Biomarker for liver ischemia/reperfusion-induced injury

描述（由申请人提供）：项目总结/摘要：肝缺血再灌注诱导的肝损伤是多发性创伤、热损伤、出血性和脓毒性休克以及肝移植过程中肝衰竭的主要致病原因，其显著导致多器官衰竭和死亡率。目前血清ALT或LDH的酶测定法要么是非特异性的，要么不够灵敏，无法检测肝损伤的早期阶段，评估实质细胞死亡的程度，或预测肝恢复。基于肝脏蛋白质组学方法，我们正在开发一组新的肝脏缺血性损伤的生物标志物。迄今为止发现的最有前途的生物标志物候选物是尿素/一氧化氮循环乙酰氨基琥珀酸合酶（ASS）的酶。本提案的目标是设计、生产和验证夹心（SW）ELISA试剂盒，用于定量测定生物液体中的ASS，以诊断缺血性肝损伤。具体目标1：设计并建立双抗体夹心ELISA定量检测氨基琥珀酸合成酶（ASS）。可从特定目标1中获得：用于定量检测生物液体中ASS的原型ELISA试剂盒的配置。我们将开发SW ELISA，优化其灵敏度检测和定量ASS，包括血清或血浆中的ASS，并生产该ELISA试剂盒用于特异性目标2的初步验证。具体目的2：初步验证ASS夹心ELISA在大鼠肝I/R损伤模型中的诊断效用。本特定目标的目的是通过测量血液中的ASS水平来检查原型夹心ELISA用于评估肝缺血性损伤的程度和结果的实用性。可从Specific Aim 2获得：用于测量血清中ASS水平的诊断ELISA试剂盒，用于非侵入性诊断和监测肝脏I/R损伤。这些努力的总体结果将是产生一个验证的夹心ELISA试剂盒检测ASS，并初步验证其在实验性缺血/再灌注肝损伤大鼠模型的实用性。在II期SBIR期间，将在腹部和多发性创伤、出血性和脓毒性休克以及肝移植患者中进一步验证该ELISA试剂盒作为人类缺血性肝损伤的可靠诊断工具。项目叙述：各种形式的肝损伤导致的肝衰竭是总体死亡率的重要来源。肝损伤的病因包括枪伤和爆炸伤造成的多发性和腹部创伤、药物和酒精中毒、出血性和感染性休克以及肝移植后移植物衰竭。肝酶的酶测定传统上被用作肝损伤的标志物。然而，这些诊断测试要么不敏感或非特异性，不允许评估肝损伤的程度，也不能预测结果。因此，临床上需要更好的缺血性肝损伤的非侵入性生物标志物，这可以导致早期诊断和更好的治疗。酶联免疫吸附试验（ELISA）等免疫检测技术可用于肝I/R损伤的诊断，是一种简便、快速、无创、可靠的检测方法。除其他用途外，它将对军事医疗或对国家灾害、败血症和多器官衰竭的紧急反应有价值。此外，它可以提供更多的信息，生化和分子机制，有助于肝损伤，功能恢复和/或潜在的新的治疗策略的目标。我们已经开发了一种特异性和敏感性的生物标志物I/R诱导的肝损伤，出现，从最初的实验，与损伤的严重程度，并可能是敏感的治疗干预。此外，该生物标志物提供了参与肝脏中一氧化氮合成和尿素循环调节的病理生物学机制的直接评估，这可能代表败血症期间肝I/R损伤、肝衰竭和代谢性脑病之间的潜在联系。也就是说，精氨琥珀酸合成酶（ASS）是一种重要的肝脏酶。ASS在缺血期间迅速上调（一小时内）并释放到循环中。该提案的总体目标是开发用于定量测定ASS的原型ELISA试剂盒，ASS作为生物流体（包括血清或血浆）中的新型诊断生物标志物。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Liver injury induced by hepatic ischemia followed by reperfusion is a major pathogenic cause of liver failure during multiple trauma, thermal injury, hemorrhagic and septic shock, and liver transplantation, which contributes significantly to multiple organ failure and mortality. Current enzymatic assays of serum ALT or LDH are either non-specific or not sensitive enough to detect early stages of liver damage, assess magnitude of parenchymal cell death, or to predict hepatic recovery. Based on liver proteomic degradomics approach, we are developing a panel of novel biomarkers for liver ischemic injury. The most promising biomarker candidate discovered to date is an enzyme of urea/nitric oxide cycle argininosuccinate synthase (ASS). The goals of this proposal are to design, produce, and validate a sandwich (SW) ELISA Kit for quantitative determination of ASS in biological fluids for diagnostics of ischemic liver injury. Specific Aim 1: Design and develop sandwich ELISA for the quantitative detection of argininosuccinate synthase (ASS). Deliverable from Specific Aim 1: Configuration of a prototype ELISA kit for the quantitative detection of ASS in biological fluids. We will develop a SW ELISA, optimize it for sensitive detection and quantification of ASS, including in serum or plasma, and produce this ELISA Kit for preliminary validation in Specific Aim 2. Specific Aim 2: Preliminary validation of the diagnostic utility of the ASS sandwich ELISA in a rat model of liver I/R injury. The objective of this Specific Aim is to examine the utility of the prototype sandwich ELISA for assessment of the magnitude of hepatic ischemic injury and outcome by measuring ASS levels in blood. Deliverable from Specific Aim 2: Diagnostic ELISA Kit for measuring ASS levels in serum for non-invasive diagnostics and monitoring liver I/R injury. The overall result of these efforts will be generation of a validated sandwich ELISA Kit for detection of ASS, and preliminary validation of its utility in experimental ischemia/reperfusion liver injury rat model. Further validation of this ELISA Kit as a reliable diagnostic tool for human ischemic liver Injury will be performed during Phase II SBIR in patients with abdominal and multiple trauma, hemorrhagic and septic shock, and liver transplantation. Project Narrative: Liver failure due to various forms of hepatic injury is a significant source of overall mortality. Etiologies of liver injury includes multiple and abdominal trauma from gunshot and blast injuries, drug and alcohol toxicity, hemorrhagic and septic shock, and graft failure after liver transplantation. Enzymatic assays of liver enzymes have traditionally been used as markers of liver injury. However, these diagnostic tests are either not sensitive or non-specific, do not allow for assessment of the magnitude of liver injury, nor do they predict outcome. Thus, there is an explicit clinical need for better non-invasive biomarkers of ischemic liver injury, which can lead to early diagnosis and better treatment. Immunoassay technologies, such as the enzyme-linked immunosorbent assay (ELISA) can be employed to develop a simple, rapid, noninvasive and reliable diagnostic test for liver I/R injury. Amongst other uses, it will be valuable for military medical or emergency responses to national disasters, sepsis and multiple organ failure. In addition, it may provide more information on biochemical and molecular mechanisms contributing to liver injury, recovery of function and/or potential targets for novel therapeutic strategies. We have developed a specific and sensitive biomarker for I/R-induced liver injury that appears, from initial experiments, to be correlated with the severity of damage and may be sensitive to therapeutic intervention. Moreover, this biomarker provides direct assessment of pathobiological mechanisms involved in regulation of nitric oxide synthesis and the urea cycle in the liver that may represent a potential link between liver I/R injury, liver failure, and metabolic encephalopathy during sepsis. Namely, argininosuccinate synthase (ASS) is a crucial liver enzyme. ASS is rapidly up-regulated (within an hour) during ischemia and is released into the circulation. The overall goal of this proposal is to develop a prototype ELISA kit for the quantitative determination of ASS as novel diagnostic biomarker in biological fluids including serum or plasma.