Rapid Detection of Antibodies to Adenovirus 36

腺病毒 36 抗体的快速检测

• 批准号: 8491482
• 负责人: Cynthia L Chappell
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-13 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8491482
• 关键词: Address; Adenovirus hexon capsid protein; Adenoviruses; Adipose tissue; Adult; Animals; Antibodies; Area; Biological Assay; Capsid Proteins; Cell Culture Techniques; Cells; Chickens; Child; Chimeric Proteins; Complementarity Determining Regions; Detection; Development; Disadvantaged; Enzyme-Linked Immunosorbent Assay; Epidemic; Epidemiologic Studies; Epitopes; Etiology; Failure; Gold; High Prevalence; Human; Human Adenoviruses; Human Development; Individual; Infection; Intervention; Laboratories; Lead; Life; Lipids; Longitudinal Studies; Maintenance; Metabolic; Methodology; Methods; Molecular Target; Mus; Non obese; Obesity; Peptides; Population; Population Study; Prevalence; Prevention; Proteins; Reagent; Recombinant Fusion Proteins; Recombinant Proteins; Recombinants; Reporting; Reproducibility; Research; Research Personnel; Risk Factors; Role; Sampling; Sensitivity and Specificity; Series; Serological; Serum; System; Technical Expertise; Testing; Time; Training; Up-Regulation; Validation; Virus; Weight Gain; Work; base; cost effective; experience; fasting glucose; glucose uptake; improved; infected vector rodent; lipid biosynthesis; neutralizing antibody; nonhuman primate; novel; polyclonal antibody; public health relevance; rapid detection; rapid technique; research study; success

DESCRIPTION (provided by applicant): Adenovirus 36 (AdV36) causes increased adiposity (leading to obesity) and metabolic changes in animals. Causation in humans has not been established, but the infection is associated with the same changes as seen in animals, i.e. increased adiposity, weight gain, and decreases in serum lipid and fasting glucose. Elucidation of AdV36's role in the current obesity epidemic will rely, in large part, on longitudinal studies o large populations. Also, understanding AdV36's effects may have implications for the planning and success of novel intervention strategies for obesity. Given the strong association of AdV36 and subsequent effects in infected humans, failure to understand AdV36's role in obesity would ignore a key risk factor in obesity and continue to follow an obesity paradigm that has largely failed to lead to effective prevention and control strategies. A major methodological barrier exists to studies of large populations needed to move the field forward. That is, the gold standard and currently the only specific method for detecting AdV36 antibodies is the serum neutralization assay (SNA)-a complicated and time-consuming method that has produced inconsistent results among laboratories. In its place, a rapid, high throughput, easy to perform, and objective assay is urgently needed to open the field to new investigators, advance the pace of current studies, and improve consistency of results. This application will develop an improved assay by identifying specific epitopes on the AdV36 hexon protein and using recombinant proteins and/or peptides in a simple ELISA-based system to detect evidence of AdV36 infection, i.e. specific (neutralizing) antibodies. The AdV hexon protein is involved in initial interactions between the virus and host cell and contains specific neutralization epitopes. The AdV36 hexon sequence has been compared to other adenoviruses, revealing nine hypervariable regions (HVRs) likely to contain one or more specific epitopes. We have cloned and expressed four recombinant fusion proteins containing AdV36 HVRs and tested each using SNA-identified positive or negative sera. Two recombinants show promise for development of a sensitive and specific assay but require further characterization and validation. Further, overlapping peptides based on HVR sequences from these recombinants will be synthesized and tested for use in an assay system. Recombinants and/or peptides identified as containing AdV36-specific epitopes will be used in an ELISA to test 300 individual sera with known SNA titers. Comparison of results between the two test systems will demonstrate the sensitivity and specificity of the new assay. In summary, a sensitive, specific, and rapid assay system that no longer depends on cell culture and live virus is essential. Without an improved methodology, studies of AdV36's role in obesity will be slow, carried out by only a few investigators, and fraught with inter-laboratory variability in results.

描述(申请人提供)：腺病毒36(AdV36)会导致动物肥胖增加(导致肥胖)和代谢变化。人类的病因尚未确定，但感染与动物相同的变化有关，即肥胖增加、体重增加以及血脂和空腹血糖下降。AdV36的S在当前肥胖流行中的作用的阐明将在很大程度上依赖于对大量人群的纵向研究。此外，了解AdV36的S效应可能对肥胖新干预策略的规划和成功具有一定的意义。鉴于AdV36在感染人群中的强大关联性和随后的影响，如果不了解AdV36在肥胖中的作用，将忽略肥胖的一个关键风险因素，并继续遵循在很大程度上未能导致有效预防和控制策略的肥胖范例。对推动这一领域向前发展所需的大量人口的研究存在一个主要的方法障碍。也就是说，检测AdV36抗体的金标准和目前唯一的特异性方法是血清中和试验(SNA)，这是一种复杂而耗时的方法，在实验室之间产生了不一致的结果。取而代之的是，迫切需要一种快速、高通量、易于操作和客观的检测方法，以向新的研究人员开放该领域，加快现有研究的步伐，并提高结果的一致性。这项应用将开发一种改进的检测方法，通过识别AdV36六邻体蛋白上的特定表位，并在基于简单ELISA的系统中使用重组蛋白和/或肽来检测AdV36感染的证据，即特异性(中和)抗体。ADV六邻体蛋白参与病毒与宿主细胞的初始相互作用，并含有特定的中和表位。AdV36六邻体序列已经与其他腺病毒进行了比较，发现了九个可能包含一个或多个特定表位的高变区(HVR)。我们克隆和表达了四个含有AdV36 HVRs的重组融合蛋白，并用SNA鉴定的阳性或阴性血清对每个融合蛋白进行了检测。两个重组体显示出开发灵敏和特异的检测方法的前景，但需要进一步的表征和验证。此外，基于来自这些重组子的HVR序列的重叠多肽将被合成并被测试用于检测系统。经鉴定含有AdV36特异性表位的重组体和/或多肽将用于检测300份已知SNA滴度的血清。两个测试系统之间的结果比较将证明新的检测方法的敏感性和特异性。总之，一个不再依赖细胞培养和活病毒的灵敏、特异和快速的检测系统是必不可少的。如果没有改进的方法，AdV36的S在肥胖中的作用的研究将是缓慢的，只有几个研究人员进行，而且结果充满了实验室之间的变异性。