Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Proteomics Clin Appl. 2007 Apr;1(4):400-9. doi: 10.1002/prca.200600805. Epub 2007 Mar 13.
Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.
目前用于钩端螺旋体病血清学诊断的方法各有其缺点，包括显微镜凝集试验（MAT）。因此，需要一种新的诊断方法来更早、更准确地诊断钩端螺旋体病。我们应用免疫蛋白质组学技术从 5 种钩端螺旋体参考株的血清型中鉴定潜在的免疫原。用每种血清型的全细胞裂解物作为抗原，与 4 例 MAT 阳性患者血清中的 IgG 和 IgM 反应。将 4 例 MAT 阴性的非钩端螺旋体病患者的血清混合作为阴性对照。2-D Western blot 分析显示，免疫反应程度与 MAT 滴度相对应。当使用混合对照血清时，未检测到免疫反应性斑点。用患者的 IgM 和/或 IgG 与 24 个蛋白斑点反应，这些免疫反应性蛋白通过 MALDI-TOF MS 鉴定，并分为 5 组，包括鞭毛蛋白、伴侣/热休克蛋白、转运蛋白、代谢酶和假设蛋白。用抗人 IgG 检测所有患者的血清和所有钩端螺旋体血清型时，检测到更多的免疫反应性斑点。一些鉴定的蛋白仅与 IgG 反应，而另一些蛋白则可与 IgM 和 IgG 同时检测到。在鉴定的免疫反应性蛋白中，FlaB 蛋白（鞭毛和鞭毛核心蛋白）已被证明在临床诊断和疫苗开发中有潜在作用。这些数据强调了免疫蛋白质组学在临床应用中的重要影响。
