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

 描述(申请人提供)：抗原检测试验与传统的血清学试验不同，它检测疾病状态，而不是宿主抗体对疾病病因的反应。因此，它既可用于诊断，也可用于疾病治疗随访。在过去的10-20年里，抗原检测方法已成功地用于各种传染病的诊断，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以及阿米巴病。虽然抗原检测试验有可能区分活动性内脏利什曼病(VL)和无症状或治愈的疾病，但矛盾的是，这种方法并没有被开发出来用于这种严重寄生虫病的诊断。利用超灵敏的质谱学技术，我们最近在来自新大陆的VL患者的尿液中发现了三种婴儿利什曼原虫/查加西蛋白。这些分子被广泛地表征并用于建立捕获ELISA抗原检测方法来诊断由婴儿乳杆菌引起的VL。使用来自新世界VL患者的人尿进行的初步验证研究表明，该测试是100%敏感和100%特异的。虽然这项试验对诊断由L.infantum引起的新世界VL是很好的，但我们最近观察到它对由L.donovani(又称kala-azar)引起的旧世界VL的诊断敏感性不够。这可能是因为这两种寄生虫在它们的基因组以及它们引起的病理和临床表现上都有很大的差异。因此，这两种寄生虫抗原库的不同宿主处理可能导致患者尿液中不同模式的生物标记物的排泄。不幸的是，观察到的婴儿乳杆菌生物标志物诊断旧世界VL的低性能是一个主要障碍，因为VL的发病率和流行率最高的是旧世界。然而，这一主要障碍可以通过破解东半球黑热病患者尿液中大量排泄的多诺瓦尼杆菌抗原来克服。因此，这个第一阶段的SBIR项目建议识别这些独特的杜氏乳杆菌生物标志物，最终目标是开发一种非侵入性的抗原检测方法，准确诊断由杜氏乳杆菌引起的VL。本研究的具体目的是：1.鉴定黑热病患者(旧世界VL)尿液中存在的杜氏乳杆菌蛋白标志物，并研制诊断试剂。目的：优化并开始用精选的纯化抗体对检测黑热病患者尿液中杜氏杆菌抗原的抗原检测试验(捕获ELISA)进行临床验证。

 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.