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A detection kit for detecting anti-aconitic acid hydratase-igg antibody

A technology of aconitate hydration and kit, which is applied in the field of biomedicine, can solve the problem that the relationship between aconitatehydratase and nephrotic syndrome has not been reported, and achieves the effects of increasing the surface area of ​​the coating, reducing the pollution, and increasing the adsorption amount of the antigen.

Active Publication Date: 2022-04-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the relationship between aconitate hydratase and nephrotic syndrome
In addition, in the prior art, research on identifying autoimmune nephrotic syndrome by detecting serum anti-aconitate hydratase-IgG antibodies is currently blank

Method used

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  • A detection kit for detecting anti-aconitic acid hydratase-igg antibody
  • A detection kit for detecting anti-aconitic acid hydratase-igg antibody
  • A detection kit for detecting anti-aconitic acid hydratase-igg antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 aconitate hydratase on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0036] (1) Extraction of total protein from glomerular podocytes: culture podocyte line (MPC5), wash 2-3 times with PBS, and then use a focused ultrasound instrument (Covaris S220, Gene) in a solution containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitors (#ab65621; Abcam, 1:200 dilution) were fully lysed on ice in lysis buffer, and then the samples were centrifuged at 12000g, 4°C for 30min. The supernatant was collected, which was the total glomerular podocyte protein collected. The total protein concentration of the collected glomerular podocytes was determined using the BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: The total protein of glomerular podocytes was extracted for two-dimensional electrophoresis, and then transferred to nitrocellulose membrane. The serum of patients with autoimmune nep...

Embodiment 2

[0037] Embodiment 2 Expression and purification of recombinant aconitate hydratase antigen protein

[0038] The gene encoding aconitate hydratase protein is used as a template to carry out PCR amplification by the method of genetic engineering, and then an expression vector is constructed to express the protein. The antigenic protein expressed in the present invention contains a His-tagged tag peptide. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc. Finally, the molecular weight of the recombinant protein aconitate hydratase was identified by SDS-PAGE as 37KDa. The results are shown in figure 2 .

Embodiment 3

[0039] Embodiment 3 The present invention adopts orthogonal experimental design to optimize the reaction conditions of the kit

[0040] According to the coating concentration of antigen aconitate hydratase (50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL four coating concentrations), each reaction time (30min, 45min) and temperature (25℃, 35℃), enzyme label The optimal dilution of secondary antibody (four dilutions of 1:100, 1:500, 1:1000, 1:1500) and other 4 factors select an orthogonal table, and each factor repeats the determination of standard positive serum and standard negative at 2 levels serum. Select the ratio (P / N) of the highest luminescence value (P) of positive serum and the lowest luminescence value (N) of negative serum. The average P / N value of the repeated determination, the optimal coating conditions and the optimal dilution of the secondary antibody were determined by statistical processing, and the orthogonal optimization conditions were carried out, which significa...

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Abstract

The invention provides a detection kit for detecting anti-aconitate hydratase-IgG antibody, comprising aconitate hydratase (aconitate hydratase) antigen protein, labeled antibody liquid, solid phase carrier coated with aconitate hydratase antigen, Sample diluent, antibody and antigen diluent, substrate chromogenic solution, washing solution, stop solution, standard, positive and negative quality control substances. The present invention contacts the target antigen aconitate hydratase polypeptide or its binding fragments with the tested serum sample to generate an immune reaction to form an antigen-antibody complex, and performs anti-aconitate hydratase-IgG autoantibody detection on the formed antigen-antibody complex. detection. The present invention identifies the anti-aconitate hydratase-IgG antibody for the first time, and provides a better detection method for the identification of autoimmune nephrotic syndrome related to the anti-aconitate hydratase-IgG antibody at home and abroad.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a detection kit for detecting anti-aconitic acid hydratase-IgG antibody. Background technique [0002] Primary nephrotic syndrome (PNS) is caused by a variety of factors that increase the permeability of the glomerular basement membrane, and then increase the filtration of plasma protein and lose it in the urine, which leads to a series of pathological changes. clinical syndrome. PNS is a common glomerular disease in children, with an annual incidence of 1.15-16.9 / 100000 according to reports. The pathological type is minimal change nephrotic syndrome (MCNS), which is the most common. According to the response effect of PNS to hormone therapy, it can be divided into steroid-sensitive type (SSNS), steroid-dependent type (SDNS), and steroid-dependent type (SDNS). Drug-resistant type (SRNS). Although the vast majority of children are sensitive to hormone therapy and have a good ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/573G01N33/543
CPCG01N33/6854G01N33/6893G01N33/58G01N33/573G01N33/54326G01N2333/988G01N2800/347
Inventor 叶青毛建华张俊峰
Owner ZHEJIANG UNIV
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