A kit for detecting anti-vinculin-igg antibody

A vinculin and kit technology, applied in the field of biomedicine, can solve the problems that the existence of Vinculin has not been reported, and achieve the effects of improving detection accuracy, simple operation, and less reagent consumption

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 Vinculin and the presence of Vinculin autoantibodies have not been reported in nephrotic syndrome
In addition, in the prior art, there is no application based on the target Vinculin or its autoantibody as a serological marker in autoimmune nephrotic syndrome
The identification of autoimmune nephrotic syndrome by detection of serum anti-Vinculin-IgG antibodies is blank

Method used

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  • A kit for detecting anti-vinculin-igg antibody
  • A kit for detecting anti-vinculin-igg antibody
  • A kit for detecting anti-vinculin-igg antibody

Examples

Experimental program
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Effect test

Embodiment 1

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

[0036] Through a large number of previous clinical and molecular mechanism studies, the present invention found for the first time that the serum IgG level of patients with nephrotic syndrome is higher, and confirmed that Vinculin on podocytes is the main target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative level of anti-Vinculin-IgG antibodies in serum is helpful for the early identification of autoimmune nephrotic syndrome, especially for screening patients with related symptoms. The specific implementation is as follows (1) extraction of 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 prot...

Embodiment 2

[0037] Embodiment 2 expression and purification of recombinant antigenic protein Vinculin

[0038] The method of genetic engineering is used to carry out PCR amplification with the gene encoding Vinculin protein as a template, and then an expression vector is constructed 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 Vinculin was identified as 37KDa 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 Vinculin 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 ( 1:100, 1:500, 1:1000, 1:1500 (four dilutions) and other four factors, select the orthogonal table, and each factor is repeatedly measured with two levels of standard positive serum and standard negative serum, and the highest positive serum is selected. The ratio (P / N) of the light signal value (P) to the lowest light signal value (N) of the negative serum. Through the orthogonal design, we obtained the optimal antigen Vinculin coating concentration of this kit is 80 μg / ml, the optimal antigen-antibody reaction temperature of the solid-phase membrane immunoassay anti-Vinculin-IgG antibody kit is 25°C, and the...

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Abstract

The invention provides a test kit for detecting anti-vinculin-IgG antibodies, comprising antigenic protein Vinculin (vinculin), a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, and a substrate display It is composed of colorant, washing solution, standard, positive quality control and negative quality control. The kit of the present invention utilizes the indirect method reaction principle combined with magnetic particle chemiluminescence immunoassay to detect the anti-Vinculin-IgG antibody in the sample to be tested. The invention firstly identifies the autoantibodies against the target antigen Vinculin in the serum of patients with autoimmune nephrotic syndrome, and provides a detection kit for the autoantibodies. The kit provided by the invention provides a basis for domestic and foreign molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to Vinculin and Vinculin-IgG autoantibodies.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a kit for detecting anti-vinculin-IgG antibodies. 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 that the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/564G01N33/543G01N33/58
CPCG01N33/564G01N33/54306G01N33/54326G01N33/581G01N33/58G01N33/582G01N2800/347G01N2333/96425G01N33/6854G01N33/6893G01N33/544G01N2333/4701
Inventor 叶青毛建华田丹丹
Owner ZHEJIANG UNIV
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