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Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum

A kit and human detection technology, applied in the field of immunization, can solve the problems of reduced specificity of anti-dsDNA antibody detection

Inactive Publication Date: 2010-07-14
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We use an optimized purification method to prepare double-stranded DNA molecules, which ensures the purity of the antigen and improves the sensitivity and specificity of detection. Since naturally purified double-stranded DNA usually has nucleoprotein components or nucleosomes and other self-antigenic substances Contamination, resulting in reduced specificity of anti-dsDNA antibody detection

Method used

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  • Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum
  • Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum
  • Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum

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preparation example Construction

[0093] Therefore, the object of the present invention is to provide a method for preparing a double-stranded DNA antigen for systemic lupus erythematosus detection, comprising:

[0094] (1) Treat the plasmid DNA with buffer P1 containing Tris, EDTA salt and RNase A, centrifuge to remove the supernatant, and then add the buffer P1 to suspend;

[0095] (2) using alkaline buffer solution P2 containing SDS (sodium dodecyl sulfate) and alkali metal ions to process the suspension obtained in step (1); and

[0096](3) Treat the product of step (2) with buffer P3 containing potassium acetate, centrifuge to obtain the supernatant, filter the supernatant, treat it with isopropanol, and centrifuge to obtain the precipitate.

[0097] In the present invention, the recombinant plasmid DNA containing the double-stranded DNA antigen for the detection of systemic lupus erythematosus can be various known recombinant plasmid DNAs containing the double-stranded DNA antigen for the detection of sy...

Embodiment 1

[0159] Example 1: Preparation of double-stranded DNA antigen

[0160] On the basis of the classic alkaline lysis method, optimization and improvement were carried out, and high-purity plasmid DNA was purified from commercially available plasmid DNA. The size of the plasmid was 2.5Kb, and it had a stable DNA double-stranded structure and strong immunogenicity. , using it as the coating antigen of the double-stranded DNA antibody ELISA kit, and the prepared kit has achieved high detection sensitivity and specificity. The plasmid prepared by the method has stable and uniform structure and convenient source, and can meet the technical requirements of the enzyme-linked immunosorbent kit for double-stranded DNA antibodies.

[0161] (1) The specific operation is as follows:

[0162] 1) Take 2mg of plasmid pET-GST (purchased from Shenzhen Qinbaosheng Biotechnology Co., Ltd.) and shake and suspend it with 10ml Buffer P1, 8000g, 4°C, 2min, remove the supernatant, and then add 10ml Buff...

Embodiment 2

[0220] Example 2: Biotin Labeling of Double-Stranded DNA

[0221] The double-stranded DNA antigen prepared in Example 1 was directly mixed with long-arm photosensitive biotin (Beijing Jiakangyuan Technology Development Co., Ltd.), and labeled with a light labeling lamp to prepare biotinylated plasmid dsDNA. The specific preparation method is as follows: add 5 μg / 10 μl of DNA to be labeled into a sterilized EP tube, add 5 μg / μl photobiotin in a dark room, mix well, and place in an ice bath.

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Abstract

The invention relates to a preparation method for a double-stranded DNA antigen and a kit for testing an anti-double-stranded-DNA antibody of human serum. Particularly, the invention provides a preparation method for a double-stranded DNA antigen used for diagnosing systemic lupus erythematosus and a preparation method for a kit used for in vitro test of an anti-double-stranded-DNA antibody of human serum. The antigen prepared in the method has the advantages of low cost and stable and reliable antigen properties, and also has high sensitivity and specificity for in vitro test of the anti-double-stranded-DNA antibody in the serum of the patient with the systemic lupus erythematosus.

Description

technical field [0001] The invention relates to the preparation of a detection double-stranded DNA antigen in the field of immune technology and the preparation method of the antigen as a kit for detecting anti-double-stranded DNA antibodies in human serum, which is used for auxiliary diagnosis of SLE and its application in other diseases . Background technique [0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that invades connective tissue throughout the body. It is more common in young women and has complex and diverse clinical manifestations. DNA antibodies are broadly divided into natural double stranded DNA (double stranded DNA, dsDNA) and denatured single stranded DNA (single stranded DNA, ssDNA) antibodies, 40% of SLE patients have anti-dsDNA antibodies in serum, 70% have anti-ssDNA antibodies, but anti-ssDNA Antibodies lack specificity and are meaningless for diagnosis. The anti-dsDNA antibody has high specificity and is called the "marker antibo...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/535G01N33/543G01N33/52C12N15/10C12N15/63
CPCG01N33/6893G01N2800/104
Inventor 张玥高成秀孙宏彬李春梅钱洁韩永俊
Owner SHANGHAI KEXIN BIOTECH
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