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Kit for detecting anti-Pelamin A/C-IgG antibody

A kit and antibody technology, applied in the field of kits for detecting anti-PrelaminA/C-IgG antibodies, to achieve the effects of high chemiluminescence quantum yield, low background and high labeling rate

Active Publication Date: 2021-10-01
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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  • Kit for detecting anti-Pelamin A/C-IgG antibody
  • Kit for detecting anti-Pelamin A/C-IgG antibody
  • Kit for detecting anti-Pelamin 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. like figure 2 shown.

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Abstract

The invention provides a kit for detecting an anti-Prelamin A / C-IgG antibody. The kit is composed of an antigen protein Prelamin A / C, a solid phase carrier, a standard substance, a positive quality control substance, a negative quality control substance, a labeled antibody, a substrate developing solution, an antibody and antigen diluent, a sample dilution buffer solution, a washing solution, a stop solution and the like. According to the invention, a target antigen Prelamin A / C corresponding to an anti-Prelamin A / C-IgG antibody is utilized, IgG of a human anti-tag peptide is taken as a standard substance for quantitative detection, and anti-Prelamin A / C-IgG antibody detection is carried out on an antigen-antibody complex formed in serum. According to the present invention, the application of the anti-Pelamin A / C-IgG antibody detection as the immune nephrotic syndrome biomarker is provided for the first time; and the kit can be used for detecting the autoantibody, and provides support for 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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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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