Novel Biomarkers for Stroke Injury

中风是全球第二大死亡原因，也是严重、长期残疾和入住疗养院的主要原因。临床上有用的诊断方法的缺乏严重阻碍了急性缺血性卒中的有效管理和治疗。目前可用的诊断是昂贵的和/或不可靠的，并且没有简单、快速、非侵入性的测试来诊断和评估急性中风的程度，以及预测中风的严重程度。 结果。免疫测定技术，如酶联免疫吸附测定（ELISA），可以用来开发这种急需的中风诊断测试。这种测试还将有助于预测治疗损伤的潜在疗法的疗效，并提供有关有助于损伤和功能恢复的精确生物化学和分子机制的信息。我们已经开发出一种特异性和敏感性的急性缺血性卒中生物标志物。全光谱蛋白 是钙蛋白酶和半胱天冬酶-3蛋白水解的主要底物，它们各自分别产生独特的150 kDa血影蛋白分解产物（SBDP 150）和独特的120 kDa蛋白水解片段（SBDP 120）。我们实验室的工作表明，除了钙蛋白酶激活外，caspase-3激活是急性缺血性卒中后的一个强有力的事件。在第一阶段的建议中，我们建议开发一种夹心ELISA，检测caspase-3介导的 SBDP 120。此外，我们正在完成检测钙蛋白酶介导的SBDP 150的ELISA的开发。该建议的主要优势在于，在急性缺血性卒中后，SBPD 120测量细胞凋亡和SPDP 150测量胀亡（坏死）和细胞凋亡的同时和差异检测，从而允许对这两种细胞死亡途径对缺血性损伤的相对贡献的重要见解。 具体目标1：用于定量和特异性检测120 kDa的胱天蛋白酶-3介导的血影蛋白分解产物（SBDP 120）的夹心ELISA的配置，该产物与神经元凋亡独特相关。 可从特定目标1中获得：定量检测SBDP 120的神经元凋亡相关诊断ELISA试剂盒的配置。（0-4个月） 具体目的2：在中风损伤的大鼠模型中初步验证SBDP 120夹心ELISA和SBDP 150夹心ELISA。 可从特异性目标2中获得：原型诊断ELISA，能够测量CSF和血清中的阿尔茨海默病相关SBDP 120水平。此外，SBDP 150和SBDP 120 ELISA将在中风损伤（MCAO损伤）的啮齿动物模型中的CSF和血清中进行验证。（4-6个月）

Stroke is the second leading cause of death worldwide and the primary cause of serious, long-term disability and nursing home admissions. The absence of clinically useful diagnostics has severely impeded effective management and treatment of acute ischemic stroke. Currently available diagnostics are either expensive and/or unreliable and there are no simple, rapid, non-invasive tests to diagnose and assess the magnitude of acute stroke, as well as to predict outcome. Immunoassay technology, such as the enzyme-linked immunosorbent assay (ELISA), can be employed to develop this much needed stroke diagnostic test. Such a test would also help predict the efficacy of potential therapies to treat injury, as well as provide information on precise biochemical and molecular mechanisms contributing to injury and recovery of function. We have developed a specific and sensitive biomarker for acute ischemic stroke. all-Spectrin is a major substrate for calpain and caspase-3 proteolysis, which each produce a unique 150 kDa spectrin breakdown product (SBDP150) and a unique 120 kDa proteolytic fragment (SBDP120), respectively. Work from our laboratories demonstrates that in addition to calpain activation, caspase-3 activation is a robust event following acute ischemic stroke. In this Phase I proposal, we propose to develop a sandwich ELISA that detects the caspase-3 mediated SBDP120. In addition, we are completing development of an ELISA that detects the calpain mediated SBDP150. A major strength of this proposal will be the concurrent and differential detection of SBPD120 measuring apoptosis and SPDP150 measuring oncosis (necrosis) and apoptosis following acute ischemic stroke, allowing important insights into the relative contributions of these two cell death pathways to ischemic injury. Specific Aim 1: Configuration of a sandwich ELISA for the quantitative and specific detection of a caspase-3 mediated spectrin breakdown product of 120 kDa (SBDP120), which is uniquely associated with neuronal apoptosis. Deliverable from Specific Aim 1: Configuration of a neuronal apoptosis-linked diagnostic ELISA kit that quantitatively detects SBDP120. (Months 0-4) Specific Aim 2: Preliminary validation of both the SBDP120 sandwich ELISA and the SBDP150 sandwich ELISA in a rat model of stroke injury. Deliverable from Specific Aim 2: Prototype diagnostic ELISA capable of measuring levels of apoptosis-linked SBDP120 in CSF and serum. In addition, both the SBDP150 and SBDP120 ELISA will be validated in CSF and serum in a rodent model of stroke injury (MCAO injury). (Months 4-6)