Identification, validation, and characterization of novel autoantigens of chronic inflammatory demyelinating polyneuropathy (CIDP)

我的项目目的是鉴定患有慢性炎症性脱髓鞘性多神经病变（CIDP）的致残性神经自身免疫性疾病患者血清中的自身抗原。此病患者表现为逐渐加重的肢体无力，肌肉运动缺乏自主协调（共济失调），肌腱反射缺失或减弱，刺痛或灼烧感（感觉异常）。识别这种疾病的自身抗原将改善其诊断、治疗和理解。最近的研究未能为大多数患者确定抗原，因为研究人员要么使用了一种有限的靶向方法，仅在罕见的患者亚组中找到感兴趣的蛋白质，要么在制备过程中丢失了感兴趣但难以处理的蛋白质。为了克服这些问题，我将采用两种经批准的针对新抗原的非靶向方法。此外，我进行了初步实验，使样品制备和电泳适应项目需要。结果，我显著提高了难处理的高分子量蛋白的可及性和分辨率，因为它们可以在一个有趣的靶区，即朗维耶节点上找到。因此，我相信我的研究代表了创新和原创的进步，可以识别缺失的候选人。我访问了当地的生物库，包括数百份珍贵的血清样本，这证明了我的方法是可行的。第一步——鉴定——将包括(1)使用人血清抗体进行免疫沉淀，以捕获大鼠和奶牛神经组织的相应抗原；(2)神经组织蛋白的1D+2D蛋白电泳，然后进行血清孵育。这两种方法都将采用高灵敏度的质谱法来识别结合抗原。通过应用适当的统计数据，我将分离出在患者组中出现，但在对照组（健康对照组和其他神经系统疾病组）中没有出现的候选蛋白质。通过酶联免疫吸附试验（ELISA），基于细胞的试验和免疫印迹，我将验证有趣的候选物，并评估其作为生物标志物的敏感性和特异性。我的项目将有助于了解自身抗体在CIDP病理生理中的作用，并将提供新的生物标志物，以改善自身免疫性疾病的诊断和治疗。此外，我将显著扩展我的技能到一个新的科学领域，即医学研究，并学习应用于任何抗体相关的自身免疫性疾病的重要方法。因此，德国的医学研究也将在未来受益于我所获得的专业知识。

The aim of my project is to identify autoantigens in the blood serum of patients with the disabling neuronal autoimmune disease named chronic inflammatory demyelinating polyneuropathy (CIDP). Patients with this disease suffer from progressively increasing limb weakness, lack of voluntary coordination of muscle movements (ataxia), absent or diminished tendon reflexes, and sensation of prickling or burning (paresthesia). Identifying the autoantigens of this disease will improve its diagnosis, treatment, and understanding. Recent studies failed to identify an antigen for the majority of patients, since the researchers either used a restricted targeted approach finding their protein-of-interest only in a rare subgroup of patients, or lost interesting but difficult-to-handle proteins during their preparation. To overcome these issues, I will apply two approved untargeted approaches aiming at novel antigens. Further, I performed preliminary experiments to adapt the sample preparation and electrophoresis to the project needs. As a result, I significantly improved the accessibility and resolution of intractable high-molecular-weight proteins, as they can be found at one of the interesting target regions, namely the node of Ranvier. Thus, I am convinced that my study represents innovative and original advancements allowing to identify the missing candidate(s). My access to the local biobank including hundreds of precious serum samples completes the feasibility of my approach. The first step -- the identification -- will include (1) immunoprecipitation using human serum antibodies to capture corresponding antigens of nerve tissue of rats and cows and (2) 1D+2D protein electrophoresis of nerve tissue proteins, followed by serum incubation. Both approaches will apply highly sensitive mass spectrometry to identify the bound antigens. By applying appropriate statistics, I will isolate protein candidates that appeared in the patient group, but not in the control group (both healthy controls and other neurological diseases). By enzyme-linked immunosorbent assays (ELISA), cell-based assays, and immunoblots, I will validate interesting candidates and assess their sensitivity and specificity as a biomarker. My project will help to understand the role of autoantibodies in CIDP's pathophysiology and will provide new biomarkers improving the diagnosis and treatment of autoimmune disorders. Further, I will significantly extend my skills to a new scientific field, i.e., medical research, and learn important methods being applied for any antibody-related autoimmune disease. Thus, also the medical research in Germany will benefit from my obtained expertise in the future.