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（由适用提供）：医院和护理设施中最常见的腹泻归因于艰难梭菌分泌的毒素。这些称为Toin A（TCDA）和毒素B（TCDB）的毒素是艰难梭菌相关腹泻（CDAD）诊断的主要标记。 CDAD诊断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，劳动力和复杂的设置，这些程序并未常规部署在许多医院环境中。基于EIA的毒素测定法，虽然具体，但并不是很敏感。毒素基因的RT-PCR分析非常敏感，但不能将菌落与活性疾病的菌落分化。结果，CDAD有过度诊断和治疗。据此，在医院或护士护理设施中检测具有高灵敏度和特异性的艰难梭菌毒素的诊断测定不足。我们的公司Saureus Inc.拥有专业知识和技术，可以为艰难梭菌毒素进行简单，廉价和快速测定的汇编。在优化的乳胶珠（尺寸，电荷密度和耦合化学）的凝集平台上发现了这项技术，该平台具有共价链接的独特IgG结合域蛋白，该蛋白结合了兔IgG更好的小鼠IgG。针对TCDB或TCDA的兔单克隆抗体将通过FC在优化的乳胶珠的表面与Z-域蛋白结合。通过使用针对TCDA和TCDB发散表位的几种单克隆抗体，粪便过滤器中的同源毒素将与固定化抗体结合，从而导致凝集反应。我们预测，基于IgG结合域蛋白在优化的乳胶珠的表面上固定的抗毒素抗体的评估将很快（<10分钟），廉价且对TCDB和TCDA高度具体。开发一个新型的凝集平台来检测艰难梭菌毒素，将消除大多数基于PCR的测定所需的昂贵设备和高运营商技能的需求。为了开发该测定法，我们有两个具体的目的：i）优化基于与艰难梭菌毒素A和B的兔单克隆抗体，该抗体与独特的IgG结合域蛋白相连，该抗体与优化的乳胶珠共价链接； ii）优化用于诊断艰难梭菌A和B的铅原型凝集试剂盒（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。这些目标完成后，我们预计将申请II期SBIR资金来完成光学阅读装置，该设备将用于评估患者的腹泻样品的临床研究。这些临床试验的完成将使我们能够申请FDA-CLIA批准诊断套件。