A test kit for detecting anti-prelamin A/C-IgG antibody

A technology for labeling antibodies and antigenic proteins, which is applied in the biological field to achieve the effects of fast reaction speed, mild labeling conditions and less reagent consumption.

Active Publication Date: 2022-04-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, studies in the urinary system have not involved

Method used

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  • A test kit for detecting anti-prelamin A/C-IgG antibody
  • A test kit for detecting anti-prelamin A/C-IgG antibody
  • A test kit for detecting anti-prelamin A/C-IgG antibody

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Experimental program
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Effect test

Embodiment 1

[0038] 1.1 Expression of Prelamin A / C protein antigen: the corresponding antigen protein was expressed by bioengineering method.

[0039] 1.2 Antigen protein immobilization method: the antigen coating solid-phase carrier method of the present invention uses direct coating method: 1, antigen is combined on polystyrene microporous plate or nitrocellulose membrane by physical adsorption or non-covalent bond mode; 2, Antigens are bound to magnetic particles containing carboxyl functional groups through chemical coupling.

[0040] 1.3 Positive quality control substance and standard substance: The positive quality control substance and standard substance selected in the present invention are IgG or human anti-tag peptide IgG extracted and quantified from patient serum, and the serum of a healthy person is a negative quality control substance.

[0041] 1.4 Labeled antibody and substrate chromogen: The selected labeled antibody of the present invention can be acridinium ester-labeled ...

Embodiment 2

[0057] Example 2 Prelamin A / C on podocytes is one of the main target antigens targeted by autoantibodies in patients with autoimmune nephrotic syndrome

[0058] Culture podocyte strain (MPC5), wash 2-3 times with PBS, then use focused ultrasonic instrument (Covaris S220, Gene) to contain 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (#ab65621; Abcam, 1:200 dilution) in the lysis buffer for sufficient lysis on ice, and then the sample was placed in a centrifuge at 12000g, 4°C for 30min. The supernatant was collected, which was the collected glomerular podocyte total protein. The BCA protein concentration assay kit was used to measure the total protein concentration of the collected glomerular podocytes to obtain purified total protein of the glomerular podocytes. Then carry out two-dimensional electrophoresis, transfer to the membrane, and then incubate with the serum of the healthy control group and the patient respectively, and then add the secondary antibody for d...

Embodiment 3

[0059] Example 3 Prelamin A / C Antigen Protein Expression

[0060] The expression and purification of antigenic protein Prelamin A / C uses the method of genetic engineering to use the gene encoding Prelamin A / C protein as a template for PCR amplification, and then constructs 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., and finally the molecular weight of the synthetic protein Prelamin A / C was identified and quantified by SDS-PAGE. Such as figure 2 shown.

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Abstract

The invention provides a test kit for detecting anti-Prelamin A / C-IgG antibodies, which is composed of antigenic protein Prelamin A / C, a solid phase carrier, a standard product, a positive quality control product, a negative quality control product, a labeled antibody, and a substrate for color development Solution, Antibody and Antigen Diluent, Sample Dilution Buffer, Washing Solution, Stop Solution, etc. In the present invention, the target antigen Prelamin A / C corresponding to the anti-Prelamin A / C-IgG antibody is used, and the IgG of the human anti-tag peptide is used as a standard for quantitative detection, and the antigen-antibody complex formed in the serum is subjected to anti-Prelamin A / C-IgG IgG antibody detection. The present invention proposes for the first time that anti-Prelamin A / C-IgG antibody detection is used as a biomarker for immune nephrotic syndrome. The invention can be used for the detection of the autoantibody, and provides support for the research and clinical diagnosis and treatment of the autoimmune nephrotic syndrome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kit for detecting anti-Prelamin A / C-IgG antibodies. The invention provides for the first time a fast, simple and highly sensitive detection kit for qualitatively or quantitatively analyzing anti-Prelamin A / C-IgG levels in serum for immune nephrotic syndrome. Background technique [0002] The diagnosis and treatment of immune nephrotic syndrome is an ongoing challenge in biomedical science. Nephrotic syndrome is a clinical syndrome characterized by a series of characteristic pathological changes of a large amount of proteinuria caused by increased filtration of plasma protein due to the increased permeability of the glomerular filtration membrane. Many of the symptoms exhibited to a large extent can be caused by various reasons (including congenital genetic causes, renal dysfunction and insufficiency, genitourinary tract infections and diseases, drug abuse, loss of appetite and malnu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/564G01N33/545G01N33/548G01N33/543
CPCG01N33/564G01N33/545G01N33/548G01N33/54326G01N2800/24G01N2800/347G01N2333/47
Inventor 叶青毛建华韩秀翠
Owner ZHEJIANG UNIV
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