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A kit for detecting anti-peroxide reductase-1-igg antibody

A peroxiredoxin-reductase technology, applied in the field of biomedicine, can solve the problems of the expression of peroxiredoxin-1 and the existence of peroxiredoxin-1, which has not been reported, and achieve the effects of increasing the surface area of ​​the coating, reducing the probability of cross-infection, and reducing pollution.

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

[0005] However, the expression of peroxiredoxin-1 and the presence of autoantibodies to peroxiredoxin-1 have not been reported in nephrotic syndrome
In addition, in the prior art, there is no application based on the target peroxiredoxin-1 or its autoantibody as a serological marker in autoimmune nephrotic syndrome
Identification of autoimmune nephrotic syndrome by detection of serum anti-peroxiredoxin-1-IgG antibodies is blank

Method used

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  • A kit for detecting anti-peroxide reductase-1-igg antibody
  • A kit for detecting anti-peroxide reductase-1-igg antibody
  • A kit for detecting anti-peroxide reductase-1-igg antibody

Examples

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Embodiment 1

[0035] Example 1 The peroxiredoxin-1 protein on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0036] Through a large number of clinical and molecular mechanism studies in the early stage, the present invention finds for the first time that the serum IgG level of patients with nephrotic syndrome is higher, and confirms that peroxiredoxin-1 on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. The specific implementation is as follows The specific implementation is as follows (1) Extraction of the total protein of glomerular podocytes: culture podocyte strain (MPC5), wash 2-3 times with PBS, then use a focused ultrasonic instrument (Covaris S220, Gene) in a medium containing 30mm Tris -HCl, 8m urea, 4% CHAPS and protease inhibitors (#ab65621; Abcam, 1:200 dilution) in the lysis buffer for sufficient lysis on ice, then put the sample in a centrifuge, 12000g, 4 ℃, centrifuge for 30...

Embodiment 2

[0037] Example 2 Expression and purification of recombinant antigenic protein peroxiredoxin-1

[0038] The method of genetic engineering is used to use the gene encoding peroxiredoxin-1 protein as a template to carry out PCR amplification, and then construct an expression vector for protein expression. The antigenic protein expressed in the present invention contains the tag peptide of the His tag. 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 peroxiredoxin-1 was identified as 27KDa by SDS-PAGE, see figure 2 .

Embodiment 3

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

[0040] According to the antigen peroxiredoxin-1 coating concentration (50μg, 80μg, 100μg, 150μg four coating concentrations), each reaction time (15min, 30min, 45min) and temperature (25°C, 37°C), the optimal dilution of the enzyme-labeled secondary antibody Degree (1:100, 1:500, 1:1000, 1:1500 four dilutions) and other 4 factors selection orthogonal table, each factor is repeated at 2 levels for standard positive serum and standard negative serum. Select the ratio (P / N) of the highest optical signal value (P) of positive serum to the lowest optical signal value (N) of negative serum, select the highest optical signal value (P) of positive serum and the lowest optical signal value of negative serum ( N) ratio (P / N). Through the orthogonal design, we obtained the optimum antigen-peroxiredoxin-1 coating concentration of this kit is 80μg / ml, the optimum antige...

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Abstract

The invention provides a test kit for detecting anti-peroxiredoxin-1-IgG antibody, which consists of antigenic protein peroxiredoxin-1 (peroxiredoxin-1), solid phase carrier, labeled antibody, antigen diluent, sample dilution Buffer, antibody diluent, substrate chromogen, washing solution, standard, positive quality control, negative quality control. The kit of the invention utilizes the indirect method reaction principle combined with magnetic particle chemiluminescence immunoassay to detect the anti-peroxide reductase-1-IgG antibody in the serum to be tested. The present invention identifies for the first time the autoantibodies against the target antigen peroxide reductase-1 in the serum of patients with autoimmune nephrotic syndrome, and the kit provided is domestic and foreign antibodies against peroxide reductase-1 and peroxide reductase ‑1‑IgG autoantibody-related autoimmune nephrotic syndrome molecular mechanism research and provide basis for clinical diagnosis and treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a kit for detecting anti-peroxide reductase-1-IgG antibody. Background technique [0002] In recent years, there are more and more types of kidney diseases in children, among which autoimmune nephrotic syndrome has the highest incidence rate, which seriously endangers children's physical and mental health. Autoimmune nephrotic syndrome is a type of autoimmune nephrotic syndrome, because the permeability of the glomerular filtration membrane increases, resulting in increased plasma protein filtration, causing a large amount of proteinuria, and thus causing patients to mainly manifest as massive proteinuria, hypoproteinemia, A clinical syndrome characterized by high levels of edema. Ali et al. observed that after transplanting the kidneys from patients with refractory minimal change nephrotic syndrome, the recipients had normal kidney function without any proteinuria, which shows...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/573G01N33/543
CPCG01N33/6854G01N33/6893G01N33/58G01N33/573G01N33/54326G01N2333/908G01N2800/347C12N9/0065C12Y111/01015
Inventor 叶青毛建华田丹丹
Owner ZHEJIANG UNIV
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