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Application of reagent for detecting anti-cytoskeleton associated protein 4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

An autoantibody, vascular endothelium technology, applied in the field of biomedicine, can solve the problems of lack of clinical detection kits, lack of quantitative detection level, etc., and achieve the effect of improving accuracy

Pending Publication Date: 2022-07-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current domestic and foreign research on Cytoskeleton-associated protein 4 and anti-Cytoskeleton-associated protein 4-IgG antibodies in patients with kidney disease is limited to the research on the molecular mechanism, and there is no quantitative detection of their levels in the serum of patients. Lack of corresponding clinical testing kits

Method used

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  • Application of reagent for detecting anti-cytoskeleton associated protein 4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury
  • Application of reagent for detecting anti-cytoskeleton associated protein 4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury
  • Application of reagent for detecting anti-cytoskeleton associated protein 4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

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Experimental program
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Effect test

Embodiment 1

[0051] Cytoskeleton-associated protein 4 on vascular endothelial cells is the main target antigen for autoantibodies in patients with nephrotic syndrome:

[0052] The present invention finds for the first time that the serum IgG level of patients with nephrotic syndrome is higher through a large number of clinical and molecular mechanism studies in the early stage, and confirms that Cytoskeleton-associated protein 4 on vascular endothelial cells is the main target of autoantibodies in patients with autoimmune nephrotic syndrome. antigen. Therefore, detecting the presence and quantitative level of anti-Cytoskeleton-associated protein 4-IgG antibody in serum is helpful to clarify vascular endothelial cell injury.

[0053] The specific implementation is as follows:

[0054] (1) Extraction of total protein from vascular endothelial cells: culture vascular endothelial cell line (ECV 304), wash 2-3 times with PBS, and then use a focused ultrasound instrument (Covaris S220, Gene) in...

Embodiment 2

[0058] Expression and purification of recombinant antigen protein Cytoskeleton-associated protein 4

[0059]The gene encoding the Cytoskeleton-associated protein 4 protein was used as a template for PCR amplification by genetic engineering, and then an expression vector was constructed for protein expression. 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, and finally, the molecular weight of the recombinant protein Cytoskeleton-associated protein 4 was identified by SDS-PAGE as 40KDa, see figure 2 (SDS-PAGE identification of the expressed recombinant protein Talin-1), wherein, lane A: supernatant of cell lysate, induced at 15°C for 16 hours; lane B: supernatant of cell lysate, at 37 Induction for 4 hours at °C.

Embodiment 3

[0061] The present invention adopts orthogonal experimental design to optimize the reaction conditions of the kit

[0062] According to the coating concentration of antigen Cytoskeleton-associated protein 4 (50μg, 80μg, 100μg, 150μg four coating concentrations), each reaction time (15min, 30min, 45min) and temperature (25℃, 37℃), the enzyme-labeled secondary antibody is the most suitable. Four factors including optimal dilution (1:100, 1:500, 1:1000, 1:1500) and other four factors were selected in an orthogonal table, and the standard positive serum and standard negative serum were repeatedly determined at 2 levels for each factor. The ratio (P / N) of the highest light signal value (P) of positive serum to the lowest light signal value (N) of negative serum. The best antigen-Cytoskeleton-associated protein 4 coating concentration of the kit is 250ug / mL, and the best antigen-antibody reaction temperature of the solid-phase membrane immunodetection anti-Cytoskeleton-associated pr...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of Cytoskeleton-associdprotein4 in preparation of a vascular endothelial cell injury detection kit, in particular to application of Cytoskeleton-associdprotein4 in preparation of a vascular endothelial cell injury detection kit. The invention further provides a kit for detecting the anti-Cytoskeleton-asserted-protein 4-IgG antibody, the anti-Cytoskeleton-asserted-protein 4-IgG antibody in a biological sample is subjected to qualitative and quantitative detection, the accuracy and the sensitivity are high, the kit is simple, convenient and rapid, and the blank of the kit for detecting the anti-Cytoskeleton-asserted-protein 4-IgG antibody in the field is filled up.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of Cytoskeleton-associated protein 4 in the preparation of a vascular endothelial cell damage detection kit. Background technique [0002] Blood, blood vessels and heart make up the blood circulatory system of the human body. The blood in the circulatory system flows in the blood vessels and flows through the whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of blood vessels. Vascular endothelial cells are a layer of mononuclear cells between blood flow and blood vessel wall tissue. They can secrete a series of NO and PGI2 through autocrine, endocrine and paracrine pathways. , ET-1 and other vasoactive substances play a role in regulating vascular tone, anti-thrombosis, inhibiting smooth muscle cell proliferation and vascular wall inflammation. NO is the most important vasodilator...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/543
CPCG01N33/6854G01N33/6893G01N33/581G01N33/582G01N33/58G01N33/54326G01N33/543G01N33/54313G01N2800/32
Inventor 叶青毛建华刘颖
Owner ZHEJIANG UNIV
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