Novel Point of Care assays for Urinary Diagnostics of Nephritis

项目总结/摘要 系统性红斑狼疮（“狼疮”）是一种导致慢性炎症的系统性自身免疫性疾病 包括肾脏在内的多个器官患者通常有疾病的恶化（肾 肾炎“发作”），其间有静止间期。早期发现并及时治疗耀斑， 对患者的健康和生存有重大影响，其中女性，西班牙裔， 或者非裔美国人肾组织的活检取样是狼疮诊断的金标准，但 侵入性，不能频繁重复。在这种情况下，可以预测血液或尿液中的生物标志物， 肾脏病理学可能非常有用，特别是如果它们支持即时检测或理想的家庭检测。我们 在一个国家中发现并广泛验证了与狼疮发作相关的血液和尿液诊断生物标志物 蛋白质筛查的规模空前，扩展到4个种族的患者群体（非洲裔美国人， 高加索人、西班牙人和中国人）。尿液标记物比传统的实验室标记物表现更好， 狼疮，并显示随着时间的推移跟踪疾病活动的潜力。虽然尿液是一种容易提供的样本， 生物标记物浓度必须校正其被尿产生稀释。传统上， 用于尿稀释、肌酐的标准化，与标准免疫测定不太兼容，但我们 已经鉴定了一种蛋白质，其在尿液中的浓度与肌酸酐的浓度密切相关， 沿着耀斑标记测量。一个点的护理（医生的办公室），或更好的是，一个家庭自我测试， 狼疮发作可以通过加快治疗来改善结果。这种测试的自然格式是 侧流试验（LFA），广泛用作家庭妊娠试验。然而，目前的LFA 需要昂贵的设备或缺乏必要的灵敏度和定量。我们建议解决这个问题 基于我们新的磷光（“在黑暗中发光”）纳米粒子的智能手机LFAs的问题 记者说动机假设：我们假设，增加的敏感性和量化能力， 纳米磷-LFA将使患者或医生能够简单地测量我们的新的肾炎发作 本发明的目的是提供尿中的生物标志物，并且因此解决了对狼疮肾炎发作的临床/家庭监测的未满足的需求。 这项工作的顺利完成也将为定量化提供一个通用的平台技术 智能手机LFA测试不需要精心的手机修改（只有10美元的幻灯片附件），和一个新的 尿标记物标准化方法非常适合LFA和ELISA中的一般使用。具体目标：我们 目的：（1）建立尿潮红标志物的定量LFA和肌酐相关标准化 （目的2）将多个标志物和标准化蛋白质测试整合到用户友好的系统中， 软件错误捕获、条形码阅读和质量控制;最后（目标3）评估性能 使用大量患者的尿液样本进行狼疮检测。

Project Summary/Abstract Systemic Lupus Erythematosus (“Lupus”) is a systemic autoimmune disease that leads to chronic inflammation in multiple organs including the kidneys. Patients commonly have exacerbations of the disease (kidney nephritis “flares”) interspersed by quiescent intervals. Early detection and the prompt treatment of flares can have a significant impact on the health and survival of patients, who are disproportionately female, Hispanic, and/or African-American. Biopsy sampling of kidney tissues is the gold standard for Lupus diagnosis, but is invasive and cannot be repeated frequently. In this context, blood or urine biomarkers that are predictive of kidney pathology could be very useful, especially if they supported point-of-care or ideally at-home testing. We have discovered and extensively validated lupus flare-correlated blood and urine diagnostic biomarkers in a protein screen of unprecedented scale, extending across 4 ethnic groups of patients (African American, Caucasian, Hispanic and Chinese). The urine markers perform better than conventional laboratory markers for Lupus and show potential to track with disease activity over time. While urine is an easy sample to give, urine biomarker concentrations must be corrected for their dilution by urine production. The molecule traditionally used for normalizing for urinary dilution, creatinine, is not very compatible with standard immunoassays, but we have identified a protein whose concentration in urine closely correlates with that of creatinine and that can be measured along with the flare markers. A point-of-care (Doctor's office), or better yet, a home self-test, for lupus flares could improve outcomes by expediting treatment. The natural format for such a test would be the lateral-flow assay (LFA), which is widely used as the home pregnancy test. Current LFAs, however, either require expensive equipment or lack the necessary sensitivity and quantitation. We propose to address this problem with smartphone-based LFAs based on our new phosphorescent (“glow-in-the-dark”) nanoparticle reporters. Motivating hypothesis: We hypothesize that the increased sensitivity and quantification ability of nanophosphor-LFAs will enable patients or doctors to simply measure our new kidney nephritis flare biomarkers in urine, and thus, address the unmet need for clinic/home monitoring of lupus nephritis flares. Successful completion of this work also will provide a generally-useful platform technology for quantitative smartphone LFA tests requiring no elaborate phone modifications (only a $10 slide-on attachment), and a new method of urine marker normalization well suited to general use in LFA and ELISA. Specific Aims: We propose: (Aim 1) To develop quantitative LFAs for urinary flare markers and creatinine-correlated normalizing protein; (Aim 2) To integrate multiple marker and normalizing protein tests into a user-friendly system with software error-catching, barcode reading, and quality controls; and finally (Aim 3) To evaluate the performance of the tests in lupus, using urine samples from broad cohorts of patients.