双抗原夹心ELISA法检测烟曲霉Afmp1cr/Afmp2cr抗体
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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 crossreactivity 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.
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 crossreactivity 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.
引文
[1]Woo PC,Chong KT,Leung AS,et al.AFLMP1 encodes an antigenic cel wall protein in Aspergillus flavus[J].J Clin Microbiol,2003,41(2):845-50.
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[3]王丽.烟曲霉基因组与致病机制[J].中国真菌学杂志,2011,6(1):1-4.
[4]Park HS,Bayram O,Braus GH,et al.Characterization of the velvet regulators in Aspergillus fumigatus[J].Mol Microbiol,2012,86(4):937-53.
[5]中华内科编辑委员会.侵袭性肺部真菌感染的诊断标准与治疗原则(草案)[J].中华内科杂志,2006,45(8):697-700.
[6]Moreira-Oliveira MS,Mikami Y,Miyaji M,et al.Diagnosis of candidemia by polymerase chain reaction and blood culture:prospective study in a high-risk population and identification of variables associated with development of candidemia[J].Eur J Clin Microbiol Infect Dis,2005,24(11):721-6.
[7]Racil Z,Kocmanova I,Lengerova M,et al.Difficulties in using 1,3-{beta}-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies--high frequency of false-positive results and their analysis[J].J Med Microbiol,2010,59(Pt 9):1016-22.
[8]Yuen KY,Chan CM,Chan KM,et al.Characterization of AFMP1:a novel target for serodiagnosis of aspergillosis[J].J Clin Microbiol,2001,39(11):3830-7.
[9]Chong KT,Woo PC,Lau SK,et al.AFMP2 encodes a novel immunogenic protein of the antigenic mannoprotein superfamily in Aspergillus fumigatus[J].J Clin Microbiol,2004,42(5):2287-91.
[10]Wang ZY,Cai JP,Qiu LW,et al.Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models[J].Eur J Clin Microbiol Infect Dis,2012,31(11):2943-50.
[11]郝卫,钟善传,徐华,等.健康人血清中烟曲霉菌特异性IgG水平及其意义[J].中华结核和呼吸杂志,2003,26(12):795-795.
[12]Chan CM,Woo PC,Leung AS,et al.Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis[J].J Clin Microbiol,2002,40(6):2041-5.
[13]Sarfati J,Monod M,Recco P,et al.Recombinant antigens as diagnostic markers for aspergillosis[J].Diagn Microbiol Infect Dis,2006,55(4):279-91.
[14]Wong SS,Wong KH,Hui WT,et al.Differences in clinical and laboratory diagnostic characteristics of penicilliosis marneffei in human immunodeficiency virus(HIV)-and non-HIV-infected patients[J].J Clin Microbiol,2001,39(12):4535-40.
[15]Brandt ME,Lockhart SR,Warnock DW.Laboratory aspects of medical mycology//Essentials of Clinical Mycology[M].2011:3-25.
[16]Shi LN,Li FQ,Lu JF,et al.Antibody specific to thioredoxin reductase as a new biomarker for serodiagnosis of invasive aspergillosis in non-neutropenic patients[J].Clin Chim Acta,2012,413(9/10):938-43.
[2]Barton RC.Laboratory diagnosis of invasive aspergillosis:from diagnosis to prediction of outcome[J].Scientifica(Cairo),2013:459405.
[3]王丽.烟曲霉基因组与致病机制[J].中国真菌学杂志,2011,6(1):1-4.
[4]Park HS,Bayram O,Braus GH,et al.Characterization of the velvet regulators in Aspergillus fumigatus[J].Mol Microbiol,2012,86(4):937-53.
[5]中华内科编辑委员会.侵袭性肺部真菌感染的诊断标准与治疗原则(草案)[J].中华内科杂志,2006,45(8):697-700.
[6]Moreira-Oliveira MS,Mikami Y,Miyaji M,et al.Diagnosis of candidemia by polymerase chain reaction and blood culture:prospective study in a high-risk population and identification of variables associated with development of candidemia[J].Eur J Clin Microbiol Infect Dis,2005,24(11):721-6.
[7]Racil Z,Kocmanova I,Lengerova M,et al.Difficulties in using 1,3-{beta}-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies--high frequency of false-positive results and their analysis[J].J Med Microbiol,2010,59(Pt 9):1016-22.
[8]Yuen KY,Chan CM,Chan KM,et al.Characterization of AFMP1:a novel target for serodiagnosis of aspergillosis[J].J Clin Microbiol,2001,39(11):3830-7.
[9]Chong KT,Woo PC,Lau SK,et al.AFMP2 encodes a novel immunogenic protein of the antigenic mannoprotein superfamily in Aspergillus fumigatus[J].J Clin Microbiol,2004,42(5):2287-91.
[10]Wang ZY,Cai JP,Qiu LW,et al.Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models[J].Eur J Clin Microbiol Infect Dis,2012,31(11):2943-50.
[11]郝卫,钟善传,徐华,等.健康人血清中烟曲霉菌特异性IgG水平及其意义[J].中华结核和呼吸杂志,2003,26(12):795-795.
[12]Chan CM,Woo PC,Leung AS,et al.Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis[J].J Clin Microbiol,2002,40(6):2041-5.
[13]Sarfati J,Monod M,Recco P,et al.Recombinant antigens as diagnostic markers for aspergillosis[J].Diagn Microbiol Infect Dis,2006,55(4):279-91.
[14]Wong SS,Wong KH,Hui WT,et al.Differences in clinical and laboratory diagnostic characteristics of penicilliosis marneffei in human immunodeficiency virus(HIV)-and non-HIV-infected patients[J].J Clin Microbiol,2001,39(12):4535-40.
[15]Brandt ME,Lockhart SR,Warnock DW.Laboratory aspects of medical mycology//Essentials of Clinical Mycology[M].2011:3-25.
[16]Shi LN,Li FQ,Lu JF,et al.Antibody specific to thioredoxin reductase as a new biomarker for serodiagnosis of invasive aspergillosis in non-neutropenic patients[J].Clin Chim Acta,2012,413(9/10):938-43.