Ultrasensitive quantitative digital ELISA for C. difficile toxins in stool

粪便中艰难梭菌毒素的超灵敏定量数字 ELISA

• 批准号: 8427747
• 负责人: Nira Pollock
• 金额: $ 27.3万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-17 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8427747
• 关键词: Antibiotics; Antibodies; Attention; Biological Assay; Buffers; Clinical; Clinical Research; Clostridium difficile; Communicable Diseases; Complex; Cytotoxin; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Diarrhea; Disease; Disease Outcome; Enterotoxins; Enzyme Immunoassay; Enzyme-Linked Immunosorbent Assay; Epidemic; Evaluation; Feces; Foundations; Future; Gene Expression; Genes; Gold; Hospitals; Incidence; Infection; Infection Control; Lead; Measurement; Methods; Monitor; Nucleic Acid Amplification Tests; Outcome; Patients; Performance; Production; Proteins; Pseudomembranous Colitis; Recurrence; Regulation; Reproduction spores; Research; Resources; Risk; Sampling; Severity of illness; Solid; Specificity; Specimen; Technology; Testing; Therapeutic; Toxin; Validation; Work; base; cost; cytotoxicity test; digital; improved; in vivo; prospective; public health relevance; response; single molecule

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.

描述（由申请人提供）：最近由产酶艰难梭菌引起的疾病的全球发病率和严重程度增加，这是一个主要的临床和诊断问题。C.艰难梭菌感染（CDI），从轻微的抗生素相关性腹泻到潜在的致死性伪膜性结肠炎，消耗了用于诊断、治疗和感染控制的医院资源的比例稳步增加。认识到这个问题的升级，除了能够毒素过度生产和增加疾病严重性的流行菌株的出现之外，已经增加了改进用于诊断CDI的方法的紧迫性。 由C.艰难梭菌是由于携带毒素基因的菌株产生毒素A和B，并且直接测试粪便中的这些毒素在临床上被认为是最重要的。然而，现有的诊断方法都有很大的局限性。特别是，最广泛使用的测试，直接检测粪便中毒素A和B的酶免疫测定（EIA），具有低灵敏度和毒素检测的高分析极限。迫切需要一种简单的粪便检测，将高分析灵敏度与临床诊断相结合。 毒素检测的特异性，使我们能够最佳地分类那些有和没有疾病。疾病严重程度与宿主中的毒素水平直接相关的事实表明，定量粪便中毒素水平的能力也可能允许使用该测试来预测疾病结果并监测对治疗的反应。 Quanterix公司（剑桥，MA）开发了一种超灵敏的定量“数字ELISA”技术，用于测量复杂临床标本中微量的蛋白质分析物。数字ELISA的检测限（LOD）始终比使用相同抗体进行捕获和检测的EIA低至少1，000倍。在这里，Pollock博士（BIDMC）建议与Quanterix合作开发和临床验证C.粪便标本中艰难梭菌毒素A和B。拟议的检测将是第一个为细菌感染性疾病应用开发的数字ELISA，也是该技术首次使用粪便进行应用。我们的具体目标是：1）开发C。艰难梭菌毒素A和B，粪便中的检测限为1 pg/mL，使用商业抗体捕获和检测纯化的天然毒素A和B，和2）使用提交的用于C的废弃临床标本临床验证两种数字ELISA。通过PCR进行艰难梭菌检测。与此临床验证同时，我们将对定量毒素A和B水平与疾病严重程度和C的相关性进行探索性评价。艰难菌株型。这项工作将为未来开发低成本自动化平台和大型前瞻性临床研究奠定坚实的技术基础，以评估用于预测临床结果和监测治疗反应的测定实用性。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。