Novel Point of Care assays for Urinary Diagnostics of Nephritis

项目摘要/摘要 系统性红斑狼疮是一种导致慢性炎症的全身性自身免疫性疾病 包括肾脏在内的多个器官。患者通常有疾病的恶化(肾脏 肾炎(“红斑”)间歇性静息。早期发现和及时处理耀斑可以 对患者的健康和生存有重大影响，这些患者中女性、西班牙裔、 和/或非裔美国人。肾组织的活检样本是狼疮诊断的金标准，但 具有侵袭性，不能频繁重复。在这种情况下，血液或尿液生物标记物可以预测 肾脏病理学可能非常有用，特别是如果它们支持护理点或理想的在家测试。我们 发现并广泛验证了狼疮发作相关的血液和尿液诊断生物标记物 规模空前的蛋白质筛查，覆盖了4个种族的患者(非裔美国人， 高加索人、西班牙人和中国人)。尿液标志物在以下方面的表现优于传统的实验室标志物 狼疮，并显示出随着时间的推移跟踪疾病活动的潜力。虽然尿液是一个容易提供的样本，但尿液 生物标志物的浓度必须根据尿液产生的稀释度进行校正。传统上，这种分子 用于尿液稀释的正常化，肌酐，与标准免疫分析不是很兼容，但我们 已经发现了一种蛋白质，它在尿液中的浓度与肌酐浓度密切相关，这可能是 与照明弹标记物一起测量。一个看护点(医生办公室)，或者更好的是，一个家庭自我测试，用于 狼疮发作可以通过加快治疗来改善预后。这种测试的自然格式是 侧向流动法(LFA)，被广泛用作家庭妊娠试验。然而，目前的LFA要么 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 基于我们新的磷光纳米颗粒的智能手机LFA的问题 记者。激励假说：我们假设，提高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新的肾炎发作 尿液中的生物标志物，从而解决了狼疮性肾炎发作的临床/家庭监测的未得到满足的需求。 这项工作的成功完成也将为定量化研究提供一个通用的平台技术 智能手机LFA测试不需要复杂的手机修改(只需10美元的滑盖附件)，以及新的 尿液标记物归一化方法适用于LFA和ELISA法的通用性。具体目标：我们 建议：(目标1)建立用于尿Flare标志物和肌酐相关归一化的LFA定量方法 蛋白质；(目标2)将多个标记和标准化蛋白质测试集成到一个用户友好的系统中， 软件错误捕获、条形码读取和质量控制；最后(目标3)评估性能 在狼疮检测中，使用了来自广泛队列患者的尿样。

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.