IDENTIFICATION OF IMMUNOGENIC GLYCOLIPIDS FROM BORRELIA BURGDORFERI

伯氏疏螺旋体免疫原性糖脂的鉴定

• 批准号: 7723061
• 负责人: ALLEN C STEERE
• 金额: $ 1.94万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723061
• 关键词: Aftercare; Antibiotics; Antibody Formation; Antigens; Arthritis; Biological Assay; Borrelia burgdorferi; Chromatography; Class; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; Development; Electrospray Ionization; Enzyme-Linked Immunosorbent Assay; Fractionation; Funding; Galactosides; Glycolipids; Goals; Grant; Immune response; Individual; Institution; Joints; Lipids; Lipoproteins; Lyme Arthritis; Lyme Disease; Mass Spectrum Analysis; Oral; Order Spirochaetales; Patients; Phase; Refractory; Research; Research Personnel; Resources; Serum; Source; Structure; Synovial Fluid; Testing; Ticks; Tissues; United States; United States National Institutes of Health; Work; immunogenic; killings; response; reversed phase chromatography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme disease is caused by the tick-transmitted spirochete Borrelia burgdorferi (Bb). In the United States, a small percentage of patients with Lyme arthritis have persistent arthritis for months or even several years after treatment with 2-3 months of oral and/or IV antibotic therapy, termed antibiotic-refractory Lyme arthritis. In these patients, the arthritis persists despite negative results for B. burgdorferi DNA in joint fluid and synovial tissue in the post-antibiotic period. This work is focused on identification of a Bb glycolipid that induces an immune response in patients with Lyme disease, but the response disappears predictably after spirochetal killing. Towards this goal, we will extract and fractionate Bb lipids, and test individual fractions using ELISA assays with sera from Lyme disease patients or control sera from uninfected donors. Normal phase chromatography will be employed for the initial lipid fractionation. This will separate lipids by (headgroup) class. Those fractions that are ELISA-positive will be sub-fractionated by reversed-phase chromatography, which separates lipids by their hydrophobic moieties. These sub-fractions will again be tested by ELISA. The goal is to fractionate the complex lipid extract, guided by serum reactivity, so that off-line mass spectrometry can be used to identify the structure of the immunogenic component. We have extracted total lipids from Bb and separated them by normal phase chromatography. We have analyzed lipid fractions by MALDI-TOF and ESI qoTOF mass spectrometry to assess their complexity and to identify intact lipids for subsequent tandem MS structural identification. To date, this analysis has identified, by intact mass, several previously identified Bb lipids, which will be used to identify possible immunogenic lipids or lipoproteins of the spirochete. We have also performed serum ELISA assays on acylated cholesteryl galactoside (ACG) purified from lipid fractions, an immunogenic glycolipid of Bb. Preliminary analysis shows that this lipid is immunogenic, and we are now working on the development of a reliable ELISA to assess the antibody response to this antigen.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 莱姆病是由tick滴的螺旋体玻璃体玻璃体burgdorferi（BB）引起的。在美国，一小部分莱姆关节炎患者患有2-3个月口服和/或IV抗抗体疗法治疗后的几个月甚至几年，称为抗生素 - 抗生素 - 抗生素 - 依赖性莱姆人关节炎。在这些患者中，尽管在抗生物后期，关节液和滑膜组织中的墨褐色​​芽孢杆菌DNA持续下降，但关节炎仍然存在。这项工作的重点是鉴定诱导莱姆病患者免疫反应的BB糖脂质，但螺旋颗粒杀死后的反应可以预见。为了实现这一目标，我们将提取和分级BB脂质，并使用来自莱姆病患者的血清或对照未感染供体的血清的ELISA测定进行测试。最初的脂质分馏将采用正常的相色谱法。这将通过（头组）类分开脂质。那些ELISA阳性的馏分将通过反相色谱法进行亚分级，从而通过其疏水部分将脂质分开。这些子裂缝将再次由ELISA测试。目的是在血清反应性的指导下对复合脂质提取物进行分割，以便可以使用离线质谱法来识别免疫原分成分的结构。 我们已经从BB中提取了总脂质，并通过正常相色谱分离它们。我们已经通过MALDI-TOF和ESI QOTOF质谱分析了脂质分数，以评估其复杂性并确定完整的脂质，以进行随后的串联MS结构鉴定。迄今为止，通过完整的质量确定了几种先前鉴定的BB脂质，这些脂质将用于鉴定螺旋体的可能的免疫原性脂质或脂蛋白。我们还对脂质级分纯化的酰化胆固醇（ACG）进行了血清ELISA分析，该脂质级分是BB的免疫原性糖脂。初步分析表明，这种脂质具有免疫原性，我们现在正在开发可靠的ELISA，以评估对该抗原的抗体反应。