Rapid Detection of Antibodies to Adenovirus 36

描述（由申请方提供）：腺病毒36（AdV 36）导致动物肥胖增加（导致肥胖）和代谢变化。人类中的因果关系尚未确定，但感染与动物中观察到的相同变化相关，即肥胖增加、体重增加以及血脂和空腹血糖降低。阐明AdV 36在当前肥胖流行中的作用将在很大程度上依赖于对大人群的纵向研究。此外，了解AdV 36的作用可能对肥胖症新干预策略的规划和成功具有影响。考虑到AdV 36与受感染人类的后续效应之间的密切联系，如果不了解AdV 36在肥胖中的作用，就会忽视肥胖的一个关键风险因素，并继续遵循肥胖范式，这种范式在很大程度上未能导致有效的预防和控制策略。一个主要的方法障碍存在于需要推动该领域向前发展的大量人群的研究中。也就是说，检测AdV 36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）-一种复杂且耗时的方法，在实验室之间产生不一致的结果。取而代之的是，迫切需要一种快速、高通量、易于执行和客观的检测方法，以向新的研究人员开放该领域，推进当前研究的步伐，并提高结果的一致性。本申请将通过鉴定AdV 36六邻体蛋白上的特异性表位并在简单的基于ELISA的系统中使用重组蛋白和/或肽来检测AdV 36感染的证据，即特异性（中和）抗体，来开发改进的测定。AdV六邻体蛋白参与病毒与宿主细胞之间的初始相互作用，并含有特异性中和表位。AdV 36六邻体序列已与其他腺病毒进行了比较，揭示了可能含有一个或多个特异性表位的九个高变区（HVR）。我们已经克隆和表达了四个重组融合蛋白含有腺病毒36高变区，并测试了每一个使用SNA鉴定的阳性或阴性血清。两个重组体显示出开发灵敏和特异性测定的前景，但需要进一步的表征和验证。此外，将合成基于来自这些重组体的HVR序列的重叠肽，并测试其在测定系统中的使用。鉴定为含有AdV 36特异性表位的澄清剂和/或肽将用于ELISA中，以测试具有已知SNA滴度的300个个体血清。两种检测系统之间的结果比较将证明新检测方法的灵敏度和特异性。总之，灵敏、特异、快速的检测系统，不再依赖于细胞培养和活病毒是必不可少的。如果没有改进的方法，AdV 36在肥胖中的作用的研究将是缓慢的，只有少数研究者进行，并且充满了实验室间的结果差异。

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.