白色念珠菌烯醇化酶双抗体夹心ELISA定量检测方法的建立
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国家自然科学基金青年科学基金项目(81302536);江苏省科技支撑计划-社会发展项目(BE2009673) ;南京军区医学科技创新重点课题(10Z027)


Development of a sandwich ELISA for quantitative detection enolase of Candida albicans
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    摘要:

    目的:建立定量检测白色念珠菌烯醇化酶(enolase,Eno)的双抗体夹心ELISA法,并应用于不同真菌培养上清液的检测,探讨其在侵袭性念珠菌感染中潜在的诊断价值-方法:将白色念珠菌Eno单抗作为包被抗体,HRP标记的羊抗Eno作为检测抗体,利用重组白色念珠菌Eno蛋白作为标准品评价该方法的精密度-线性范围-最低检测下限等性能指标-用建立的双抗体夹心ELISA法定量检测不同真菌培养上清和白色念珠菌在不同温度下培养上清中Eno水平-结果:Eno浓度为20 ng/ml和5 ng/ml时,批内和批间精密度分别为6.61%-9.19%和6.98%-13.81%,最低检出下限为1.25 ng/ml,线性范围为1.25~50.00 ng/ml-白色念珠菌37℃培养24 h时的上清中Eno含量为3.06 ng/ml,培养120 h时Eno水平上升到33.43 ng/ml,Eno水平与白色念珠菌菌丝含量呈正相关-本方法与近平滑念珠菌有微弱交叉反应,与热带念珠菌-光滑念珠菌-季也蒙念珠菌-新型隐球菌和酿酒酵母均无交叉反应-结论:成功建立了定量检测白色念珠菌Eno的双抗体夹心ELISA法,在侵袭性念珠菌感染研究中具有潜在的应用价值-

    Abstract:

    Objective:To develop a sandwich ELISA for quantitative detection enolase of Candida albicans,and apply the assay to detect enolase levels in supernatant of common fungi cultures. To investigate the potential diagnostic value of enolase in invasive candidiasis. Methods:Anti-enolase of Candida albicans monoclonal antibody was employed as coating antibody,HRP-conjugated goat polyclonal antibody against Candida albicans enolase was used as detecting antibody. The performance parameters of the sandwich ELISA including precision,linear range and limit of detection were verified by using recombinant Candida albicans enolase. Then the developed assay was applied to determine enolase levels in supernatant of pathogenic fungi cultures such as common Candida spp. Cryptococcus neoformans and Saccharomyces cerevisiae for preliminary evaluation. Results:The intra and inter-coefficient of variation was 6.61%,9.19% and 6.98%,13.81% at the concentration of 20 ng/ml and 5 ng/ml respectively. The limit of detection was 1.25 ng/ml. The linear range was 1.25~50.00 ng/ml. The level of enolase in Candida albicans culture after incubated in 37℃ for 24 h was 3.06 ng/ml and gradually increased to 33.43 ng/ml at 120 h after incubated in 37℃,consistent with growth of hyphae. The sandwich ELISA was weak cross-reactive to Candida parapsilosis and no cross-reactivity to Candida tropicalis,Candida guilliermondii,Candida glabrata,Cryptococcus neoformans and Saccharomyces cerevisiae was found. Conclusion:A sandwich ELISA for quantitative detection enolase of Candida albicans was developed,which had the potential to be applied to stndy invasive candidasis.

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胡毓安,史利宁,李芳秋,李 伟,马春芳.白色念珠菌烯醇化酶双抗体夹心ELISA定量检测方法的建立[J].南京医科大学学报(自然科学版),2014,(6):826-830

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  • 收稿日期:2014-02-23
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  • 在线发布日期: 2014-06-19
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