Rabbit InMAD Discovery of Novel Biomarkers for POC Tuberculosis Diagnostic Assay

描述（由申请人提供）：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940万结核新病例中，85%发生在亚洲和非洲资源有限的国家。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又有35万人死于结核病。在一些结核病流行地区，由于缺乏准确和易于使用的诊断分析，只有不到40%的结核病病例得到诊断。目前，诊断依赖于通过连续痰涂片镜检和培养在临床标本中发现结核分枝杆菌。这些方法缺乏灵敏度，耗时，昂贵，并且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法来检测结核分枝杆菌。抗原体液中的抗原，如血清或尿液目前用于检测结核分枝杆菌的商业免疫测定试剂盒。糖脂阿拉伯甘露聚糖（LAM）在尿液中表现出较差的敏感性，可能是由于丰度低。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。生物标记物在感染期间进入尿液和/或血清中，并且在家兔体内可以产生针对这些生物标记物的高亲和力单克隆抗体。随后，我们假设通过使用这些单克隆抗体，可以构建一种敏感和特异性的免疫测定方法来检测患者体内的这些生物标志物。第一个具体目标是鉴定新型结核分枝杆菌。感染期间生物标志物进入血清和尿液。这将通过用感染兔子的血清和尿液免疫感染兔子来实现，这种技术被称为InMAD。不像用原始抗原免疫的兔子，比如结核分枝杆菌。全细胞裂解液或培养滤液，使用这种方法免疫的兔子将产生仅针对临床相关生物标志物的抗体，这些抗体脱落到血清或尿液中。实现这一目标的关键是，兔子将感染一种高毒力的结核分枝杆菌菌株。（w - beijing菌株5097 RD207亚系），传播率高，代表地方性疾病。该多克隆抗体将用于感染家兔血清和尿液样本的蛋白质组学分析，以验证鉴定的生物标志物。第三个具体目标是产生针对验证结核分枝杆菌的兔单克隆抗体。生物标志物。而一些结核分枝杆菌。免疫反应蛋白以前已经被描述过，我们将在所有三个目标中使用的方法将促进新的生物标志物的鉴定。将兔用于疾病模型和免疫，将提供相关的疾病状态，血清和尿液以及抗体，这些抗体将具有更高的亲和力和更大的表位识别

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.