Multiplex Diagnostic for Enteric Category B Pathogens and Intestinal Biomarkers

描述（由申请人提供）：我们的目标是开发一种特异于贾第鞭毛虫、隐孢子虫、大肠杆菌的多重免疫诊断组合。溶组织菌、产滋贺毒素志贺菌和E.粪便大肠杆菌检测。进一步的努力将量化粪便生物标志物乳铁蛋白和血红蛋白，肠道炎症和出血的指标。完整的多重检测板将提供腹泻/痢疾的主要B类肠道致病性病原体的快速诊断，并描述疾病的严重程度。 该检测采用了灵敏和特异的免疫荧光Luminex技术。Luminex允许使用独特可检测微珠组阵列对单个样品中的分析物进行多重检测。分析物特异性抗体选自现有的FDA批准的免疫测定，确保其性能和稳定性。初步数据表明，Luminex的灵敏度肠道原生动物超过ELISA，并与RT-PCR，纯化的寄生虫曲线。粪便中的检测确定了108个FDA批准的ELISA阳性反应中的108个，但仍需要完整的临床灵敏度验证。Luminex检测乳铁蛋白和血红蛋白与商业ELISA检测相关，并提供了扩大的定量范围，提高了ELISA的粪便分析效率。 目的#1详细描述了贾第鞭毛虫、隐孢子虫和E.溶组织剂目标#2扩展了该腹泻/痢疾检测板，以包括滋贺毒素、志贺样毒素（Stx 1）和志贺样毒素2（Stx 2）的检测。目标#3增加了乳铁蛋白和血红蛋白的检测，作为腹泻/痢疾严重程度和患者状况的生物标志物。 这些研究的成功完成将提供能够同时检测B类肠道病原体和描述感染严重程度的肠道生物标志物的原型诊断。多重检测试剂盒将竞争NIH小型企业II期资金，以完成生产转移和FDA 510（k）许可验证。基于Luminex的多重检测是商业化的极好候选者，并且它允许将来包括其他肠道病原体和肠道功能生物标志物。 公共卫生相关性：本研究提出了一个诊断面板，以确定类别B生物防御肠道病原体贾第鞭毛虫，隐孢子虫，E。溶组织菌、产滋贺毒素志贺菌和E.腹泻和痢疾患者中的大肠杆菌。该小组还将量化粪便生物标志物，以描述肠道炎症和出血，提供有关疾病严重程度和患者状况的新信息。

DESCRIPTION (provided by applicant): Our goal is development of a multiplex immunodiagnostic panel specific for Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli detection in stool. Additional effort will quantify the fecal biomarkers lactoferrin and hemoglobin, indicators of intestinal inflammation and hemorrhage. The full multiplex panel will provide rapid diagnosis of major Category B enteropathogenic agents of diarrhea/dysentery and describe severity of disease. The assay utilizes sensitive and specific immunofluorescence-based Luminex technology. The Luminex allows multiplexed detection of analytes in a single sample using an array of uniquely detectable bead sets. Analyte-specific antibodies are selected from existing FDA-cleared immunoassays, providing assurance of their performance and stability. Preliminary data indicate Luminex sensitivity for the enteric protozoa exceeds that of ELISA, and is comparable to RT-PCR, with purified parasite curves. Detection in stool identified 108 of 108 parasite FDA-cleared ELISA positive reactions, but still requires complete validation of clinical sensitivity. Luminex detection of lactoferrin and hemoglobin correlate with commercial ELISA detection and provide an expanded range of quantification, increasing efficiency of fecal analysis over ELISA. Aim #1 details an enteric protozoan parasite panel for Giardia, Cryptosporidium, and E. histolytica. Aim #2 expands this diarrhea/dysentery panel to include detection of shiga toxin, shiga-like toxin (Stx1) and shiga-like toxin 2 (Stx2). Aim #3 adds detection of lactoferrin and hemoglobin as biomarkers of diarrhea/dysentery severity and patient condition. Successful completion of these studies will provide a prototype diagnostic capable of the simultaneous detection of Category B enteropathogens and intestinal biomarkers that describe severity of infection. The multiplex assay will be competitive for Phase II NIH small business funding to complete manufacturing transfer and FDA 510(k) clearance validations. The Luminex-based multiplex detection is an excellent candidate for commercialization, and it allows for future inclusion of other enteric pathogens and gut function biomarkers. PUBLIC HEALTH RELEVANCE: This study proposes a diagnostic panel to identify the Category B Biodefense intestinal pathogens Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli in patients with diarrhea and dysentery. The panel will also quantify fecal biomarkers to describe intestinal inflammation and hemorrhage, providing novel information regarding disease severity and patient condition.