Rabbit InMAD Discovery of Novel Biomarkers for POC Tuberculosis Diagnostic Assay

Rabbit InMAD 发现用于 POC 结核病诊断分析的新型生物标志物

• 批准号: 8603728
• 负责人: SEAN BAUMAN
• 金额: $ 29.96万
• 依托单位: IMMUNO-MYCOLOGICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8603728
• 关键词: Affinity; Africa; Africa South of the Sahara; Antibodies; Antigens; Area; Asia; Bacillus (bacterium); Bacteria; Biological Assay; Biological Markers; Biological Process; Body Fluids; Carbohydrates; Cells; Cessation of life; Chronic; Clinic; Clinical; Communicable Diseases; Country; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Disease model; Early Diagnosis; Endemic Diseases; Enzyme Immunoassay; Epitopes; Exhibits; Goals; HIV; HIV Seropositivity; Health Care Costs; Human; Human Resources; Immunization; Immunoassay; Infection; Lateral; Liquid substance; Methods; Microscopy; Modification; Monoclonal Antibodies; Morbidity - disease rate; Mus; Mycobacterium tuberculosis; Oryctolagus cuniculus; Patients; Phase; Polysaccharides; Population; Production; Proteins; Proteomics; Research Project Grants; Resources; Sampling; Serum; Small Business Technology Transfer Research; Specimen; Sputum; Techniques; Technology; Testing; Time; Training; Translational Research; Tuberculosis; Urine; Validation; Virulent; World Health Organization; clinically relevant; cost; immunogenic; immunogenicity; lipoarabinomannan; meetings; microbial; mortality; novel; phase 1 study; point of care; point-of-care diagnostics; polyclonal antibody; public health relevance; research study; success; tool; transmission process; tuberculosis treatment

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a chronic infectious disease that infects approximately one-third of the world's population. Eighty-five percent of the estimated 9.4 million new cases of TB in 2008 occurred in resource-limited countries located in Asia and Africa. In 2010, there were 1.1 million deaths from TB among HIV-negative patients and 350,000 additional TB deaths among HIV-positive patients. In some tuberculosis-endemic areas, fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis, in clinical specimens by serial sputum smear microscopy and culture. These methods lack sensitivity, are time consuming, are expensive, and require trained personnel. An alternative approach is to develop an efficient immunoassay to detect M. tb. antigens in bodily fluid, such as serum or urine. Current commercial immunoassay kits that detect M. tb. glycan lipoarabinomannan (LAM) in urine exhibit poor sensitivity perhaps due to low abundance. Alternative biomarkers need to be identified to facilitate accurate diagnosis and treatment of tuberculosis. Our overall hypothesis is that M. tb. biomarkers are shed into the urine and/or serum during infection and that high-affinity monoclonal antibodies can be generated in rabbits to these biomarkers. Subsequently, we hypothesize that through the use of these mAbs, a sensitive and specific immunoassay can be constructed to detect these biomarkers in patients. The first specific aim is to identify novel M. tb. biomarkers shed into serum and urine during infection. This will be accomplished by immunizing na¿ve rabbits with serum and urine from infected rabbits, a technique known as InMAD. Unlike rabbits immunized with crude antigens, such as M. tb. whole cell lysate or culture filtrate, rabbits immunized using this approach will create antibodies to only clinically relevant biomarkers that are shed into the serum or urine. The key to this aim is that rabbits will be infected with a highly virulent strain of M. tb. (W-Beiing strain 5097 sublineage RD207) that has high transmission rates and represents endemic disease. The polyclonal antibodies will be used in proteomic analysis of serum and urine samples of infected rabbits in the second aim, which is to validate the identified biomarkers. The third specific aim is to produce rabbit monoclonal antibodies to the validated M. tb. biomarkers. While several M. tb. immunoreactive proteins have been previously described, the approach that we will use in all three aims will facilitate the identification of new biomarkers. Using the rabbit for both the disease model and immunization will provide relevant disease state serum and urine as well as antibodies that will have higher affinity and greater epitope recognition than their mouse counter-parts. If the goals of this Phase I are achieved, Phase II will use mAbs from Phase I to construct and evaluate an immunoassay in POC format. Our preferred assay platform would be the lateral flow immunochromatographic (dipstick) assay, as it will meet all of the World Health Organization's A.S.S.U.R.E.D. criteria for developing world diagnostic assays. If successful, this translational research project could dramatically decrease mortality from tuberculosis through accurate diagnosis, allowing for appropriate treatment. Importantly, this can be done at the low cost needed in resource-limited countries.

描述(申请人提供)：结核病(TB)是一种慢性传染病，感染了大约三分之一的世界人口。在2008年估计的940万新结核病病例中，85%发生在位于亚洲和非洲的资源有限的国家。2010年，艾滋病毒阴性患者中有110万人死于结核病，艾滋病毒阳性患者中死于结核病的人数增加了35万人。在一些结核病流行地区，由于缺乏准确和易于使用的诊断方法，只有不到40%的结核病病例被诊断出来。目前，诊断依赖于通过连续痰涂片显微镜和培养在临床标本中证实结核分枝杆菌。这些方法缺乏敏感性，耗时长，费用昂贵，需要训练有素的人员。另一种方法是开发一种有效的免疫分析方法来检测结核分枝杆菌。体液中的抗原，如血清或尿液。目前用于检测结核分枝杆菌的商业免疫分析试剂盒。尿液中的葡聚糖脂阿拉伯甘露聚糖(LAM)的敏感性较差，可能是由于其丰度低所致。需要确定替代生物标志物，以促进结核病的准确诊断和治疗。我们的总体假设是结核分枝杆菌。在感染期间，生物标记物被排入尿液和/或血清中，并可在兔体内产生针对这些生物标记物的高亲和力单抗。随后，我们假设通过使用这些单抗，可以构建一种灵敏和特异的免疫分析方法来检测患者的这些生物标记物。第一个具体目标是确定新的结核分枝杆菌。在感染过程中，生物标志物会散落到血清和尿液中。这将通过用感染兔的血清和尿液免疫初生兔来完成，这项技术被称为InMAD。与用粗制抗原免疫的兔子不同，如结核分枝杆菌。无论是全细胞裂解液还是培养滤液，使用这种方法免疫的兔子将只产生针对临床相关生物标志物的抗体，这些抗体被释放到血清或尿液中。实现这一目标的关键是让兔子感染一种高毒力的结核分枝杆菌。(W-北京株5097亚种RD207)，传播率高，代表地方病。第二个目的是将多克隆抗体用于感染兔的血清和尿液样本的蛋白质组学分析，以验证所识别的生物标志物。第三个具体目标是生产针对已确认的结核分枝杆菌的兔单克隆抗体。生物标志物。而几个结核分枝杆菌。免疫活性蛋白以前已经描述过，我们将在所有三个目标中使用的方法将有助于识别新的生物标记物。将兔用于疾病模型和免疫，将提供相关的疾病状态血清和尿液，以及将具有更高亲和力和更大表位识别能力的抗体 它们的鼠标对应物。如果第一阶段的目标实现，第二阶段将使用第一阶段的单抗构建和评估POC格式的免疫分析。我们首选的检测平台将是横向流动免疫层析(试纸)检测，因为它将满足世界卫生组织关于发展中国家诊断检测的所有ASS.U.R.E.D.标准。如果成功，这个转化性研究项目可以通过准确的诊断大幅降低结核病的死亡率，从而实现适当的治疗。重要的是，这可以在资源有限的国家以所需的低成本完成。