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Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay

A technology of chemiluminescent immunity and magnetic particles, which is applied in the direction of measuring devices, scientific instruments, instruments, etc., can solve the problems of cumbersome operation and long time consumption, and achieve the effects of high detection accuracy, easy operation and high precision

Inactive Publication Date: 2020-11-13
THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] At present, WB method and ELISA method are mainly used in scientific research, and have the disadvantages of cumbersome operation and long time-consuming, and their specificity, sensitivity and high-throughput processing capacity need to be further improved

Method used

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  • Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay
  • Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay
  • Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay

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Experimental program
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Effect test

Embodiment 1

[0054] Preparation of biotinylated anti-DNASE1L3 antibody: Mouse anti-human anti-DNASE1L3 antibody (purchased) was diluted to 1 mg / mL with 0.5 mmol / L borate buffer (pH=8.6), and then diluted with 0.5 mmol / L boric acid buffer (pH=8.6) in a dialysis bag (MW CO 8000) L boric acid buffer (pH = 8.6) was alternately dialyzed 3 times; N-hydroxysuccinimide biotin (NHSB, purchased) was dissolved in dimethyl sulfoxide (DMSO) at a ratio of 1 mL:1 mg, and the final concentration was 1 mg / mL; Add 120 μL of the above NHSB solution to 1 mL of the antibody solution, stir magnetically for 2 hours at room temperature, add 9.6 μL of 1mol / L ammonium chloride and stir for 10 minutes; dialyze three times with PBS buffer at 4°C to remove free Biotin; put the above-mentioned dialysis sample on the molecular sieve column (G25), slowly elute with PBS, and collect the biotinylated anti-DNASE1L3 antibody; finally add the biotinylated antibody to sodium azide (final concentration 0.5g / L) And 1.0% (w / w) bo...

Embodiment 2

[0057] Step 1: Add 50 μL of sample to 100 μL of the biotinylated anti-DNASE1L3 antibody reaction tube prepared by the method in Example 1, and react at 37°C for 5 minutes to form a biotinylated anti-DNASE1L3 antibody-antigen complex;

[0058] Step 2: Add 60 μL 1mg / mL streptavidin-coated magnetic particles (purchased, particle size 3 μm) to the above reaction tube, and react at 37°C for 5 minutes, through the specific binding reaction of streptavidin and biotin , immobilizing the anti-DNASE1L3 antibody-antigen complex on magnetic particles;

[0059] Step 3: Remove the liquid part from the above-mentioned reaction tube under the action of magnetic force; then wash it with Tris buffer solution, remove the liquid under the action of magnetic force, and repeat the operation 5 times;

[0060] Step 4: Add 100 μL of ALP-labeled anti-DNASE1L3 antibody reagent to the above reaction tube, react at 37°C for 5 minutes, magnetically separate, and wash with Tris buffer. Repeat the operation...

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Abstract

The invention relates to the technical field of immunological detection, in particular to a method for detecting DNASE1L3 based on a magnetic particle chemiluminescence immunoassay. The method comprises the following steps: preparing a biotinylated anti-DNASE1L3 antibody; preparing an enzyme-labeled anti-DNASE1L3 antibody; combining the biotinylated anti-DNASE1L3 antibody with the anti-DNASE1L3 antibody in a sample to be detected; adding streptavidin-coated magnetic particles, reacting, washing under the action of magnetic force, and removing uncombined substances; adding an enzyme-labeled anti-DNASE1L3 antibody, reacting, washing under the action of magnetic force, and removing uncombined substances; adding a buffer solution and a luminous substrate solution for reaction, detecting the luminous intensity on a luminoscope, and calculating the concentration of the DNASE1L3 in the sample. The preparation method and application of the mortise and tenon joint structure g-c3n4moo3 compositephotocatalytic material can rapidly realize quantitative analysis of DNASE1L3, and have the advantages of high detection sensitivity and strong specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay. Background technique [0002] DNASE1L3, DNase 1-like protein 3, is an endonuclease in the DNASE superfamily. It is a secreted protein that is mainly produced by myeloid cells such as dendritic cells and macrophages and enters the blood. It has DNA hydrolysis activity. Cleaves single- and double-stranded DNA, and also cleaves chromatin into nucleosomal units, and cleaves nucleosome- and liposome-coated DNA. Currently, studies have demonstrated that DNASE1L3 deficiency can lead to anti-DNA responses and autoimmunity in humans and mice, and blood DNASE1L3 participates in circulating plasma DNA homeostasis by enhancing fragmentation and affecting terminal motif frequency. The reduction or lack of DNASE1L3 level is related to the pathogenesis, clinical features and disease activity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/543G01N33/535G01N33/532
CPCG01N33/573G01N33/54326G01N33/535G01N33/532
Inventor 郭永灿程渝杨加贝奚宇戈
Owner THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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