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Kit for detecting anti-peroxide reductase 1-IgG antibody

一种过氧化物、抗原抗体的技术，应用在生物医学领域，能够解决peroxiredoxin-1表达情况peroxiredoxin-1存在情况未见报道等问题，达到增加包被表面积、减少污染、增加吸附量的效果

Pending Publication Date: 2022-08-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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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  • Kit for detecting anti-peroxide reductase 1-IgG antibody
  • Kit for detecting anti-peroxide reductase 1-IgG antibody
  • Kit for detecting anti-peroxide reductase 1-IgG antibody

Examples

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Embodiment 1

[0036] Example 1 The peroxiredoxin-1 protein on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0037] The present invention finds for the first time that the serum IgG level of patients with nephrotic syndrome is relatively high through a large number of clinical and molecular mechanism studies in the early stage, 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: (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, then centrifuged the samples at 12000g, 4°C for 30min . The supernatant was collected, which was the tot...

Embodiment 2

[0038] Example 2 Expression and purification of recombinant antigen protein peroxiredoxin-1

[0039] The gene encoding peroxiredoxin-1 protein was used as a template to carry out PCR amplification by using the method of genetic engineering, and then an expression vector was 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 peroxiredoxin-1 was identified by SDS-PAGE as 27KDa, see figure 2 .

Embodiment 3

[0040] Embodiment 3 The present invention adopts orthogonal experimental design to optimize the reaction conditions of the kit

[0041] According to the antigen peroxiredoxin-1 coating concentration (50μg, 80μg, 100μg, 150μg four coating concentrations), each reaction time (15min, 30min, 45min) and temperature (25℃, 37℃), the optimal dilution of enzyme-labeled secondary antibody Four factors including the degree of dilution (1:100, 1:500, 1:1000, 1:1500) and other four factors were selected in the orthogonal table, and the standard positive serum and standard negative serum were repeatedly determined at 2 levels for each factor. Select the ratio (P / N) of the highest optical signal value (P) of the positive serum and the lowest optical signal value (N) of the negative serum, and select the highest optical signal value (P) of the positive serum and the lowest optical signal value of the negative serum ( N) ratio (P / N). Through orthogonal design, we obtained the best antigen-per...

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Abstract

The invention provides a kit for detecting an anti-peroxiredoxin-1 (peroxiredoxin-1) antibody. The kit consists of antigen protein peroxiredoxin-1 (peroxiredoxin-1), a solid-phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer solution, an antibody diluent, a substrate color developing agent, a washing solution, a standard substance, a positive quality control substance and a negative quality control substance. According to the kit disclosed by the invention, the anti-peroxide reductase 1-IgG antibody in serum to be detected is detected by utilizing an indirect method reaction principle in combination with magnetic particle chemiluminescence immunoassay. According to the invention, the autoantibody aiming at the target antigen peroxide reductase 1 in the serum of the patient with the autoimmune nephrotic syndrome is identified for the first time; the kit provided by the invention provides a basis for molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndromes related to peroxidereductase-1 and peroxidereductase-1-IgG autoantibodies at home and abroad.

Description

[0001] This application is a divisional application with an application number of 202110743523.5, an application date of July 1, 2021, and the title of the invention is a kit for detecting anti-peroxidoreductase-1-IgG antibodies. technical field [0002] The invention belongs to the technical field of biomedicine, and relates to a kit for detecting anti-peroxidase reductase-1-IgG antibody. Background technique [0003] In recent years, there are more and more types of kidney diseases in children. Among them, autoimmune nephrotic syndrome has the highest incidence, which seriously endangers children's physical and mental health. Autoimmune nephrotic syndrome is a type of autoimmune nephrotic syndrome due to the increase in the permeability of the glomerular filtration membrane, which increases the filtration of plasma proteins and causes a large amount of proteinuria. Clinical syndrome characterized by high edema. Ali and other scholars observed that after transplanting kidn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/573G01N33/543C12N9/08
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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