Mycobacterium Tuberculosis Lipoarabinomannan In Vivo

体内结核分枝杆菌脂阿拉伯甘露聚糖

• 批准号: 8243889
• 负责人: Libin Shi
• 金额: $ 21.47万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8243889
• 关键词: Acid Fast Bacillae Staining Method; Address; Animal Model; Antibodies; Antigens; Arabinose; Bacillus (bacterium); Biological; Biological Assay; Biological Markers; Blood Circulation; Carbohydrates; Cavia; Cell Wall; Charge; Chemicals; Chromatography; Clinical; Communicable Diseases; Communities; Country; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Digestion; Disease; Early Diagnosis; Electrospray Ionization; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitope Mapping; Epitopes; Failure; Fingerprint; Future; Genetic screening method; Glycoside Hydrolases; Goals; HIV Infections; Health Services Accessibility; Human; Hydrophobic Interactions; Immunodominant Antigens; In Vitro; Infection; Kidney; Lead; Leprosy; Liquid Chromatography; Liquid substance; Liver; Maps; Mass Fragmentography; Methods; Microscopy; Modification; Mycobacterium tuberculosis; Mycobacterium tuberculosis H37Rv; Neuraminidase; Oligosaccharides; Organ; Patients; Performance; Phenotype; Polysaccharides; Protocols documentation; Reaction; Research; Resources; Running; Sepharose; Serum; Specimen; Spleen; Sputum; Structure; Succinates; Testing; Time; Tissues; Tuberculosis; Urine; Validation; Virulent; Western Blotting; Work; World Health Organization; analytical method; araban; arabinogalactan; base; cost; in vivo; innovation; ionization; lipoarabinomannan; pulmonary granuloma; research study

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a treatable infectious disease, however, the lack of efficient diagnostic tests are important bottleneck impeding access to treatment. Thus, simple, sensitive and specific diagnostic approaches are desperately needed. An antigen detection assay for TB is uniquely attractive because tests can be performed using a variety of patient specimens. The urine lipoarabinomannan- enzyme-linked immunosorbent assay (LAM-ELISA) detections are attractive, especially for the patients with HIV infection, unfortunately, the sensitivity of these assays is low, which makes this test suboptimal. The question we ask is why the sensitivity of LAM-ELISA detection is inefficient? LAM from Mtb grown in vivo may have a structure quite unique and distinct from its in vitro counterpart and it could have distinct modifications such as succinylation, mannosylation and substitution with methythioxylofuranose which will lend credence to its sensitivity. If so, it will explain the failure in reaction with antibodies included in the commercial kits to capture LAM in biofluid as all are raised against in vitro grown Mtb or LAM isolated from Mtb grown in vitro. Thus in this proposal we propose to exploit our unique ability to extract LAM from lung, granulomas, liver and kidneys from experimentally infected guinea pigs with different clinical strains, and to define the nonreducing end exhaustively focusing on the terminal residue modifications since they are the epitopes for the anti-LAM antibodies recognition. Initial proof of concept experiments has shown that the yield of LAM in vivo will not be sufficient for the structural analysis using the methods for the structural analysis on LAM from the in vitro grown bacilli. Therefore, our structural studies will rely heavily on use of more sensitive tests such as Gas Chromatography-Mass Spectrometry (GC-MS), enzyme digestion, and mapping of the oligosaccharides with Electrospray Ionization. As a fingerprint, in vitro grown bacilli from clinical isolates with definite phenotype will be included in the protocol. The definition of the terminal ends of LAM in vivo will impact the future when a sensitive and specific anti-LAM antibody can be generated based on this study to enhance LAM based existing assays for use in resource poor regions. PUBLIC HEALTH RELEVANCE: Lipoarabinomannan (LAM) from Mycobacterium tuberculosis (Mtb) in vivo is distinct to LAM from Mtb in vitro. The structural analysis of the nonreducing terminal motifs of LAM from Mtb in vivo will be the major goal in this proposed project, which will be beneficial for generating sensitive antibodies against LAM from Mtb in vivo to detect the LAM in specimens like urine and will have a profound effect on the TB diagnosis in the future.

描述（由申请人提供）：结核病（TB）是一种可治疗的传染病，然而，缺乏有效的诊断测试是阻碍获得治疗的重要瓶颈。因此，迫切需要简单、灵敏且特异的诊断方法。结核病抗原检测分析具有独特的吸引力，因为可以使用各种患者样本进行检测。尿液阿拉伯脂甘露聚糖酶联免疫吸附测定 (LAM-ELISA) 检测很有吸引力，特别是对于 HIV 感染患者，但不幸的是，这些测定的灵敏度较低，这使得该测试不是最理想的。我们要问的问题是为什么LAM-ELISA检测的灵敏度低下？来自体内生长的 Mtb 的 LAM 可能具有非常独特的结构，与体外对应物不同，并且可能具有不同的修饰，例如琥珀酰化、甘露糖基化和甲硫氧基呋喃糖取代，这将增强其敏感性。如果是这样，这将解释为什么与商业试剂盒中包含的抗体反应未能捕获生物液中的 LAM，因为所有抗体都是针对体外生长的 Mtb 或从体外生长的 Mtb 中分离出的 LAM 产生的。因此，在本提案中，我们建议利用我们独特的能力，从不同临床毒株的实验感染豚鼠的肺、肉芽肿、肝脏和肾脏中提取 LAM，并详尽地定义非还原端，重点关注末端残基修饰，因为它们是表位用于抗LAM抗体识别。初步概念验证实验表明，使用体外生长的杆菌对 LAM 进行结构分析的方法，体内 LAM 的产量不足以进行结构分析。因此，我们的结构研究将在很大程度上依赖于使用更灵敏的测试，例如气相色谱-质谱法（GC-MS）、酶消化和电喷雾电离寡糖图谱。作为指纹，来自具有明确表型的临床分离株的体外生长的杆菌将包含在方案中。体内 LAM 末端的定义将影响未来，届时可以根据本研究生成敏感且特异的抗 LAM 抗体，以增强基于 LAM 的现有检测方法，以便在资源匮乏地区使用。 公共健康相关性：体内结核分枝杆菌 (Mtb) 的脂阿拉伯甘露聚糖 (LAM) 与体外 Mtb 的 LAM 不同。体内 Mtb 的 LAM 非还原末端基序的结构分析将是本项目的主要目标，这将有利于体内产生针对 Mtb 的 LAM 的敏感抗体，以检测尿液等样本中的 LAM，并将具有对今后结核病的诊断产生深远的影响。