"Development of a Laboratory Test for TTP"

描述(由申请人提供)：这是一项第一阶段的STTR应用程序，旨在开发一种用于诊断和监测血栓性血小板减少性紫癜(TTP)患者的实验室测试。由于没有可靠的、可控的实验室检测，TTP必须仅在临床上诊断。因此，一些病例没有得到诊断，而另一些病例被误诊并接受昂贵的治疗，包括每天进行治疗性血浆交换(每次约1500美元，每次治疗4小时×7-14天)，使用新鲜冰冻血浆作为血浆交换替代液(与过敏反应和包括艾滋病毒、丙型肝炎和乙肝病毒在内的输血传播疾病有关)。最近，一种金属蛋白酶(MP[ADAMTS13])被“鉴定”出来，它能裂解大的von Willebrand因子(VWF)蛋白多聚体。抑制这种MP的活性被认为是获得性TTP的主要原因。只有基于实验室的研究测试才能确定这种MP的存在和/或量化其活性。这些测试繁琐、缓慢、耗费时间和劳动力，而且成本高昂。此外，基于实验室的MP检测没有可以标准化或广泛使用的标准或对照。因此，在我们自己的改良的、基于实验室的MP检测方面积累了相当多的经验后，我们开发了一个概念，它将允许一种廉价、快速和特异的基于EL ISA的检测方法，并可用于商业用途。正是这一概念，我们寻求第一阶段的资金。具体地说，我们已经/将设计(Ed)一个类似于vWF的A2结构域的氨基酸目标蛋白，其中包含MP的特定切割位点；当存在MP活性时，MP会将目标蛋白切割成两个不相等的蛋白质片段，最初将通过高效液相色谱进行鉴定。然后，我们将设计一种ELISA，其中目标蛋白将在切割位置包含猝灭的荧光色素，当存在分解产物时，该荧光将发出荧光，从而产生MP活性。最后，我们将设计一种目标蛋白，它将包含一种酶，该酶在切割后将被激活，并转换为允许自动检测的颜色底物。这3种分析形式(高效液相色谱、荧光显色法和比色法)将允许对MP活性进行定性和定量测定。在可行性被证明后，将寻求第二阶段资金，将上述原型测试优化并商业化，使其成为一种快速、廉价、灵敏和特异的、临床实验室、自动化、诊断和监测TTP测试的格式，并将其与临床诊断为TTP的患者以及其他相关血液病患者的参考样本进行测试。

DESCRIPTION (provided by applicant): This is a Phase I STTR application to develop a laboratory test for the diagnosis and monitoring of patients with thrombotic thrombocytopenic purpura (TTP). As there is no reliable, controlled laboratory test, TTP must be diagnosed on clinical grounds alone. Thus, some cases go undiagnosed, while others are misdiagnosed and subjected to costly therapy including daily therapeutic plasma exchange (about $1500 per treatment, 4 hrs per treatment x 7-14 days), that utilizes fresh frozen plasma as the plasma exchange replacement fluid (associated with allergic reactions and transfusion transmitted diseases including HIV, HCV and HBV). Recently, a metalloprotease (MP [ADAMTS13]) has been "identified" that cleaves large von Willebrand factor (vWF) protein multimers. Inhibition of the activity of this MP is thought to be the primary cause of acquired TTP. Only research laboratory based tests are available to determine the presence and/or quantify activity of this MP. These tests are cumbersome, slow, time and labor-intensive, and expensive. Additionally, there are no standards or controls for which the lab-based MP assay against which can be normalized or made widely available. Therefore, after considerable experience with our own modified, lab-based MP assay, we have developed a concept that would allow for an inexpensive, rapid, and specific, ELISA based test that could be made commercially available. It is this concept for which we seek Phase I funding. Specifically, we have/will design(ed) an amino acid target protein analogous to the A2 domain of vWF containing the specific cleavage site for the MP; when present, MP activity would cleave the target protein into 2 unequal protein fragments, which, initially, will be identified by HPLC. We will then design an ELISA wherein the target protein will contain a quenched fluorochrome at the cleavage site, which will fluoresce when the breakdown products, and thus MP activity, are present. Finally, we will design a target protein which will contain an enzyme that will become activated after its cleavage and convert a color substrate allowing automated detection. These 3 assay formats (HPLC, fluorochrome and colorimetric) will allow both qualitative and quantitative determination of MP activity. After feasibility is demonstrated, Phase II funding will be sought to optimize and commercialize the above prototype tests into a rapid, inexpensive, sensitive and specific, ELISA-format, clinical laboratory, automated, diagnostic and monitoring test for TTP, and to test it against reference samples from patients with clinically diagnosed TTP as well as others with related hematologic disorders.