Identification of The Membranous Nephropathy Antigen

膜性肾病抗原的鉴定

• 批准号: 6865436
• 负责人: DAVID J SALANT
• 金额: $ 23.35万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865436
• 关键词: antigen antibody reaction; antigens; autoantibody; cell membrane; clinical research; complementary DNA; disease /disorder model; gel electrophoresis; human subject; human tissue; immune adherence reaction; immunologic substance development /preparation; immunoprecipitation; laboratory rabbit; laboratory rat; mass spectrometry; membranous glomerulonephritis; molecular biology information system; podocyte; protein purification; protein quantitation /detection; protein sequence; proteomics; serum; western blottings

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.

描述（由申请人提供）：