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Kit for detecting antigen myosin 1-IgG antibody

An antigen protein and kit technology, which is applied in the field of kits for detecting antigenic myosin 1-IgG antibodies in serum, can solve problems such as the presence or absence, and achieve simple operation, high chemiluminescence quantum yield, and strong adsorption capacity. Effect

Active Publication Date: 2021-11-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although there are studies showing the application of Tropomyosin 1 expression in urological diseases, there are no studies on the presence of Tropomyosin 1 autoantibodies, especially in immune nephrotic syndrome, let alone their value

Method used

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  • Kit for detecting antigen myosin 1-IgG antibody
  • Kit for detecting antigen myosin 1-IgG antibody
  • Kit for detecting antigen myosin 1-IgG antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Expression of Tropomyosin 1 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 the direct coating method: (1) the antigen is bound to the polystyrene microwell plate or the nitrocellulose membrane by physical adsorption or non-covalent bond mode; ( 2) The antigen is bound to the 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.

[0041] 1.4 Labeled antibody and substrate chromogenic reagent: the selected labeled antibody of the present invention can be acridinium ester-labeled anti-human IgG, horseradish peroxidase-labele...

Embodiment 2

[0056] Example 2 Tropomyosin 1 on podocytes is one of the main target antigens targeted by autoantibodies in patients with autoimmune nephrotic syndrome

[0057] 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 ...

Embodiment 3

[0058] Example 3 Expression of Tropomyosin 1 antigen protein

[0059] The expression and purification of the antigenic protein Tropomyosin 1 was carried out by PCR amplification using the gene encoding the Tropomyosin 1 protein as a template, and then an expression vector was constructed for protein expression by means of genetic engineering. 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 synthetic protein Tropomyosin 1 was identified and quantified by SDS-PAGE. like figure 2 shown.

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Abstract

The invention provides a kit for detecting an antigen myosin 1-IgG antibody. The kit consists of an antigen protein Tropomyosin 1 (tropomyosin 1), 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 diluent, an antigen diluent, a sample dilution buffer solution, a washing solution, a stop solution and the like. According to the invention, a target antigen Tropomyosin 1 corresponding to an anti-Tropomyosin 1-IgG antibody is utilized, and IgG of human anti-tag peptide is used as a standard substance for quantitative detection, so that the anti-Tropomyosin 1-IgG antibody detection is carried out on an antigen-antibody compound formed in serum. The kit disclosed by the invention is simple and convenient to operate, very good in reagent stability and convenient to store, and fills the blank of identifying biomarkers of immune nephrotic syndrome patients at home and abroad.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kit for detecting antigen myosin 1-IgG antibody in serum. The invention provides for the first time a fast, simple and highly sensitive detection kit for qualitatively or quantitatively analyzing the level of antigen myosin 1-IgG 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 mal...

Claims

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Application Information

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IPC IPC(8): G01N33/564G01N33/543G01N33/545G01N33/548
CPCG01N33/564G01N33/54393G01N33/54326G01N33/548G01N33/545G01N2333/4712G01N2800/24G01N2800/347
Inventor 叶青毛建华韩秀翠
Owner ZHEJIANG UNIV
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