Developing a Z-domain based latex agglutination assay for C. difficile toxins

 描述(申请人提供)：医院和护理机构中最常见的腹泻归因于艰难梭菌分泌的毒素。这些毒素被称为Toin A(TcdA)和毒素B(Tcdb)，是艰难梭菌相关性腹泻(CDAD)诊断的主要标志。诊断CDAD的金标准是分离细菌，然后使用培养的人类细胞进行细胞毒素检测。由于时间、人力和复杂的设置，这些程序在许多医院环境中并不是常规部署。基于EIA的毒素检测虽然具有特异性，但并不十分灵敏。毒素基因的RT-PCR检测非常敏感，但不能区分定殖者和疾病活动期的人。因此，CDAD存在过度诊断和过度治疗。因此，医院或护理机构对检测艰难梭菌毒素的快速和廉价的诊断方法的需求尚未得到满足，该方法具有高灵敏度和高特异性。我们的公司Saureus Inc.拥有专业知识和技术，能够编制一种简单、廉价和快速的艰难梭菌毒素检测方法。这项技术是建立在优化的乳胶珠(大小、电荷密度和偶联化学)的凝集平台上的，该乳胶珠具有共价连接的独特的免疫球蛋白结合结构域蛋白，可以更好地结合兔的免疫球蛋白和小鼠的免疫球蛋白。兔抗TcdB或TcdA的单抗通过Fc结合到优化乳胶珠表面的Z结构域蛋白上。通过使用几种针对TcdA和TcdB不同表位的单抗，粪便滤液中的同源毒素将与固定化抗体结合，导致凝集反应。我们预测，基于优化乳胶珠表面的免疫球蛋白结合结构域蛋白固定的抗毒素抗体，我们的检测将是快速的(&lt；10分钟)，廉价，并且对TcdB和TcdA具有高度的特异性。一种检测艰难梭菌毒素的新型凝集平台的开发将消除对昂贵设备的需求，并消除大多数基于聚合酶链式反应的分析所需的高操作员技能。为了开发这一检测方法，我们有两个具体的目标：i)基于艰难梭菌毒素A和B的兔单抗优化凝集平台，艰难梭菌毒素A和B与独特的免疫球蛋白结合结构域蛋白共价连接到优化的乳胶珠上；ii)优化用于艰难梭菌毒素A和B诊断的主要原型凝集试剂盒(即敏感性、特异性、定量、试剂稳定性和易用性)。我们的目标是开发一种比现有EIA更灵敏的凝集反应。在完成这些目标后，我们预计将申请第二期SBIR拨款，以完成一种光学读数设备，该设备将用于评估对患者腹泻样本的临床研究。完成这些临床试验将使我们能够向FDA-CLIA申请批准诊断试剂盒。

 DESCRIPTION (provided by applicant): The most common diarrhea in hospitals and nursing care facilities are attributed to toxins secreted by Clostridium difficile. These toxins, called Toin A (TcdA) and Toxin B (TcdB), are the primary markers for the diagnosis of C. difficile associated diarrhea (CDAD). The gold standard for diagnosis of CDAD is isolation of the bacteria followed by cytotoxin detection using cultured human cells. Due to time, labor and sophisticated setup, these procedures are not routinely deployed in many hospital settings. EIA-based assays for toxins, while specific, is not very sensitive. RT-PCR assay for toxin genes is very sensitive but cannot differentiate colonizers from those with active disease. As a result, there is over-diagnosis and treatment of CDAD. Accordingly, there is an unmet need for a rapid and cheaper diagnostic assay for detecting C. difficile toxins with high sensitivity and specificity at hospitas or nursing care facilities. Our company, Saureus Inc., has the expertise and technology to enable compilation of a simple, cheap and rapid assay for C. difficile toxins. This technology is found on an agglutination platform of optimized latex beads (size, charge density and coupling chemistry) that has covalently-linked unique IgG-binding domain protein which binds rabbit IgG better mouse IgG. Rabbit monoclonal antibodies against TcdB or TcdA would bind to Z-domain protein on the surface of optimized latex beads via the Fc. By using several monoclonal antibodies against divergent epitopes of TcdA and TcdB, cognate toxins in stool filtrates will bind to immobilized antibodies, leading to an agglutination reaction. We predict that our assay, based on anti-toxin antibodies immobilized by IgG-binding domain protein on the surface of optimized latex beads, will be rapid (< 10 min), cheap and highly specific for TcdB and TcdA. The development of a novel agglutination platform to detect C. difficile toxins will eliminate the need for expensive equipment and high operator skill required for most PCR-based assays. For development of this assay, we have two specific aims: I) optimize the agglutination platform based on rabbit monoclonal antibodies to C. difficile toxins A and B attached to unique IgG-binding domain protein that is covalently linked to optimized latex bead; II) optimize the lead prototype agglutination kit for the diagnosis of C. difficile toxins A and B (i.e. sensitivity, specificity, quantitation, reagent stability and ease of use). Our goal is to develop an agglutination reaction that is more sensitive than existing EIA. Upon completion of these aims, we expect to apply for Phase II SBIR funding to complete an optical reading device which will be used to evaluate clinical studies on diarrheal samples from patients. Completion of these clinical trials will enable us to apply to the FDA-CLIA for approval of the diagnostic kit.