白念珠菌烯醇化酶双抗体夹心ELISA检测方法的建立

Development of a sandwich ELISA for the detection of enolase from Candida albicans

目的建立检测白念珠菌烯醇化酶(enolase,Eno)的双抗体夹心ELISA法,并试用于几种真菌培养上清液的检测。方法将抗白念珠菌Eno单抗作为包被抗体,辣根过氧化物酶标记的羊抗Eno抗体作为检测抗体,用方阵滴定法确定包被抗体和酶标抗体浓度,建立检测Eno的ELISA法,对方法的精密度、特异性、最低检测限等指标进行评价。用该法检测白念珠菌、热带念珠菌、光滑念珠菌、近平滑念珠菌、季也蒙念珠菌、新生隐球菌和酿酒酵母等不同真菌培养上清中Eno水平。结果 Eno浓度为20 ng/mL和5 ng/mL时,批内和批间变异系数(CV)分别为6.61%、9.19%和6.98%、13.81%;最低检测限为1.25ng/mL。本法可从白念珠菌37℃培养24 h后的上清中检出Eno,Eno含量与白念珠菌菌丝含量呈正相关。本法对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、新生隐球菌和酿酒酵母均无交叉反应。结论建立的检测白念珠菌Eno的双抗体夹心ELISA方法有较好的特异性,可用于评价Eno检测在侵袭性白念珠菌感染中的诊断价值。

Objective To develop a sandwich ELISA for the detection of enolase from Candida albicans, and verify it with the super- natant from 7 fungi cultures. Methods The monoclonal antibody to Candida albicans enolase and HRP-eonjugated goat polyclonal an- tibody to enolase were used as coating antibody and detecting antibody, respectively. Then, the optimal concentrations of coating anti- body and detecting antibody were determined with the square matrix titration. Next, the precision, specificity and limit of detection of the established sandwich ELISA to detect the enolase from Candida albicans were evaluated. Finally, the levels of enolase in the super- natant from Candida albicans, Candida tropicalis, Candida guilliermondii, Candida glabrata, Candida parapsilosis, Cryptococcus neo- formans and Saccharomyces cerevisiae cultures were detected with the established sandwich ELISA. Results The intra-coefficient of variation （CV） and inter-CV were 6.61% and 9.19%, respectively, for 20 ng/mL of enolase, and 6.98% and 13.81% for 5 ng/mL of enolase. The limit of detection was 1.25 ng/mL. The established sandwich ELISA could detect the enolase from the Candida albi- cans culture incubated at 37 ℃ for 24 h, and the level of enolase increased with the hyphae growth. The ELISA method to detect Can- dida albicans had no cross-reactivity with Candida tropicalis, Candida guilliermondii, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae except Candida parapsilosis with weak cross reactivity. Conclusion The established sandwich ELISA method can specifically detect the enolase from Candida albicans, which will be helpful to assess the diagnostic value of enolase in invasive candidiasis.