Point-of-care immunoassay for diagnosis of histoplasmosis in HIV/AIDS

描述（由申请人提供）：进行性播散性组织胞浆菌病是美国和拉丁美洲HIV/AIDS患者中常见的危及生命的真菌感染。艾滋病毒/艾滋病的发病率可能> 20%，在资源有限的国家，组织胞浆菌是地方病，死亡率>30%。早期诊断和治疗对于降低这一高死亡率至关重要。诊断目前依赖于文化或组织病理学。这些方法灵敏度低，耗时，昂贵，并且需要训练有素的人员。另一种方法是免疫测定来检测H。血清或尿液中的荚膜多糖。靶抗原被认为是细胞壁半乳甘露聚糖。迄今为止的研究已经发现这种测定的高灵敏度。然而，组织胞浆菌抗原的免疫测定i）目前仅通过使用参考实验室以ELISA形式提供，ii）尚未在资源有限的国家进行商业分销，iii）取决于多克隆兔抗体（pAbs）的使用，和iv）不适合护理点（POC）使用。我们的总体假设是，可以用靶向H的多糖抗原的高亲和力单克隆抗体（mAb）构建用于HIV/AIDS中播散性组织胞浆菌病POC诊断的免疫测定法。在感染过程中脱落到血清和尿液中的包囊。该假设的推论是，使用适当的mAb对进行测定构建将能够靶向常见和独特的H。荚膜抗原表位，提供了对pAb不可能的测定特异性的控制。第一个具体目标是生产适用于杂交瘤筛选和mAb特异性评价的多糖抗原。第二个具体目标是产生与在脱落到血清和尿液中的组织胞浆菌多糖抗原上发现的表位谱反应的mAb文库。如果实现了第一阶段的目标，第二阶段将使用第一阶段的mAb构建和评价POC形式的免疫测定。我们优选的测定平台是侧流免疫层析（试纸）测定。如果成功，这项转化研究项目可以通过早期诊断和治疗大大降低组织胞浆菌病的死亡率。重要的是，这可以在资源有限的国家以所需的低成本完成。

DESCRIPTION (provided by applicant): Progressive disseminated histoplasmosis is a common and life-threatening fungal infection among patients with HIV/AIDS in the United States and Latin America. Incidence rates in HIV/AIDS can be >20%, with mortality rates >30% in resource-limited countries where the fungus, Histoplasma capsulatum, is endemic. Early diagnosis and treatment are essential to reducing this high mortality rate. Diagnosis currently depends on culture or histopathology. These methods have low sensitivity, are time consuming, are expensive, and require trained personnel. An alternative approach is an immunoassay to detect H. capsulatum polysaccharide in serum or urine. The target antigen is believed to be a cell wall galactomannan. Studies to date have found a high sensitivity for such assays. However, immunoassay for Histoplasma antigen i) is currently available only in ELISA format through use of a reference laboratory, ii) is not commercially available for distribution in resource-limited countries, iii) is dependent on the use of polyclonal rabbit antibodies (pAbs), and iv) is not amenable to point-of-care (POC) use. Our overall hypothesis is that an immunoassay for POC diagnosis of disseminated histoplasmosis in HIV/AIDS can be constructed with high-affinity monoclonal antibodies (mAbs) that target the polysaccharide antigen of H. capsulatum that is shed into serum and urine during infection. A corollary to this hypothesis is that the use of appropriate pairs of mAbs for assay construction will enable targeting of common and unique H. capsulatum epitopes, providing a control over assay specificity that is not possible with pAbs. The first Specific Aim is to produce polysaccharide antigens suitable for screening of hybridomas and evaluation of mAb specificity. The second Specific Aim is to produce a library of mAbs reactive with the spectrum of epitopes found on the Histoplasma polysaccharide antigen that is shed into serum and urine. If the goals of this Phase I are achieved, Phase II will use mAbs from Phase I to construct and evaluate an immunoassay in POC format. Our preferred assay platform would be the lateral flow immunochromatographic (dipstick) assay. If successful, this translational research project could dramatically decrease mortality from histoplasmosis through earlier diagnosis and treatment. Importantly, this can be done at the low cost needed in resource-limited countries.