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.

性状（由申请方提供）：耐甲氧西林沙门氏菌。金黄色葡萄球菌已经在世界范围内引起了重大的公共卫生问题。沙门氏菌耐甲氧西林的基础。金黄色葡萄球菌菌株的青霉素结合蛋白是用与大多数2-内酰胺结合较差的替代青霉素结合蛋白PBP 2a功能性替代先天青霉素结合蛋白（PBPs）。甲氧西林耐药沙门氏菌诊断的一个主要难题是：金黄色葡萄球菌感染是入院后诊断所需的时间，因此使这些危险的病原体能够在医院内传播。传统的耐甲氧西林沙门氏菌的培养诊断。金黄色葡萄球菌至少需要两天。一种更新，更快的诊断方法是基于PCR的测试。虽然这种方法比传统的培养快，但需要昂贵的设备和更高的单位成本。因此，耐甲氧西林链球菌的快速诊断仍存在空白。金黄色葡萄球菌，其需要具有更快周转时间和更便宜成本的简单独立测定。索雷乌斯公司是一家专注于快速诊断重要医疗相关病原体的技术公司。利用我们在革兰氏+分子遗传学方面的知识，我们已经改造了L。lactis细菌，使异源表达的SPA，一种表面蛋白的S。金黄色。由于Spa能有效结合哺乳动物IgG，我们证明兔抗PBP 2a抗体能以高亲和力结合锚定在L.乳酸。为了促进快速细胞裂解，溶葡萄球菌酶，一种高度特异性裂解S。金黄色葡萄球菌（S. epidermidis），将以仅裂解S.金黄色葡萄球菌有效，导致细胞内和膜结合的内容物，包括PBP 2a溢出。将几种表位特异性的兔抗PBP 2a单克隆抗体固定在L.乳酸菌将与PBP 2a凝集，从而导致凝集反应，该凝集反应可以在没有任何外部视觉辅助的情况下容易地可视化。该测定法简单且具有成本效益，不依赖于操作员技能或复杂的实验室设备;这些特征通常在基于PCR的测定法中不存在。因此，我们计划开发两个具体的目标，为这个建议：一）建立基于兔单克隆抗体的凝集平台PBP 2a固定在斯帕表达L。乳酸菌和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，其将增强用于MRSA的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。在完成这些研究后，我们计划申请第II期SBIR资助，以进行有关沙门氏菌临床样本的研究。金黄色。临床研究的成功结论将证明向FDA-CLIA申请批准诊断试剂盒的合理性。