Iga nephropathy detection method and detection kit

An inspection method and kit technology, which can be used in disease diagnosis, biological testing, material inspection products, etc., can solve problems such as no practical methods, and achieve good detection sensitivity and specificity.

Inactive Publication Date: 2011-05-04
EISIA R&D MANAGEMENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In addition, as a diagnostic method for IgA nephropathy, a method using an IgA-fibronectin complex is known (for example, refer to Japanese Patent Application Laid-Open No. 2000-241431, Japanese Patent No. 2592121, Cederholm et al., Proc. Natl .Acad.Sci., 85:4865-8, 1988), but so far there is no practical method

Method used

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  • Iga nephropathy detection method and detection kit
  • Iga nephropathy detection method and detection kit
  • Iga nephropathy detection method and detection kit

Examples

Experimental program
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Embodiment 1

[0090]

[0091] Normal human mesangial cells (Cryo NHMC: Cambrex) were cultured, and BALB / c mice (female) were immunized with the cells or fractions extracted from cytoskeletal components as immunogens. After confirming that the antibody titer against the immunogen has risen, the splenocytes were fused with P3U-1 cells to prepare hybridomas. The first screening of hybridomas was carried out by the EIA method of immobilizing two kinds of immune antigens (normal human glomerular mesangial cells or their cytoskeleton component extraction fractions) in the cup of the microplate, and selecting for both or either Hybridomas whose antigens showed high chromogenic values. In addition, the cytoskeleton component extraction fraction was prepared using ProteoExtract Subcellular ProteomeExtraction Kit (manufactured by Calbiochem).

[0092] The selected hybridomas were monoclonalized by the limiting dilution method, and these were used for secondary screening to select hybridomas specif...

Embodiment 2

[0115]

[0116] ~Detection of IgA-4H3-recognized antigen complex by ELISA~

[0117] The 4H3-1D12-1D2 antibody (hereinafter referred to as "4H3") was purified with PROSEP-A (manufactured by MILLIPORE), dialyzed against 50 mM Tris / HCl (pH 7.5), 0.15 M NaCl, and the obtained purified antibody was washed with 50 mM After diluting Tris / HCl (pH 7.5) and 0.15M NaCl to a concentration of 5 μg / ml to 10 μg / ml, 50 μl / well was injected into a PolySorp cup (manufactured by NUNC). Place the cups in a humid box and cover overnight at 4°C. After coating, wash 3 times with washing solution (50mM Tris / HCl (pH7.5), 150mM NaCl, 0.01% Tween20), inject blocking solution (50% N102 (manufactured by NOF Corporation), 25mM Tris / HCl) at 150 μl / well (pH 7.5), 75mM NaCl, 2% Block Ace (manufactured by Dainippon Sumitomo Pharmaceutical Co., Ltd.), and then blocked at room temperature for 2 hours or at 4°C for more than 1 day (hereinafter referred to as "4H3 coated cup").

[0118] The 4H3 coated cup was ...

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Abstract

Disclosed is a renal disease detection method which involves a complex detection step of detecting a complex of an antigen derived from a human renal mesangial cell and human IgA in a sample derived from urine collected from a subject. It is preferred that the renal disease detection method further involves a determination step of determining the renal disease as IgA nephropathy based on the ratio of the quantity of the complex detected in the complex detection step to the quantity of a urinary protein in the sample. The renal disease detection method has good detection sensitivity and specificity, and can determine the occurrence of a renal disease (preferably, IgA nephropathy) conveniently and safely.

Description

technical field [0001] The present invention relates to an examination method and an examination kit for IgA nephropathy. Background technique [0002] IgA nephropathy was described by Berger et al. in 1968, and is the most frequent nephropathy among chronic glomerulonephritis other than diabetic nephropathy (for example, see Non-Patent Document 1). IgA nephropathy is a disease with poor long-term prognosis, and it is estimated that about 25% of patients with advanced renal failure requiring dialysis treatment are caused by IgA nephropathy. In the past, there was no curative therapy, even if there was a therapy to delay the progression of IgA nephropathy. However, in recent years, Horita et al. have established a therapy that can achieve complete remission if treatment is started early. [0003] On the other hand, according to "Joint Committee of the Specified Progressive Diseases Investigative Research Division for Progressive Renal Diseases of the Ministry of Health and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/6854G01N2800/347
Inventor 小原隆沟口贞明堀田修
Owner EISIA R&D MANAGEMENT CO LTD
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