Identification of The Membranous Nephropathy Antigen

描述(由申请人提供)： 在这个项目的R21阶段，我们建议使用我们以前成功鉴定的肾性足细胞抗原的蛋白质组学技术来分离和鉴定膜性肾病(MN)的靶抗原/S。来自经活检证实的MN患者的血清将被筛选出与肾小球抗原/S的反应性，阳性样本将与正常人肾小球的提取物一起使用，采用蛋白质组学方法，包括免疫沉淀、凝胶电泳和质谱仪，以分离和测序免疫反应性多肽。肽序列将被用于搜索蛋白质数据库以识别已知蛋白质，搜索EST数据库以寻找匹配的cDNA克隆，或设计寡核苷酸以筛选cDNA表达文库。当一个假定的抗原被识别后，将产生一种单特异性抗体，用于确定该抗原是否适当地位于足细胞质膜上，并进行测试，以确定它是否能够在实验动物体内或在体外灌流的人肾中产生膜免疫沉积。将获得或生产全长的cDNA，并用于产生细菌或哺乳动物的融合蛋白，用于抗体生产和未来在R33阶段使用。在确定了与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，我们将进入R33阶段。重组抗原将用于建立一种诊断酶联免疫分析法(ELISA)，用于MN的血清学检测，并与其他常见的肾病综合征原因，即局灶性和节段性肾小球硬化(FSGS)和糖尿病肾病(DN)进行比较。正常志愿者的血清将被用来确定ELISA的正常限，用重组抗原免疫印迹阳性的MN血清将被用来建立该方法的检测下限。一旦建立了ELISA，将对新诊断的、经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FSGS和糖尿病肾病患者以及正常志愿者的血清进行比较，以评估该检测方法的诊断实用价值。最后，将使用标准技术检查MN自身抗体的肾炎特性，以评估它们的类别和亚类特征，以及使用表达MN抗原的细胞来评估它们的细胞病变效应。

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.