"Development of a Laboratory Test for TTP"

描述（由申请人提供）：这是一项I期STTR申请，旨在开发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必须单独根据临床诊断。因此，一些病例未得到诊断，而另一些则被误诊，并接受昂贵的治疗，包括每日治疗性血浆交换（每次治疗约1500美元，每次治疗4小时，7-14天），使用新鲜冷冻血浆作为血浆交换替代液（与过敏反应和输血传播疾病有关，包括艾滋病毒、丙型肝炎病毒和乙型肝炎病毒）。最近，一种金属蛋白酶（MP [ADAMTS13]）被“鉴定”为可切割vWF蛋白多聚体。这种MP活性的抑制被认为是获得性TTP的主要原因。只有基于研究实验室的测试可用于确定该MP的存在和/或量化其活性。这些测试繁琐、缓慢、耗时费力，而且价格昂贵。此外，没有标准或对照，可使实验室MP测定标准化或广泛使用。因此，经过我们自己的改进的，基于实验室的MP测定的大量经验，我们已经开发了一个概念，将允许廉价，快速，特异性，基于ELISA的测试，可以商业化。我们正是为这个概念寻求第一阶段的资金。具体来说，我们已经/将设计（ed）一个氨基酸靶蛋白，类似于vWF的A2结构域，含有MP的特定切割位点；当存在时，MP活性会将目标蛋白切割成2个不相等的蛋白片段，最初将通过HPLC进行鉴定。然后，我们将设计一种酶联免疫吸附试验，其中目标蛋白将在裂解位点含有一个淬灭的荧光染料，当分解产物存在时，该荧光染料将发出荧光，从而产生MP活性。最后，我们将设计一个目标蛋白，该蛋白将包含一种酶，该酶将在其裂解后被激活，并转化为允许自动检测的颜色底物。这3种分析格式（高效液相色谱法、荧光色法和比色法）将允许对MP活性进行定性和定量测定。在证明可行性后，将寻求二期资金来优化和商业化上述原型测试，使其成为快速、廉价、敏感和特异性、elisa格式、临床实验室、自动化的TTP诊断和监测测试，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.