Afmp1cr and Afmp2cr antibodies

作　　者：杨梅[1] 王卓娅[1] 郝卫[1] 王艳芳[1] 黄莉[1] 蔡建飘[2] 江凌晓[1] 车小燕[1] 钟晓祝[1] 余楠[1] 

机构地区：[1]南方医科大学珠江医院,广东广州510282 [2]香港大学微生物系

出　　处：《南方医科大学学报》2014年第5期646-650,共5页Journal of Southern Medical University

基　　金：国家自然科学基金青年项目(81101320;81101314);广东省医学科研基金(B2012173)~~

摘　　要：目的建立检测烟曲霉特异性抗体的双抗原夹心ELISA方法。方法用毕赤酵母系统表达重组的烟曲霉特异性甘露聚糖蛋白片段Afmp1cr和Afmp2cr,经棋盘滴定法建立可检测烟曲霉特异性抗体的双抗原夹心ELISA方法。用Afmp1cr和Afmp2cr分别免疫兔血清评估方法灵敏度,健康人血清、其它微生物感染者血清评估特异性,并检测曲霉感染者血清。结果 Afmp1cr双抗原夹心ELISA法可捕获1∶800稀释兔多抗血清中的抗体,Afmp2cr双抗原夹心ELISA法可捕获1∶3200稀释兔多抗血清中的抗体,两者均与其他真菌及细菌无交叉反应。2例确诊曲霉感染和1例拟诊曲霉感染患者血清中能检测出抗体。结论初步建立了检测anti-Afmp1cr/Afmp2cr抗体的双抗原夹心ELISA方法,可辅助诊断临床烟曲霉感染。Objective To establish two double-antigen sandwich ELISA systems to detect anti-Afmp1cr and Afmp2cr antibodies of Aspergillus fumigatus. Methods Recombinant Afmp1cr and Afmp2cr proteins of A.fumigatus expressed in Pichia pastoris were obtained. Double-antigen sandwich ELISA systems for detecting specific anti-Afmp1cr and anti-Afmp2cr antibodies were developed after chessboard titrating to determine the appropriate concentrations of the recombinant proteins and HRP-labeled proteins. The sensitivity of the assay was evaluated using serum samples of rabbits immunized with Afmp1cr and Afmp2cr. The specificity of the assay was evaluated by detecting serum samples from healthy donors and patients with other pathogenic fungal and baterial infections. The performance of the two ELISA kits was furthered evaluated using serum samples from patients with suspected Aspergillus infection. Results The established ELISA kits were capable of detecting anti-Afmp1cr and anti-Afmp2cr antibodies in immunized rabbit serum at the maximum dilutions of 800 and 3200, respectively. No cross-reactivity was observed in detecting serum from patients with other pathogenic fungal or bactetial infections. Both of the two kits yielded positive results in sera from two established Aspergillus-infected cases and a suspected case. Conclusion Two antibody-capture ELISA kits were developed for the laboratory diagnosis of A.fumigatus infection and can be potentially useful in the clinical diagnosis of Aspergillosis infections.

关 键 词：烟曲霉 双抗原夹心 ELISA法 抗体检测 Afmp1p Afmp2cr Afmp2cr 

分 类 号：R446.6[医药卫生—诊断学]