摘要
目的用制备好的兔和大鼠PP65多克隆抗体建立检测人巨细胞病毒PP65抗原的ELISA方法。方法优化ELISA检测条件,建立检测PP65抗原的标准曲线,确定检测方法的精密度及检出下限。通过对101份临床疑似HCMV感染标本的检测,并与HCMV-IgM(ELISA)以及HCMV-DNA(FQ-PCR)检测结果的比较,分析了该方法的敏感性和特异性。分别检测HSV-1、HSV-2以及EB病毒感染血标本各5份,分析该方法是否非特异结合疱疹病毒科其他病毒抗原。结果以1∶2 000大鼠pAb包被酶标板,以1∶20 000兔pAb为检测一抗,建立了检测PP65抗原血症的ELISA方法。批间精密度与批内精密度均小于10%,检出下限为5 ng/ml。本方法检测的敏感性和特异性与HCMV-DNA(FQ-PCR)无差别(P>0.05),但敏感性高于HCMV-IgM(ELISA)方法(P<0.05)。对HSV-1、HSV-2以及EB病毒感染标本的检测结果均为阴性,排除了该方法非特异结合疱疹病毒科其他病毒抗原的可能。结论建立了检测PP65抗原血症的间接双抗夹心ELISA方法,该方法具有较好的敏感性、特异性、稳定性和重复性。
This study designed to establish and evaluate a double-antibody sandwich ELISA for detecting HCMV-PP65 antigen. The method of ELISA was established by using 1：2 000 rat anti-PP65 polyclonal antibodies （pAb） and 1：20 000 rabbit anti-PP65 pAb as coating antibody and sandwich antibody respectively. Then the ELISA was optimized and the standard curve for PP65 detection was established. The precision and minimum detectable quantity of the method was analyzed by applying to 101 clinical suspected specimens and comparing with HCMV- DNA detection assay （FQ-PCR） and HCMV-IgM detection assay （ELISA）. The constructed method also used to detect HSV-1, HSV-2 and EB virus infecting samples for analyzing the specificity. Result showed the coefficients of variation within assay and between assays were all lower than 10%, while the minimum detectable quantity was 5 ng/ ml; the sensitivity and specificity did not differ from that of HCMV-DNA （FQ-PCR） （P〉 0.05）, and the sensitivity was higher than that of HCMV-IgM （ELISA） （P〈 0.05）. HSV-1, HSV-2 and EB virus infecting samples detection suggested that the assays did not react with other herpes virus. Hereto, the indirect sandwich ELISA detecting HCMV-PP65 antigen is established with higher sensitivity, specificity, stability and repeatability.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2014年第6期545-549,共5页
Immunological Journal
基金
山西省科技开发基金资助项目(033068)
