Development of a protein-based antigen detection test for kala-azar

 描述（由申请方提供）：与常规血清学试验相比，抗原检测试验检测疾病状态，而不是对疾病病原体的宿主抗体应答。因此，它可用于诊断和疾病治疗随访目的。在过去的10-20年中，抗原检测试验已成功用于诊断不同的传染病，包括化脓性链球菌引起的咽喉痛、肝炎、肺炎链球菌或嗜肺军团菌引起的肺炎和阿米巴病。虽然抗原检测试验有可能区分活动性内脏利什曼病（VL）无症状或治愈的疾病，矛盾的是，这种方法还没有开发用于诊断这种严重的寄生虫病。使用超灵敏的质谱技术，我们最近确定了三个婴儿利什曼原虫/恰加斯蛋白质的存在下，从新世界的VL患者的尿液。这些分子被广泛表征并用于开发用于由L.婴儿使用来自新世界VL患者的人尿液进行的该检测的初步验证研究表明，该检测具有100%的灵敏度和100%的特异性。虽然该试验对诊断新世界VL是极好的，但由L。我们最近观察到，其诊断由L. donovani也被称为黑热病是不够的。这可能是因为这两种寄生虫在基因组以及它们引起的病理和临床表现方面都有很大的差异。因此，两种寄生虫抗原库的不同宿主处理可导致患者尿液中生物标志物的不同模式的排泄。不幸的是，观察到的低性能的L。婴儿生物标志物诊断旧世界VL是一个主要障碍，因为VL的最高发病率和患病率发生在旧世界。尽管如此，这个主要的障碍可以通过解开L来克服。在旧世界黑热病患者的尿液中大量排泄的donovani抗原。因此，第一阶段SBIR项目建议识别这些独特的L。donovani生物标志物，最终目标是开发一种非侵入性的抗原检测方法，可以准确诊断由L. donovani。具体目标是：目标1。鉴定L.本发明涉及黑热病患者（旧世界VL）的尿液中存在的杜氏蛋白生物标志物，并生产诊断测定试剂。目标2.优化并开始临床验证抗原检测试验（捕获ELISA），使用选择的纯化抗体对检测L。黑热病患者尿液中的Donovani抗原。

 DESCRIPTION (provided by applicant): Antigen detection assays in contrast to conventional serological test, detects disease status and not the host antibody response to the disease etiological agent. It can therefore be used for both diagnosis and disease treatment follow-up purposes. Antigen detection assay has been successfully used for the past 10-20 years for the diagnosis of different infectious diseases including sore throat caused by Streptococcus pyogenes, hepatitis, pneumonia caused by Streptococcus pneumoniae or Legionella pneumophilla, and amoebiasis. Although antigen detection assay has the potential to discriminate active visceral leishmaniasis (VL) from asymptomatic or cured disease, paradoxically this approach has not been developed for the diagnosis of this serious parasitic disease. Using the ultra-sensitive mass spectrometry technology we have recently identified the presence of three Leishmania infantum/chagasi proteins in urine of VL patients from the New World. These molecules were extensively characterized and used to develop a capture ELISA antigen detection assay for VL diagnosis caused by L. infantum. A pilot validation studies with this assay using human urine from New World VL patients indicated that the test was 100% sensitive and 100% specific. Although this test was excellent for the diagnosis of New World VL, caused by L. infantum, we have recently observed that its sensitivity for the diagnosis of Old World VL caused by L. donovani also known as kala-azar was not adequate. This is likely explained by the fact that these two parasites have substantial differences both in their genomes as well as in the pathologies and clinical manifestations that they cause. Therefore, the different host handling of the two parasites antigenic repertoires can result in excretion of distinct patterns of biomarkers in the patients' urines. Unfortunately, the observed low performance of the L. infantum biomarkers to diagnose Old World VL is a major hindrance because the highest incidence and prevalence of VL occurs in the Old World. Nonetheless, this major hindrance can be overcome by unravelling L. donovani antigens that are abundantly excreted in the urine of patients with Old World kala-azar. Hence, this Phase I SBIR project proposes to identify these unique L. donovani biomarkers with the ultimate goal of developing a non-invasive, antigen detection assay that will accurately diagnose VL caused by L. donovani. The Specific Aims are: Aim 1. To identify L. donovani protein biomarkers present in the urine of kala-azar patients (Old World VL) and to produce diagnostic assay reagents. Aim 2. To optimize and to begin the clinical validation of an antigen detection test (capture ELISA) using select pairs of purified antibodies for the detection of L. donovani antigens in the urine of kala-azar patients.