Developing a rapid agglutination assay for MRSA.

开发 MRSA 快速凝集测定方法。

• 批准号: 8277211
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8277211
• 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.

描述（由申请人提供）：耐甲氧西林S.金黄色葡萄球菌在全球构成了主要的公共卫生问题。金黄色葡萄球菌菌株中甲氧西林耐药性的基础是用替代性青霉素结合蛋白PBP2A的先天青霉素结合蛋白（PBP）的功能替代，该蛋白与大多数2-酰基酰胺的结合很差。诊断甲氧西林的金黄色葡萄球菌感染的主要困境是入院后诊断所需的时间，从而实现了这些危险病原体的院内扩散。诊断甲氧西林的耐甲氧西葡萄球菌的常规培养物至少需要两天。基于PCR的测试是一种更新，更快的诊断方法。尽管这种方法比传统文化更快，但该测定需要昂贵的设备和更高的单位成本。因此，耐甲氧西林的金黄色葡萄球菌的快速诊断存在差距，这需要一个简单的独立测定，其周转时间更快，成本更便宜。 Saureus Inc.是一家专注于启用技术来快速诊断重要医疗保健相关病原体的公司。利用我们在革兰氏+分子遗传学方面的知识，我们已经设计了乳酸乳杆菌细菌，以实现Spa的异源表达，Spa是金黄色葡萄球菌的表面蛋白。随着水疗中心与哺乳动物IgG有效结合，我们证明了兔抗PBP2A抗体在高亲和力下与固定在乳酸乳杆菌表面的重组水疗中心的高亲和力结合。为了促进快速细胞裂解，溶血蛋白是一种高度特异性的金黄色葡萄球菌裂解（对于表皮链球菌的裂解），将以有效的裂解金黄色葡萄球菌的方式部署，导致金黄色葡萄球菌有效地裂解，导致细胞内和膜结合的含量包括PBP2A。固定在乳酸乳杆菌细菌表面上的几种表位特异性兔抗PBP2A单克隆抗体将用PBP2A凝结，从而导致结块反应，可以轻松地可视化，而无需任何外在可视化。该测定简单且具有成本效益，不依赖操作员技能或精致的实验室设备；这些特征通常在基于PCR的测定中不存在。因此，我们计划针对该提案开发两个具体目标：i）基于兔子单克隆抗体建立固定在Spa表达乳酸乳杆菌的PBP2A的凝集平台，以及基于乳脂蛋白的乳乳杆菌和基于lysostaphin介导的细胞介导的细胞含量的裂解平台。 ii）优化将增强MRSA铅原型凝集试剂盒的灵敏度，特异性，试剂稳定性和易用性的变量。这些研究完成后，我们计划向金黄色葡萄球菌的临床样本申请II期SBIR资金。临床研究的成功结论将证明对FDA-CLIA的应用合理以批准诊断套件。