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

本课题的目的是鉴定慢性炎症性脱髓鞘多神经病(CIDP)患者血清中的自身抗原。这种疾病的患者的症状是逐渐加重的四肢无力，肌肉运动缺乏自主协调(共济失调)，肌腱反射缺失或减弱，以及刺痛感或灼热感(感觉异常)。识别这种疾病的自身抗原将提高其诊断、治疗和了解。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有限的靶向方法，仅在少数患者中找到他们感兴趣的蛋白质，要么在准备过程中丢失了有趣但难以处理的蛋白质。为了克服这些问题，我将针对新的抗原应用两种已获批准的非靶向方法。此外，我还进行了初步的实验，以使样品的制备和电泳适应项目的需要。结果，我显著提高了难处理的高分子量蛋白质的可及性和分辨率，因为它们可以在感兴趣的靶区之一，即Ranvier的结点上找到。因此，我相信我的研究代表了创新和原创性的进步，可以确定失踪的候选人(S)。我接触到了当地的生物库，包括数百份珍贵的血清样本，这完成了我的方法的可行性。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)神经组织蛋白质的一维+二维蛋白质电泳，然后进行血清孵育。这两种方法都将应用高灵敏的质谱仪来识别结合的抗原。通过应用适当的统计数据，我将分离出出现在患者组中但不出现在对照组中的候选蛋白质(包括健康对照组和其他神经疾病)。通过酶联免疫吸附分析(ELISA)、基于细胞的分析和免疫印迹，我将验证感兴趣的候选对象，并评估它们作为生物标记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病理生理学中的作用，并将提供新的生物标记物，改善自身免疫性疾病的诊断和治疗。此外，我将把我的技能大大扩展到一个新的科学领域，即医学研究，并学习应用于任何与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受益于我未来获得的专业知识。

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.