Developing a rapid agglutination assay for MRSA.

描述（由申请人提供）：耐甲氧西林金黄色葡萄球菌在世界范围内造成了重大的公共卫生问题。金黄色葡萄球菌耐甲氧西林的基础是先天青霉素结合蛋白（PBPs）被替代的青霉素结合蛋白PBP2a功能性替代，而PBP2a与大多数2-内酰胺结合不良。诊断耐甲氧西林金黄色葡萄球菌感染的一个主要难题是住院后诊断所需的时间，从而使这些危险病原体在医院内传播。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。一种更新和更快的诊断方法是基于聚合酶链反应的检测。虽然这种方法比传统的培养更快，但化验需要昂贵的设备和更高的单位成本。因此，在耐甲氧西林金黄色葡萄球菌的快速诊断方面存在空白，因为耐甲氧西林金黄色葡萄球菌需要一种简单的独立检测方法，周转时间更快，成本更低。Saureus Inc.是一家专注于快速诊断重要医疗保健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，我们设计了乳酸乳杆菌，使金黄色葡萄球菌表面蛋白Spa能够异源表达。由于Spa与哺乳动物igg有效结合，我们证明了兔抗pbp2a抗体与乳杆菌表面固定的重组Spa具有高亲和力。为了促进细胞的快速裂解，溶葡萄球菌酶，一种对金黄色葡萄球菌裂解具有高度特异性的酶（对表皮葡萄球菌裂解的特异性要低得多），将以一种只能有效裂解金黄色葡萄球菌的方式进行配置，导致细胞内和膜结合的内容物溢出，包括PBP2a。几种固定在乳酸菌表面的表位特异性兔抗PBP2a单克隆抗体会与PBP2a凝集，导致凝集反应，无需任何外部视觉辅助即可轻松可视化。这种分析方法简单，成本效益高，不依赖于操作人员的技能或复杂的实验室设备；这些特征在基于pcr的分析中通常不存在。因此，我们计划制定两个具体目标：1)建立固定在表达spa的L. lactis上的PBP2a兔单克隆抗体的凝集平台和基于溶葡萄球菌素介导的MRSA细胞内容物释放的裂解平台；II)优化可提高MRSA先导原型凝集试剂盒的敏感性、特异性、试剂稳定性和易用性的变量。在完成这些研究后，我们计划申请二期SBIR资金用于金黄色葡萄球菌临床样本的研究。临床研究的成功结论将证明向FDA-CLIA申请批准诊断试剂盒是合理的。

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.