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Kit for detecting anti-proteasome subunit alpha1-IgG antibody

An anti-protease and kit technology, applied in the field of biomedicine, can solve the problems of the existence of Proteasomesubunitalphatype1 expression and nephrotic syndrome, which has not been reported, and achieve the effects of increasing the coating surface area, easy cleaning and separation, and increasing the adsorption capacity.

Active Publication Date: 2021-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the expression of Proteasome subunit alpha type 1 and the presence of autoantibodies to Proteasome subunit alpha type 1 have not been reported in nephrotic syndrome
In addition, in the prior art, there is no application based on the target Proteasome subunit alpha type 1 or its autoantibody as a serological marker in autoimmune nephrotic syndrome
There are no studies identifying autoimmune nephrotic syndrome by detecting serum anti-Proteasome subunit alpha type1-IgG antibodies

Method used

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  • Kit for detecting anti-proteasome subunit alpha1-IgG antibody
  • Kit for detecting anti-proteasome subunit alpha1-IgG antibody
  • Kit for detecting anti-proteasome subunit alpha1-IgG antibody

Examples

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Embodiment 1

[0034] Example 1 Proteasome subunit alpha type 1 protein on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0035] Through a large number of clinical and molecular mechanism studies in the early stage, the present invention found for the first time that the serum IgG level of patients with nephrotic syndrome is higher, and confirmed that Proteasome subunit alpha type 1 on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative level of anti-Proteasome subunit alpha type 1-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, Ge...

Embodiment 2

[0036] Example 2 Expression and purification of recombinant antigenic protein Proteasome subunit alpha type 1

[0037] Using the method of genetic engineering, the gene encoding Proteasome subunit alpha type 1 protein was used as a template for PCR amplification, and then an expression vector was constructed for protein expression. The antigenic protein expressed in the present invention contains a GST 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 Proteasome subunit alpha type 1 was identified by SDS-PAGE as 56KDa, see figure 2 .

Embodiment 3

[0038] Embodiment 3 The present invention adopts orthogonal test design to optimize the reaction conditions of the kit

[0039]According to the antigen Proteasome subunit alpha type 1 coating concentration (50μg / ml, 90μg / ml, 120μg / ml, 150μg / ml four coating concentrations), each reaction time (15min, 30min, 45min) and temperature (25℃, 37 ℃), the optimal dilution of the enzyme-labeled secondary antibody (1:100, 1:500, 1:1000, 1:1500 four dilutions) and other four factors to select the orthogonal table, and each factor is based on 2 levels of repeated determination standards For 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. Through the orthogonal design, we obtained the optimal antigen package of this kit, the concentration of Proteasome subunit alpha type 1 is 90 μg / ml, and the optimal antigen-antibody reaction temperature of the solid-phase m...

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Abstract

The invention provides a kit for detecting an anti-proteasome subunit alpha1-IgG antibody. The kit consists of an antigen protein Proteasome subunit alpha type 1, a solid phase carrier, a labeled antibody, an antigen and antibody diluent, a sample dilution buffer solution, 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-proteasome subunit alpha1-IgG antibody in serum to be detected is detected by combining an indirect method reaction principle with magnetic particle chemiluminescence immunoassay. According to the invention, the autoantibody aiming at the target antigen proteasome subunit alpha1 in the serum of the patient with the autoimmune nephrotic syndrome is identified for the first time. The invention provides a basis for molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndromes related to proteasome subunit alpha1 and proteasome subunit alpha1-IgG autoantibodies at home and abroad.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a kit for detecting anti-proteasome subunit α1-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 show...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/573G01N33/543
CPCG01N33/6854G01N33/6893G01N33/58G01N33/573G01N33/54326G01N2333/96425G01N2800/347G01N33/564
Inventor 叶青毛建华田丹丹
Owner ZHEJIANG UNIV
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