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针对粪便中贾第鞭毛虫、隐孢子虫、溶组织芽胞杆菌、志贺毒素产志贺菌和大肠杆菌检测的多重免疫诊断试剂盒。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。完整的多重诊断将提供腹泻/痢疾的主要B类肠致病性病原体的快速诊断，并描述疾病的严重程度。该检测采用敏感和特异性的基于免疫荧光的Luminex技术。Luminex允许使用一系列独特的可检测的头集对单个样品中的分析物进行多路检测。分析物特异性抗体是从现有的fda批准的免疫测定中选择的，保证了它们的性能和稳定性。初步数据表明，Luminex对肠道原生动物的敏感性超过ELISA，与RT-PCR相当，具有纯化的寄生虫曲线。在粪便中检测出108例经fda清除的寄生虫ELISA阳性反应，但仍需完全验证临床敏感性。Luminex检测乳铁蛋白和血红蛋白与商业ELISA检测相关，并提供了更大的定量范围，提高了粪便分析的效率。目标1详细介绍了贾第鞭毛虫、隐孢子虫和溶组织芽胞杆菌的肠道原生动物寄生虫小组。目标#2扩展了腹泻/痢疾小组，包括志贺毒素、志贺样毒素（Stx1）和志贺样毒素2 （Stx2）的检测。Aim #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病情的生物标志物。这些研究的成功完成将提供一种原型诊断，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。该多重分析将有竞争力获得II期NIH小型企业资金，以完成生产转移和FDA 510(k)许可验证。基于luminex的多重检测是一个很好的商业化候选，它允许未来包含其他肠道病原体和肠道功能生物标志物。

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.