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Kit and method for detecting myositis and myasthenia gravis autoantibodies in human body fluid

A myasthenia gravis and autoantibody technology, applied in the field of biomedicine, can solve the problems of time-consuming detection process, large antibody demand, and low detection sensitivity, and achieve the effects of short detection cycle, reduced pollution, and simple operation

Active Publication Date: 2020-09-22
SHAANXI MYBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a detection kit and method for autoantibodies in myositis and myasthenia gravis in human body fluid, so as to solve the problem of high detection cost and antibody demand of existing detection methods or detection kits. Large size, low detection sensitivity, time-consuming detection process and other problems

Method used

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  • Kit and method for detecting myositis and myasthenia gravis autoantibodies in human body fluid
  • Kit and method for detecting myositis and myasthenia gravis autoantibodies in human body fluid
  • Kit and method for detecting myositis and myasthenia gravis autoantibodies in human body fluid

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preparation example Construction

[0091] The target antigen Ag in the detection kit of the present invention can be obtained by the prior art, and can also be obtained by the following method of the present invention, preferably the preparation method of the present invention. Specifically, the preparation method of the material for detecting myositis and myasthenia gravis autoantibody in human body fluid of the present invention includes the following steps:

[0092] Step 1. Obtain the CDS sequence of the target antigen Ag as the target gene, insert the target gene with the restriction site into the pET28a plasmid vector to obtain the recombinant plasmid vector pET28a-Ag; Ag represents the autoantibody corresponding to myositis and myasthenia gravis disease One of the target antigens;

[0093] Step 1.1: Obtain the CDS sequence of the autoantibody target antigen Ag by artificial synthesis or PCR as the target gene, and add NheI / NotI restriction sites at both ends of the target gene;

[0094] Step 1.2, insert the tar...

Embodiment 1

[0127] This example discloses a preparation method of a detection material for myositis and myasthenia gravis autoantibodies in human body fluid. The target antigen in this example is the target antigen MDA5 for myositis disease, and the negative control antigen is GAPDH. The preparation method is specific include:

[0128] 1. Plasmid construction:

[0129] Obtain the CDS sequence of autoantibody target antigen HuB and GAPDH by artificial synthesis or PCR method as the target gene, and add NheI / NotI restriction sites at both ends of the target gene;

[0130] Insert the target gene with restriction site into the pET28a vector, the insertion site is NheI / NotI, to obtain the recombinant vector, the recombinant vector is named pET28a-HuB and pET28a-GAPDH, specifically:

[0131] The glycerol strain of pET28a plasmid was inoculated into 3mL LB liquid medium resistant to kanamycin, and placed in a 37°C constant temperature incubator, 220r / min, overnight shaking. On the second day, 500 μL of...

Embodiment 2

[0150] This embodiment discloses a detection kit, which includes a test strip, a working solution and a blocking protein solution;

[0151] The test strip is the test strip corresponding to the target antigen in Example 1. Specifically, the test strip includes a backing plate, a carrier film provided with a control line and a test line, an absorbent pad, a sample pad and a gold label pad; carrier film detection The target antigen Ag is solidified on the dot, and Ag represents one of the target antigens corresponding to autoantibodies of myositis and myasthenia gravis disease; a negative control antigen is solidified on the control line. The absorbent pad, carrier film, sample pad, and gold-labeled pad are all laid on the pad, the absorbent pad is set at the end of the carrier film control line, and the sample pad or gold-labeled pad is set at the end of the carrier film detection point, such as figure 1 , figure 2 Shown.

[0152] Among them, the working solution is a mixed solutio...

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Abstract

The invention discloses a kit and a method for detecting myositis and myasthenia gravis autoantibodies in human body fluid. The kit comprises a test strip, a working solution and a blocking protein solution, the detection method comprises the following steps: mixing the working solution and the blocking protein solution to form a sample diluent, and adding a to-be-detected sample into the sample diluent; dropwise adding the to-be-detected sample onto the sample pad of the test strip; taking down the water absorption pad and the sample pad on the test strip, washing the carrier membrane, and fixing the water absorption pad and the gold-labeled pad at the two ends of the carrier membrane after the washing is finished; and immersing the gold-labeled pad end of the test strip into the workingsolution, and observing the color development condition. In the detection process, a sample chromatography method is an inverse chromatography method, and a carrier membrane washing method is ultrasonic washing so that the detection sensitivity and specificity are effectively improved, and the antibody can still be detected after strong positive serum is diluted by more than ten thousand times.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a kit and method for detecting myositis and myasthenia gravis autoantibodies in human body fluids. Background technique [0002] Idiopathic inflammatory myopathies (IIMs) is a rare autoimmune disease. The disease is characterized by muscle inflammation, leading to proximal muscle atrophy and disability, different skin rashes, ulcers, malignant tumors, etc. symptom. Autoantibodies can be detected in the body fluids of more than 50% of IIM patients, so autoimmunity is considered to play an important role in the pathogenesis of myositis. Current studies have shown that these autoantibodies target nuclear and cytoplasmic proteins. They are usually divided into two subgroups, Myositis-specific autoantibodies (MSAs) and Myositis-associated autoantibodies (Myositis-associated autoantibodies, MAAs). A large number of clinical related studies have shown that MSAs and MAAs are...

Claims

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Application Information

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IPC IPC(8): G01N33/564G01N33/558C07K14/47C12N15/70
CPCG01N33/564G01N33/558C07K14/47C12N15/70C07K1/22C07K1/18C07K1/20G01N2800/2878G01N2800/10
Inventor 闫亚平李怡婷冯昆封雪裴元元李科
Owner SHAANXI MYBIOTECH CO LTD
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