Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach.
Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach.
复制标题
DOI：
10.1371/journal.pntd.0002499
复制
发表时间：
2013
影响因子：
3.8
通讯作者：
Felgner PL
中科院分区：
文献类型：
--
作者：
Lessa-Aquino C;Borges Rodrigues C;Pablo J;Sasaki R;Jasinskas A;Liang L;Wunder EA Jr;Ribeiro GS;Vigil A;Galler R;Molina D;Liang X;Reis MG;Ko AI;Medeiros MA;Felgner PL
钩端螺旋体病是一种世界性的人畜共患病。缺乏足够的实验室检查是诊断的主要障碍，特别是在疾病的早期阶段，抗生素治疗是最有效的。因此，迫切需要一种针对这种威胁生命的疾病的有效诊断测试。为了确定新的目标，可用作诊断标志物钩端螺旋体病，我们构建了一个蛋白质芯片，包括61%的问号钩端螺旋体蛋白质组和调查的IgG反应从274个人，包括80个急性期，80个恢复期患者和114个健康对照受试者从地方性，高度地方性和地方性传播的钩端螺旋体病。进行硝酸纤维素线印迹分析以验证蛋白质微阵列结果的准确性。我们发现了16种抗原，可以区分急性病例和健康个体从一个地区的地方性传播的钩端螺旋体病，和18个抗原，区分恢复期病例。本研究中鉴定的一些抗原，如LipL32、Lig蛋白的不同结构域、GroEL和Loa22，已知可被来自人类患者的血清识别，因此可用作血清诊断抗原发现方法的概念验证。鉴定了几种新的抗原，包括假设的蛋白质LIC 10215，其对急性期和恢复期患者均显示出良好的灵敏度和特异性。我们的研究是第一次大规模的评估与自然获得性钩端螺旋体感染相关的免疫显性抗原，并确定了新的以及已知的血清诊断钩端螺旋体抗原，这些抗原被钩端螺旋体病病例血清中的抗体所识别。这里确定的新抗原可能有潜在的用途，在开发新的测试和改善目前可用的检测方法诊断这种被忽视的热带疾病。需要进一步的研究来评估这些抗原在更可部署的诊断平台中的效用。钩端螺旋体病是一种人畜共患传染病，在人类中引起非特异性体征和症状，这阻碍了医生的临床诊断和治疗。如果在病程早期没有开始适当的治疗，可能会发生并发症。虽然早期诊断对于预防不必要的并发症至关重要，但目前可用的测试在疾病开始时没有表现出足够的诊断灵敏度。我们利用高通量技术进行了一个拥抱的体液免疫反应的细菌，以确定抗原，可用于诊断钩端螺旋体病的新测试的研究。构建了包含2，241个钩端螺旋体蛋白的蛋白质微阵列芯片，并使用来自患者和健康个体的血清样品进行探测。我们鉴定了24种被患者血清识别但不被健康个体识别的蛋白质。这些蛋白质是潜在的诊断标志物，特别是急性期患者的诊断标志物，可以在症状出现后几天内区分阳性和阴性钩端螺旋体病。这项工作建立了蛋白质微阵列的方法，以提高我们对钩端螺旋体病的血清学反应的理解。需要进一步的研究来评估这些抗原在临床环境中的性能。
Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms. Leptospirosis is an infectious zoonotic disease that causes non-specific signs and symptoms in humans, which hampers the clinical diagnosis and treatment by physicians. Complications can occur if the proper treatment is not initiated early in the course of illness. Although the early diagnosis is critical for preventing unnecessary complications, currently available tests do not exhibit sufficient diagnostic sensitivity in the beginning of disease. We took advantage of high throughput techniques to perform an embracing study of the humoral immune response to the bacteria in order to identify antigens that could be used in a new test for the diagnosis of leptospirosis. A protein microarray chip containing 2,241 leptospiral proteins was constructed and probed with serum samples from patients and healthy individuals. We identified 24 proteins that are recognized by patients' sera but not by healthy individuals. These proteins are potential diagnostic markers, especially the ones identified for acute-phase patients, which can discriminate between a positive and a negative leptospirosis case within a few days after onset of symptoms. This work establishes the protein microarray approach for improving our understanding of the serological response to leptospirosis. Further research is needed to assess the performance of these antigens in the clinical setting.
登录
查看更多内容
影响因子：
64.8
作者：
Malmström J;Beck M;Schmidt A;Lange V;Deutsch EW;Aebersold R
通讯作者：
Aebersold R
影响因子：
9.4
作者：
Flannery, B;Costa, D;Ko, AI
通讯作者：
Ko, AI
影响因子：
5.5
作者：
Koizumi, N;Watanabe, H
通讯作者：
Watanabe, H
影响因子：
3.1
作者：
Guerreiro, H;Croda, J;Haake, DA
通讯作者：
Haake, DA
DOI：
10.1073/pnas.0408782102
发表时间：
2005-01-18
影响因子：
11.1
作者：
Davies, DH;Liang, XW;Felgner, PL
通讯作者：
Felgner, PL