Novel Multiplex Assay for Sepsis Biomarkers

描述(申请人提供)：严重败血症是重症监护病房的主要死亡原因，每年占美国总死亡人数的9.3%(Angus，2001)。这种复杂的疾病很难诊断和治疗，非常需要一种可以描述患者免疫功能障碍的测试。在这笔赠款的第一阶段，我们将证明一种新的快速多重检测脓毒症生物标记物的可行性。我们开发了一种专利方法，使用实时聚合酶链式反应(PCR)技术以非常高的灵敏度和重复性以多重方式测量蛋白质浓度。该方法被称为多路实时免疫-聚合酶链式反应(MRI-PCR)，由夹心免疫分析组成，其中检测抗体被不同的DNA序列标记。然后使用实时荧光聚合酶链式反应对所有DNA标记进行并行量化，从而揭示生物样本中同源蛋白质的数量。初步数据表明，与标准的酶联免疫吸附试验相比，使用核磁共振-聚合酶链式反应检测的多重细胞因子检测的灵敏度提高了~2个数量级，动态范围增加了2个数量级，标准偏差非常低(CV&lt；10%)，并且具有很高的特异性。在第一阶段的研究中，将准备一组试剂来检测脓毒症的小鼠标志物，并在MRI-PCR测试中进行评估。然后将目标蛋白添加到正常小鼠血清中，以在生理样本中验证该分析。内毒素血症的体外模型将被用来测量内毒素血症小鼠全血和体内内毒素血症模型中细胞因子的产生。无菌烧伤诱导的全身免疫炎症反应综合征(SIRS)的小鼠模型，与烧伤加细菌感染诱导的脓毒症进行比较，将被用来确定在临床相关环境中，MRI-PCR检测是否能够区分细菌引起的SIRS和非感染性原因引起的SIRS。在第二阶段，该分析将在脓毒症的啮齿动物模型(盲肠结扎和穿孔)中得到验证，并将制备检测人类败血症生物标志物的试剂。人类败血症核磁共振-聚合酶链式反应测试将得到验证，并用于分析正常和脓毒症患者的血清。这些研究的结果将导致两个产品：用于临床诊断、预后和治疗监测的人类败血症测试，以及用于研究人员的啮齿动物败血症测试。目前，脓毒症患者的诊断和监测治疗非常困难。这项建议将开发一种血清学测试，以测量脓毒症的多个生物标志物，并将有助于脓毒症患者的诊断、预后和治疗监测。

DESCRIPTION (provided by applicant): Severe sepsis is the leading cause of death in intensive care units and accounts for 9.3% of overall deaths in the United States annually (Angus, 2001). This complex disorder is difficult to diagnose and treat, and there is a great need for a test that can profile patients' immune dysfunction. In Phase I of this grant, we will demonstrate the feasibility of a novel rapid multiplex assay for biomarkers of sepsis. We have developed a proprietary method for using the real-time-polymerase chain reaction (PCR) technology to measure protein concentrations in a multiplexed fashion with very high sensitivity and reproducibility. The method, Multiplexed Real-time-Immuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary data have demonstrated that multiplexed cytokine measurements employing MRI-PCR assays provides ~2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase I studies, a panel of reagents to detect murine markers of sepsis will be prepared and evaluated in the MRI-PCR assay. The target proteins will then be spiked into normal mouse serum to validate the assay in a physiological sample. An ex vivo model of endotoxemia will be used to measure production of cytokines in response to lipopolysaccharide stimulation in whole mouse blood as well as in vivo in a mouse model of endotoxemia. A mouse model of sterile burn-induced systemic immunoinflammatory response syndrome (SIRS) as compared to burn injury plus bacterial infection-induced sepsis will be performed to determine if the MRI-PCR assay can distinguish between SIRS induced by bacterial as opposed to non-infectious causes in a clinically relevant setting. In Phase II, the assay will be validated in rodent models of sepsis (cecal ligation and puncture), and reagents to detect human biomarkers of sepsis will be prepared. The human sepsis MRI-PCR test will be validated and used to analyze serum from normal and septic humans. The results of these studies will lead to two products: the human sepsis test for clinical diagnosis, prognosis, and monitoring of therapy, and the rodent sepsis test for researchers. Currently it is very difficult to diagnose and monitor therapy in patients with sepsis. This proposal will develop a serological test that measures multiple biomarkers of sepsis, and will be useful for the diagnosis, prognosis and monitoring of therapy in septic patients.