Ultrasensitive quantitative digital ELISA for C. difficile toxins in stool

描述(由申请人提供)：最近由产毒艰难梭菌引起的全球发病率和疾病严重程度的增加是一个主要的临床和诊断问题。艰难梭菌感染(CDI)，从轻微的抗生素相关性腹泻到潜在的致命性伪膜性结肠炎，消耗了越来越多的医院资源用于诊断、治疗和感染控制。认识到这一问题的升级，加上出现了能够高产毒素的流行菌株和疾病严重性增加，增加了改进CDI诊断方法的紧迫性。艰难梭菌引起的疾病是由于携带毒素基因的菌株产生毒素A和B，而直接检测粪便中的这些毒素被认为是临床上最重要的。然而，现有的每一种诊断方法都有很大的局限性。特别是，最广泛使用的直接检测粪便中毒素A和B的酶免疫分析法(EIA)存在灵敏度低和毒素检测的分析下限高的问题。迫切需要一种简单的粪便检测方法，将高分析灵敏度和临床应用结合起来。 毒素检测的特异性使我们能够对有病和无病的人进行最佳分类。疾病的严重程度与宿主的毒素水平直接相关，这一事实表明，对粪便中毒素水平进行量化的能力也可能使该测试用于预测疾病结果和监测治疗反应。Quanterix公司(马萨诸塞州剑桥)开发了一种超灵敏和定量的“数字酶联免疫吸附试验”技术，用于测量复杂临床标本中的微量蛋白质分析物。数字酶联免疫吸附试验的检测限(LOD)始终比使用相同抗体捕获和检测的EIA低至少1000倍。在这里，波洛克博士(BIDMC)建议与Quanterix合作，开发并在临床上验证粪便标本中艰难梭菌毒素A和B的数字ELISA。该检测方法将是首个针对细菌感染性疾病应用开发的数字酶联免疫吸附试验，也是该技术首次应用于粪便检测。我们的具体目标是1)开发艰难梭菌毒素A和B的数字化ELISA，其检测极限为粪便中1 pg/毫升，使用商业抗体捕获和检测纯化的天然毒素A和B，以及2)使用废弃的临床标本通过聚合酶链式反应检测艰难梭菌，在临床上验证这两种数字ELISA。在临床验证的同时，我们将对定量毒素A和B水平与疾病严重程度和艰难梭菌菌株类型的相关性进行探索性评估。这项工作将为未来开发一个低成本的自动化平台和大型前瞻性临床研究奠定坚实的技术基础，以评估预测临床结果和监测治疗反应的化验效用。最终，这项工作有可能导致我们诊断和管理CDI的方式发生范式转变。

DESCRIPTION (provided by applicant): The recent increase in global incidence and severity of disease caused by toxigenic Clostridium difficile presents a major clinical and diagnostic problem. C. difficile infection (CDI), ranging from mild antibiotic- associated diarrhea to potentially lethal pseudomembranous colitis, consumes a steadily increasing proportion of hospital resources for diagnosis, treatment, and infection control. Recognition of the escalation of this problem, in addition to the emergence of epidemic strains capable of toxin hyper-production and increased disease severity, have increased the urgency of improving methods for diagnosis of CDI. Disease caused by C. difficile is due to production of toxins A and B by strains harboring the toxin genes, and testing stool directly for these toxins is considered most important clinically. However, each of the existing diagnostic methods have major limitations. In particular, the most widely used tests, enzyme immunoassays (EIAs) that directly detect toxins A and B in stool, suffer from poor sensitivity and high analytical limits of toxin detection. Thereis a critical need for a simple stool test that combines high analytical sensitivity with the clinical specificity of toxin detection to allow us to optimally classify those with and without disease. Th fact that disease severity has been directly correlated to toxin levels in the host suggests that the ability to quantify toxin levels in stool could also potentially allow use of the test to predit disease outcomes and to monitor response to therapy. Quanterix Corporation (Cambridge, MA) has developed an ultrasensitive and quantitative "digital ELISA" technology for measurement of minute amounts of protein analytes in complex clinical specimens. Digital ELISA has limits of detection (LOD) that are consistently at least 1,000-fold lower than EIAs using the same antibodies for capture and detection. Here, Dr. Pollock (BIDMC) proposes to collaborate with Quanterix to develop and clinically validate digital ELISAs for C. difficile toxins A and B in stoo specimens. The proposed assay will be the first digital ELISA developed for a bacterial infectious diseases application and the first application of the technology using stool. Our specific aims are to 1) develop digital ELISAs for C. difficile toxins A and B with limits of detection of 1 pg/mL in stool, using commercial antibodies for capture and detection of purified native toxins A and B, and 2) clinically validate both digital ELISAs using discarded clinical specimens submitted for C. difficile testing by PCR. Concurrently with this clinical validation we will do an exploratory evaluation of the correlation of quantitative toxin A and B levels with disease severity and C. difficile strain type. This work will establish a solid technological foundation for future development of a low- cost, automated platform and for large prospective clinical studies to evaluate assay utility for predicting clinical outcome and monitoring therapeutic response. Ultimately, this work has the potential to lead to a paradigm shift in the way we diagnose and manage CDI.