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.
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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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