Developing a rapid agglutination assay for MRSA.

开发 MRSA 快速凝集测定方法。

• 批准号: 8124324
• 负责人: Niles Patrick Donegan
• 金额: $ 30万
• 依托单位: SAUREUS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8124324
• 关键词: Acquired Immunodeficiency Syndrome; Admission activity; Affinity; Age; Agglutination; Agglutination Tests; Agglutinins; Antibiotics; Antibodies; Area; Axilla; Bacteremia; Bacteria; Binding; Biological Assay; Cell Wall; Cells; Cessation of life; Characteristics; Cheese; Clinical; Clinical Research; Collection; Communities; Cytolysis; Dairy Products; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early Diagnosis; Engineering; Enzymes; Epitopes; Equipment; Extravasation; Funding; Geographic state; Gold; Health care facility; Healthcare; Hospitalization; Hospitals; Immunoglobulin G; Incidence; Individual; Infection; Intracellular Membranes; Knowledge; Lactams; Lactococcus lactis; Lead; Legal patent; Lysostaphin; LytA enzyme; Mediating; Membrane Proteins; Methicillin Resistance; Methods; Molecular Genetics; Monobactams; Monoclonal Antibodies; Mupirocin; Nose; Oryctolagus cuniculus; Patients; Penicillin-Binding Proteins; Phase; Population; Production; Proteins; Public Health; Reaction; Reagent; Recombinants; Resistance development; Risk; Sampling; Sensitivity and Specificity; Skin; Small Business Innovation Research Grant; Specificity; Staphylococcus aureus; Surface; Surveillance Program; Technology; Testing; Time; United States; Visual Aid; Yogurt; base; clinically relevant; cost; cost effective; environmental change; methicillin resistant Staphylococcus aureus; microorganism; novel; novel diagnostics; open wound; pathogen; point of care; prevent; prototype; rapid diagnosis; resistant strain; skills

DESCRIPTION (provided by applicant): Methicillin resistant S. aureus has posed major public health problems worldwide. The basis of methicillin resistance in S. aureus strains is the functional replacement of innate penicillin binding proteins (PBPs) with alternative penicillin binding protein PBP2a which binds poorly to most 2-lactams. A major dilemma in the diagnosis of methicillin resistant S. aureus infections is the time required for diagnosis after hospital admission, thus enabling intra-hospital spread of these dangerous pathogens. Conventional cultures for the diagnosis of methicillin resistant S. aureus take at least two days. A newer and faster method for diagnosis is the PCR-based test. Although this method is faster than conventional cultures, the assay entails expensive equipment and higher per-unit cost. Therefore, there is a gap in the rapid diagnosis of methicillin-resistant S. aureus which requires a simple standalone assay with a faster turnaround time and at a cheaper cost. Saureus Inc. is a company that focuses on enabling technology for rapid diagnosis of important healthcare- related pathogens. Taking advantage of our knowledge in Gram+ molecular genetics, we have engineered L. lactis bacteria to enable heterologous expression of Spa, a surface protein of S. aureus. As Spa binds efficiently to mammalian IgGs, we demonstrated that rabbit anti-PBP2a antibodies bind at a high affinity to the recombinant Spa anchored on the surface of L. lactis. To facilitate rapid cell lysis, lysostaphin, an enzyme highly specific for lysis of S. aureus (much less so for S. epidermidis), will be deployed in a way that only lyses S. aureus efficiently, resulting in spillage of intracellular and membrane-bound contents including PBP2a. Several epitope-specific rabbit anti-PBP2a monoclonal antibodies immobilized on the surface of L. lactis bacteria will agglutinate with PBP2a to result in a clumping reaction that can be easily visualized without any extraneous visual aid. This assay is simple and cost effective and does not rely on operator skill or sophisticated lab equipment; these characteristics are typically absent in PCR-based assays. Accordingly, we plan to develop two specific aims for this proposal: I) establishing the agglutination platform based on rabbit monoclonal antibodies to PBP2a immobilized on Spa-expressing L. lactis and the lysing platform based on lysostaphin-mediated release of cellular contents from MRSA; II) optimization of variables that would enhance sensitivity, specificity, reagent stability and ease of use for the lead prototype agglutination kit for MRSA. Upon completion of these studies, we plan to apply for Phase II SBIR funding for studies on clinical samples of S. aureus. Successful conclusion of clinical studies will justify an application to the FDA-CLIA for approval of the diagnostic kit. PUBLIC HEALTH RELEVANCE: Rapid diagnosis of MRSA is costly and time consuming. We have developed a technology to enable compilation of a cheap, simple and rapid diagnostic test. This technology is based on a novel agglutination reaction between PBP2a rapidly released from MRSA cells and rabbit antibodies immobilized by protein A on Lactococcus lactis, an innocuous bacteria normally found in dairy products such as yogurt and cheese.

描述(申请人提供)：耐甲氧西林金黄色葡萄球菌已经在世界范围内造成了重大的公共卫生问题。金黄色葡萄球菌对甲氧西林耐药的基础是天然的青霉素结合蛋白(PBPs)被替代的青霉素结合蛋白PBP2a所取代，该蛋白与大多数2-内酰胺类抗生素结合能力较差。诊断耐甲氧西林金黄色葡萄球菌感染的一个主要难题是入院后诊断所需的时间，从而使这些危险病原体能够在医院内传播。诊断耐甲氧西林金黄色葡萄球菌的常规培养至少需要两天时间。一种更新、更快的诊断方法是基于聚合酶链式反应的检测。虽然这种方法比传统的培养方法更快，但这种分析需要昂贵的设备和更高的单位成本。因此，在快速诊断耐甲氧西林金黄色葡萄球菌方面存在一个空白，这需要一种简单的独立检测方法，具有更快的周转时间和更低的成本。Saureus Inc.是一家专注于支持快速诊断重要医疗保健相关病原体的技术的公司。利用我们在Gram+分子遗传学方面的知识，我们已经改造了乳酸乳杆菌，使其能够异源表达金黄色葡萄球菌的表面蛋白SpA。由于SpA能有效地与哺乳动物免疫球蛋白结合，我们证明了兔抗PBP2a抗体与乳酸乳杆菌表面的重组SpA有很高的亲和力。为了促进细胞的快速裂解，溶葡萄球菌酶，一种高度特异的金黄色葡萄球菌裂解酶(对表皮葡萄球菌就更少了)，将以一种只有效裂解金黄色葡萄球菌的方式部署，导致包括PBP2a在内的细胞内和膜结合内容物的溢出。固定在乳酸乳杆菌表面的几种抗原表位特异性的兔抗PBP2a单抗将与PBP2a发生凝集反应，无需任何额外的视觉辅助，就可以很容易地观察到这种凝集反应。这种分析方法简单，成本效益高，不依赖于操作员的技能或复杂的实验室设备；这些特征在基于聚合酶链式反应的分析中通常是不存在的。因此，我们计划为这项建议制定两个具体目标：i)建立基于兔抗PBP2a单抗的凝集平台，该凝集平台基于SpA表达的乳酸乳杆菌，以及基于溶葡萄球菌酶介导的从MRSA释放细胞内容物的裂解平台；ii)优化变量，以提高MRSA主要原型凝集试剂盒的灵敏度、特异性、试剂稳定性和易用性。在这些研究完成后，我们计划申请第二期金黄色葡萄球菌临床样本研究的SBIR拨款。临床研究的成功结束将证明向FDA-CLIA申请批准该诊断试剂盒是合理的。 与公共卫生相关：快速诊断MRSA既昂贵又耗时。我们已经开发出一种技术，能够编制一种廉价、简单和快速的诊断测试。这项技术是基于MRSA细胞迅速释放的PBP2a与蛋白A固定在乳酸乳球菌上的兔抗体之间的一种新型凝集反应。乳酸乳球菌是一种无害的细菌，通常存在于酸奶和奶酪等乳制品中。