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

开发基于 Z 结构域的艰难梭菌毒素乳胶凝集测定

• 批准号: 8831924
• 负责人: Niles Patrick Donegan
• 金额: $ 22.49万
• 依托单位: SAUREUS, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8831924
• 关键词: Affect; Affinity; Agglutination; Agglutination Tests; Algorithms; Allergic; Antibiotics; Antibodies; Antibody Specificity; Antigens; Bacteria; Binding; Biological Assay; Care given by nurses; Caring; Cells; Charge; Chemistry; Clindamycin; Clinical; Clinical Research; Clinical Trials; Clostridium difficile; Colon; Coupled; Coupling; Cytotoxin; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Diarrhea; Disease; Disease Marker; Epitopes; Equipment; Exotoxins; Fc Immunoglobulins; Feces; Fluoroquinolones; Funding; Genes; Genetic Engineering; Glutamate Dehydrogenase; Goals; Gold; Hospital Nursing; Hospitals; Human; Human Cell Line; Immunoglobulin G; Individual; Infection; Lateral; Latex; Latex Bead; Lead; Length; Length of Stay; Link; Marketing; Messenger RNA; Metronidazole; Monoclonal Antibodies; Mus; Nursing Homes; Optics; Oral Administration; Oryctolagus cuniculus; Particulate; Patient Isolation; Patients; Phase; Procedures; Production; Protein Binding Domain; Pseudomembranous Colitis; Reading; Reagent; Relapse; Reproduction spores; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Sensitivity and Specificity; Small Business Innovation Research Grant; Surface; Symptoms; Technology; Tertiary Protein Structure; Testing; Time; Toxic Megacolon; Toxin; Vancomycin; accurate diagnosis; antigen binding; assay development; base; beta-Lactams; clinically relevant; cost; cytotoxic; cytotoxicity; density; diagnosis standard; diagnostic assay; fluid flow; image processing; methicillin resistant Staphylococcus aureus; novel; novel diagnostics; pathogen; prototype; public health relevance; rapid diagnosis; retinal rods; skills

 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.

 描述（由申请人提供）：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。这些毒素称为Toin A（TcdA）和Toin B（TcdB），是诊断C的主要标志物。艰难梭菌相关性腹泻（CDAD）。诊断CDAD的金标准是分离细菌，然后使用培养的人细胞进行细胞毒素检测。由于时间、劳动力和复杂的设置，这些程序在许多医院环境中不是常规部署的。基于EIA的毒素检测虽然具有特异性，但并不十分敏感。RT-PCR检测毒素基因非常敏感，但不能区分定植者和活动性疾病患者。因此，存在CDAD的过度诊断和治疗。因此，对于用于检测C.在医院或护理设施中，艰难梭菌毒素具有高灵敏度和特异性。我们公司，索瑞斯公司，拥有专业知识和技术，能够编制一个简单，廉价和快速的C。艰难梭菌毒素该技术是在优化的乳胶珠（尺寸、电荷密度和偶联化学）的凝集平台上发现的，该乳胶珠具有共价连接的独特IgG结合结构域蛋白，其更好地结合兔IgG和小鼠IgG。抗TcdB或TcdA的兔单克隆抗体将通过Fc与优化的乳胶珠表面上的Z结构域蛋白结合。通过使用几种针对TcdA和TcdB不同表位的单克隆抗体，粪便滤液中的同源毒素将与固定的抗体结合，导致凝集反应。我们预测，我们的测定，基于通过IgG结合结构域蛋白固定在优化的乳胶珠表面上的抗毒素抗体，将是快速的（< 10分钟），廉价和高度特异性的TcdB和TcdA。建立了一种新的检测C.艰难梭菌毒素将消除对大多数基于PCR的测定所需的昂贵设备和高操作员技能的需要。本研究的主要目的是：1）优化兔抗C.艰难梭菌毒素A和B连接到共价连接到优化的乳胶珠的独特IgG结合结构域蛋白; II）优化用于诊断C.艰难梭菌毒素A和B（即灵敏度、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更敏感的凝集反应。在完成这些目标后，我们预期会申请第二阶段SBIR资助，以完成一个光学阅读装置，该装置将用于评估对患者粪便样本进行的临床研究。这些临床试验的完成将使我们能够向FDA-CLIA申请批准诊断试剂盒。