Identification of The Membranous Nephropathy Antigen

描述（由申请人提供）： 在该项目的R21阶段，我们建议使用蛋白质组学技术，我们已经成功地识别致肾炎足细胞抗原分离和表征膜性肾病（MN）的靶抗原。将对活检证实的MN患者的血清进行肾小球抗原反应性筛选，并将阳性样本与正常人肾小球提取物一起用于蛋白质组学方法，包括免疫沉淀、凝胶电泳和质谱法，以分离和测序免疫反应性肽。肽序列将用于搜索蛋白质数据库以鉴定已知蛋白质，搜索EST数据库以匹配cDNA克隆，或设计寡核苷酸以筛选cDNA表达文库。当鉴定出推定的抗原时，将产生单特异性抗体，并用于确定抗原是否适当地位于足细胞质膜上，并进行测试以确定其是否能够在实验动物体内或在离体灌注的人肾脏中产生膜性免疫沉积物。将获得或产生全长cDNA，并用于产生细菌或哺乳动物融合蛋白，用于抗体生产和将来用于R33期。在鉴定了与MN患者血清反应的靶肾小球抗原并确定其具有致病相关性后，我们将开始R33阶段。重组抗原将被用来开发一种诊断酶联免疫测定（ELISA）的血清学检测MN相比，其他常见的原因肾病综合征，即局灶性和节段性肾小球硬化症（FSGS）和糖尿病肾病（DN）。正常志愿者的血清将用于确定ELISA的正常限值，重组抗原蛋白质印迹法阳性的MN血清将用于确定试验的检测下限。一旦开发了ELISA，将使用来自新诊断的、活检证实的特发性和狼疮MN患者的血清进行前瞻性病例对照研究，并与来自FSGS和DN患者以及正常志愿者的血清进行比较，以评估该测定的诊断效用。最后，将使用标准技术检查MN自身抗体的致肾炎特性，以评估其类别和亚类特征，以及使用表达MN抗原的细胞的细胞病变效应。

DESCRIPTION (provided by applicant): In the R21 phase of this project we propose to use proteomic techniques with which we have had previous success in identifying nephritogenic podocyte antigens to isolate and characterize the target antigen/s of membranous nephropathy (MN). Sera from patients with biopsy-proven MN will be screened for reactivity with glomerular antigen/s and positive samples will be used with extracts of normal human glomeruli in a proteomic approach involving immunoprecipitation, gel electrophoresis, and mass spectrometry to isolate and sequence immunoreactive peptides. Peptide sequences will be used to search protein databases to identify known proteins, search EST databases for matching cDNA clones, or to design oligonucleotides to screen cDNA expression libraries. When a putative antigen is identified, a monospecific antibody will be generated and used to determine if the antigen is appropriately located on the podocyte plasma membrane and tested to establish if it is able to produce membranous immune deposits in vivo in experimental animals or in ex vivo perfused human kidneys. A full-length cDNA will be acquired or produced and used to generate a bacterial or mammalian fusion protein for antibody production and for future use in the R33 phase. Having identified a target glomerular antigen that is reactive with sera from patients with MN and established that it has pathogenic relevance, we will embark on the R33 phase. The recombinant antigen will be used to develop a diagnostic enzyme-linked immunoassay (ELISA) for the serological detection of MN as compared with other common causes of nephrotic syndrome, namely focal and segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Serum from normal volunteers will be used to define the normal limits of the ELISA and MN sera that are positive on western blotting with the recombinant antigen will be used to establish the lower limits of detection of the assay. Once the ELISA has been developed, a prospective case-control study will be conducted with serum from patients with newly-diagnosed, biopsy-proven idiopathic and lupus MN and compared to serum from patients with FSGS and DN as well as that of normal volunteers to assess the diagnostic utility of the assay. Finally, the nephritogenic properties of MN autoantibodies will be examined using standard techniques to assess their class and subclass characteristics, as well as their cytopathic effects using cells expressing the MN antigen.