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SIMPLIFIED MEASUREMENT OF ANTI-PHOSPHOLIPASE A2 RECEPTOR ANTIBODY

外国特許コード F160008708
整理番号 (S2014-1186-N0)
掲載日 2016年3月30日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2015JP069961
国際公開番号 WO 2016009971
国際出願日 平成27年7月10日(2015.7.10)
国際公開日 平成28年1月21日(2016.1.21)
優先権データ
  • 特願2014-144583 (2014.7.14) JP
発明の名称 (英語) SIMPLIFIED MEASUREMENT OF ANTI-PHOSPHOLIPASE A2 RECEPTOR ANTIBODY
発明の概要(英語) The present invention addresses the problem of providing a novel means for measuring anti-phospholipase A2 receptor antibodies. In the present invention, anti-phospholipase A2 receptor antibodies in a specimen are detected using, as an antigen, phospholipase A2 receptors in which antigenicity is maintained by treatment in a trehalose solution.
従来技術、競合技術の概要(英語) BACKGROUND ART
Phospholipase A2 receptor (Phospholipase A2 receptor: PLA2R) is, cells expressing human glomerular foot-I of the molecular weight of about 180kDa are transmembrane proteins. Anti-PLA2R antibodies in the blood, the main cause of idiopathic nephrotic syndrome is a disease of the membranous nephropathy is the only disease biomarker prediction distinguishing and are promising as a (non-patent document 1). Anti-PLA2R antibodies are found to idiopathic membranous nephropathy was established by the diagnosis is precluded. More specifically, to membranous nephropathy evoked known factors and secondary membranous nephropathy is unknown factors cause of idiopathic membranous nephropathy and, membranous nephropathy in kidney biopsies of renal pathology images were acquired of the patient factors cause secondary membranous nephropathy (for example, anti-rheumatic drug dose, autoimmune diseases, such as a tumor) if they have a secondary membranous nephropathy is diagnosed, does not have an idiopathic membranous nephropathy was diagnosed. Idiopathic membranous nephropathy anti-PLA2R antibody as a biomarker for discrimination can be utilized in the finding that, in the blood of the patient if the anti-PLA2R antibody (in some cases without a renal biopsy) idiopathic membranous nephropathy can be diagnosed as well, and secondary membranous nephropathy is idiopathic membranous nephropathy and very different from those of the treatment method of, the operation of the paper currency and nephrotic syndrome condition of the patient an anti-PLA2R antibody measurements, particularly an anti-PLA2R antibody (qualitative measurements) confirmation of presence or absence, is very important.
Generally, in measurement of the anti-PLA2R antibody, the protein PLA2R presented as an antigen, the analyte in the patient's serum as the primary antibody, anti-human IgG-chemically labeled as a secondary antibody by sequential reaction of the antibody, anti-PLA2R contained in the sample to detect the antibodies. PLA2R Anti-PLA2R antibody recognizes an epitope of the antigen for the assembly, and the antigen is PLA2R when the measurement of the protein (native form) of a naturally-occurring three-dimensional structure needs to be maintained. PLA2R Anti-PLA2R antibody is disrupted the three-dimensional structure may not be coupled to the PLA2R is found (Non-Patent Document 1), such as a crystal structure analysis is still no detailed study.
Anti-PLA2R antibodies have been developed so far as the measurement method using Western blotting, indirect fluorescent antibody method and the ELISA is established. ELISA for the indirect fluorescent antibody method, a dedicated kit (manufactured by Euroimmun) is commercially available.
Is a western blot, glomerular lysate or recombinant human PLA2R (recombinant) PLA2R after polyacrylamide gel electrophoresis, transferred to PVDF membrane was immobilized on the film surface is used as an antigen protein PLA2R (non-patent document 1). Western blotting in addition to a cost-effective, and capable of measuring the most sensitive, but it is also excellent in stability, the operation is complicated and, since the PVDF film can not be efficiently stored in the operation is difficult. In addition, the previously (Non-Patent Document 1) is a western blot, at the final step of the assay step of the signal acquisition step, HRP-labeled secondary antibody used for the chemical expensive imager and complicated in a darkroom (LAS4000 manufactured by GE and the like) if operation is needed.
Indirect fluorescent antibody method, the recombinant PLA2R expressed on the cell membrane was prepared as the recombinant mammalian cell (for example HEK293 cells) 96 - well microplates or chamber such as a slide glass with the adhesive after culture, for each cell on the cell surface can be a formalin-fixed solid phase recombinant PLA2R was presented (Non-Patent Document 2) is used as an antigen. Indirect fluorescent antibody method can be carried out at a low cost, as compared to in the western blot procedure since it may be convenient. On the other hand, the qualitative property as compared with the lateral Western blot is inferior, the measurement sensitivity is not so good. Thereon, the speculum is the final step of the assay step in the fluorescence microscope is required which is expensive.
In the ELISA, 96 - well ELISA plate was immobilized highly purified recombinant PLA2R was used as an antigen (Euroimmun website http://www.euroimmun.com/index.php?id=aak_gegen_pla2r see&L=1). ELISA is simple and easy quantification is excellent in terms of the features on the other hand, the need for high purity recombinant PLA2R is remarkably expensive. In addition, the measurement sensitivity and stability as compared with the western blot method is poor, the signal acquisition step leads to a plate reader is required.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY
  • 発明者(英語)
  • AKIYAMA Shin'ichi
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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