一种用于嗜肺军团菌血清学诊断的新型酶联免疫吸附测定（ELISA）方法：使用五种纯化蛋白，即鞭毛蛋白（FLA）、主要外膜蛋白（MOMP）、巨噬细胞感染增强蛋白（MIP）、免疫原性蛋白（IP）和嗜肺军团菌脂蛋白（PILE）作为诊断抗原。

A new ELISA method for serological diagnosis of Legionella pneumophila: use of five purified proteins, FLA, MOMP, MIP, IP, and PILE, as diagnostic antigen.

作者信息

Sun Hui, Ma Haifeng, Liu Li, Cao Xiuqin, Yang Zhiwei

出版信息

Clin Lab. 2015;61(3-4):275-82. doi: 10.7754/clin.lab.2014.140908.

DOI:10.7754/clin.lab.2014.140908

PMID:25974993

Abstract

BACKGROUND

Legionella pneumophila plays an important role in human infection. Commercial ELISA kits commonly used, which take Legionella pneumophila whole-cell protein as the coating antigen, often have cross-reactivity among serogroups or species. In this study, five Legionella pneumophila proteins FLA, MOMP, MIP, IP, and PILE were purified and further applied in serological diagnosis of Legionella pneumophila infections compared with R & D Legionella ELISA kits.

METHODS

The five recombinant plasmids pET-fla, pET-momp, pET-mip, pET-ip, and pET-pile were transformed into E. coli BL21 and then induced them with IPTG. The expression products were analyzed by SDS-PAGE and purified by affinity chromatography. Indirect enzyme-linked immunosorbent assays (ELISAs) were established with the five purified proteins FLA, MOMP, MIP, IP, and PILE altogether as the coating antigen and tested for the presence of IgG, IgM, and IgA antibody independently from 50 positive sera and 40 negative sera, compared with R & D IgG, IgM, and IgA Legionella ELISA kits.

RESULTS

The FLA protein about 42 kDa in size, the MOMP protein about 45 kDa, the MIP protein about 40 kDa, the IP protein about 46 kDa, and the PILE protein about 35.7 kDa were separately expressed and purified. Compared with R & D IgG, IgM, and IgA Legionella ELISA kit, the outcome of indirect ELISAs set up with the five purified proteins showed that for IgG the sensitivity was 90.4%, the specificity was 97.4%, the area under ROC curve was 0.939, the kappa value was 0.865, the 95% confidence interval was 0.883 - 0.995. For IgM the sensitivity was 91.8%, the specificity was 95.1%, the area under ROC curve was 0.935, the kappa value was 0.866, the 95% confidence interval was 0.876 - 0.994. For IgA the sensitivity was 93.6%, the specificity was 95.3%, the area under ROC curve was 0.945, the kappa value was 0.889, the 95% confidence interval was 0.890 - 0.999.

CONCLUSIONS

The proteins FLA, MOMP, MIP, IP, and PILE were successfully expressed and purified, and they seemed to be suitable coating antigens for the serological diagnosis of Legionella pneumophila.

摘要

背景

嗜肺军团菌在人类感染中起重要作用。常用的商业ELISA试剂盒以嗜肺军团菌全细胞蛋白作为包被抗原，在血清群或菌种间常有交叉反应。在本研究中，对嗜肺军团菌的5种蛋白FLA、MOMP、MIP、IP和PILE进行了纯化，并与研发的军团菌ELISA试剂盒相比，进一步应用于嗜肺军团菌感染的血清学诊断。

方法

将5种重组质粒pET-fla、pET-momp、pET-mip、pET-ip和pET-pile转化至大肠杆菌BL21中，然后用IPTG诱导。通过SDS-PAGE分析表达产物，并通过亲和层析进行纯化。以纯化的FLA、MOMP、MIP、IP和PILE这5种蛋白作为包被抗原，建立间接酶联免疫吸附测定（ELISA），并分别检测50份阳性血清和40份阴性血清中IgG、IgM和IgA抗体的存在情况，与研发的IgG、IgM和IgA军团菌ELISA试剂盒进行比较。

结果

分别表达并纯化了大小约为42 kDa的FLA蛋白、约45 kDa的MOMP蛋白、约40 kDa的MIP蛋白、约46 kDa的IP蛋白和约35.7 kDa的PILE蛋白。与研发的IgG、IgM和IgA军团菌ELISA试剂盒相比，用这5种纯化蛋白建立的间接ELISA结果显示，对于IgG，敏感性为90.4%，特异性为97.4%，ROC曲线下面积为0.939，kappa值为0.865，95%置信区间为0.883 - 0.995。对于IgM，敏感性为�1.8%，特异性为95.1%，ROC曲线下面积为0.935，kappa值为0.866，95%置信区间为0.876 - 0.994。对于IgA，敏感性为93.6%，特异性为95.3%，ROC曲线下面积为0.945，kappa值为0.889，95%置信区间为0.890 - 0.999。