Membranous Nephritis Antigen Defined by Phage Antibodies

描述（由申请人提供） 人类特发性膜性肾小球肾炎（MGN）被认为是肾脏的器官特异性自身免疫性疾病，其中疾病诱导的自身抗体针对位于足细胞表面上的自身抗原。然而，迄今为止，在患者血清中未检测到自身抗体，并且自身抗原的身份仍然未知。因此，这一重要的研究领域仍然处于停滞状态。当患者首次出现完全发作的肾病综合征时，自身抗体被肾脏迅速吸收，随后降低至低水平或不可检测水平的可能性得到了海曼肾炎（一种MGN大鼠模型）中类似观察结果的支持。我们建议通过一种新的方法来获得这些自身抗体存在的证据：筛选特发性MGN患者的噬菌体展示抗体库。噬菌体展示提供了几个优于血清的优点;（1）抗体库的表达不依赖于可能影响血清抗体表达的任何调节机制，（2）与组织切片中的稀有抗原结合的噬菌体颗粒可以与残留的正常IgG区分开，（3）可以回收并扩增与选择性底物结合的噬菌体抗体，和（4）可以容易地获得单克隆抗体片段并测序。噬菌体展示抗体将用于人肾切片染色。文库将富集特异性噬菌体抗体，并选择和表达单克隆抗体片段。选择的克隆将用于通过免疫沉淀、蛋白质印迹分析和N-末端微测序从肾匀浆中分离和鉴定自身抗原。高亲和力克隆的肽配体将从随机肽噬菌体文库中选择并测序以定义共有序列，其可以鉴定蛋白质数据库中的潜在自身抗原。人MGN自身抗原的鉴定将为阐明MGN的发病机制提供新的努力，并为进一步的临床和临床前研究提供试剂和数据。针对抗原或其片段的人单克隆抗体将可用于体外研究或开发更接近模拟人类疾病的被动模型。肾抗原的知识将有助于临床诊断试验的发展，并可能在新的方法来治疗或预防MGN

DESCRIPTION (provided by applicant) Human idiopathic membranous glomerulonephritis (MGN) is assumed to be an organ-specific autoimmune disease of the kidney in which disease-inducing autoantibodies are directed against an autoantigen located on the surface of podocytes. However, to date no autoantibodies have been detected in patients' sera and the identity of the autoantigen remains unknown. Thus this important area of research remains at a standstill. The possibility that autoantibodies are quickly absorbed by the kidney and subsequently abate to low or undetectable levels when patients first present with full-blown nephrotic syndrome is supported by similar observation in Heymann nephritis, a rat model of MGN. We propose to obtain evidence for the existence of these autoantibodies by a novel approach: screening of phage display antibody libraries of patients with idiopathic MGN. Phage display provides several advantages over sera; (1) expression of the antibody repertoire is independent of any regulatory mechanisms that may affect expression of serum antibodies, (2) phage particles binding to rare antigens in tissue sections can be distinguished from residual normal IgG, (3) phage antibodies that bind to the selective substrate can be recovered and amplified and (4) monoclonal antibody fragments can be readily obtained and sequenced. Phage-displayed antibodies will be used for staining of human kidney sections. Libraries will be enriched for specific phage antibodies and monoclonal antibody fragments will be selected and expressed. The selected clones will be used to isolate and identify the autoantigen from renal homogenates by immunoprecipitation, western blot analysis and N-terminal microsequencing. Peptide ligands for the high affinity clones will be selected from random peptide phage libraries and sequenced to define a consensus sequence, which could identify potential autoantigens in the protein database. Identification of the autoantigen of human MGN will lead to renewed efforts to delineate the mechanism of pathogenesis and provide reagents and data to support further clinical and preclinical studies. Human monoclonal antibodies against the antigen or its fragments will be useful for in vitro studies or development of a passive model that more closely mimics the human disease. Knowledge of the renal antigen will assist in the development of a clinical diagnostic test and potentially in new approaches to therapy or prevention of MGN