阅读说明：本技术 一种抗双链dna抗体检测试剂 (Anti-double-chain DNA antibody detection reagent ) 是由 盛伟 裴中平 徐小点 于 2019-09-27 设计创作，主要内容包括：本发明提供了一种抗双链DNA抗体检测试剂盒,包括包被双链DNA抗原的磁微粒、碱性磷酸酶标记抗体和化学发光底物；所述磁微粒为二氧化硅磁性复合微粒。本发明提供的抗双链DNA抗体检测试剂具有较高的检测灵敏度,较低的检测误差并且检测时间较短的优点,可快速而精确地进行检测。(The invention provides an anti-double-stranded DNA antibody detection kit, which comprises magnetic particles coated with double-stranded DNA antigens, an alkaline phosphatase labeled antibody and a chemiluminescent substrate; the magnetic particles are silicon dioxide magnetic composite particles. The anti-double-chain DNA antibody detection reagent provided by the invention has the advantages of higher detection sensitivity, lower detection error and shorter detection time, and can be used for quickly and accurately detecting.)

1. An anti-double-stranded DNA antibody detection reagent is characterized by comprising magnetic particles coated with double-stranded DNA antigens, alkaline phosphatase labeled antibodies and a chemiluminescent substrate;

the magnetic particles are silicon dioxide magnetic composite particles.

2. The anti-double-stranded DNA antibody detection reagent according to claim 1, wherein the silica magnetic composite particle comprises a silica shell layer and a magnetic iron sesquioxide nano-core;

preferably, the magnetic fine particles have a particle diameter of 0.5 to 2 μm, more preferably 1 to 1.5 μm.

3. The reagent for detecting an anti-double-stranded DNA antibody according to claim 1 or 2, wherein the chemiluminescent substrate is 3- (2-helical adamantane) -4-methoxy-4- (3-phosphoxyacyl) -phenyl-1, 2-dioxetane disodium salt.

4. The anti-double stranded DNA antibody detection reagent according to any one of claims 1 to 3, wherein the luminescent substrate further comprises an enhancer;

preferably, the enhancer is selected from polyquaterniums, further preferably bis-decyl dimethyl ammonium bromide and/or bis-decyl dimethyl ammonium chloride.

5. The anti-double stranded DNA antibody detection reagent according to any one of claims 1 to 4, wherein the double stranded DNA antigen is selected from a plasmid DNA antigen and/or an escherichia coli double stranded DNA antigen, further preferably a plasmid DNA antigen;

preferably, the antibody is selected from any one of anti-human IgG, IgA or IgM or a combination of at least two thereof, further preferably anti-human IgG.

6. The anti-double-stranded DNA antibody detection reagent according to any one of claims 1 to 5, wherein the magnetic particle is coupled with streptavidin;

preferably, the double stranded DNA antigen is coated on the magnetic particle by streptavidin.

7. The reagent for detecting an anti-double-stranded DNA antibody according to any one of claims 1 to 6, wherein the magnetic fine particles coating the double-stranded DNA antigen are prepared by the following method:

adding an antigen solution into the magnetic particles, and incubating and coating to obtain the magnetic particles coated with the double-stranded DNA antigen;

preferably, the preparation method is as follows:

(1) adding a streptavidin solution into an enzyme label plate for coupling reaction to obtain streptavidin coupled magnetic particles;

(2) and adding the antigen solution into streptavidin coupled magnetic particles for incubation coating to obtain the magnetic particles coated with the double-stranded DNA antigen.

8. The reagent for detecting an anti-double-stranded DNA antibody according to any one of claims 1 to 7, wherein the alkaline phosphatase-labeled antibody is prepared by the following method:

and mixing the activated antibody with the activated alkaline phosphatase, and purifying to obtain the alkaline phosphatase labeled antibody.

9. The anti-double stranded DNA antibody detection reagent according to any one of claims 1 to 8, wherein in the anti-double stranded DNA antibody detection reagent, the alkaline phosphatase-labeled antibody is present in the form of a solution;

preferably, the concentration of the solution is 0.1-2. mu.g/mL.

10. The anti-double stranded DNA antibody detection reagent according to any one of claims 1 to 9, wherein in the anti-double stranded DNA antibody detection reagent, the magnetic fine particles coating the double stranded DNA antigen are present in the form of a dispersion;

preferably, the concentration of the dispersion is 1-4. mu.g/mL.

Technical Field

The invention belongs to the technical field of detection, and relates to an anti-double-chain DNA antibody detection reagent.

Background

Anti-double-stranded DNA antibody is one of the anti-DNA antibodies, is a serological marker of Systemic Lupus Erythematosus (SLE), and is a laboratory detection index which is most frequently performed clinically. For the diagnosis of systemic lupus erythematosus, the anti-double-stranded DNA antibody has quite high specificity and is easy to detect, and simultaneously has certain relation with the systemic lupus erythematosus and can change along with the activity degree of the disease, so that the detection of the double-stranded DNA antibody can quickly and accurately judge the systemic lupus erythematosus.

The current diagnostic detection methods mainly comprise chemiluminescence analysis, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunofluorescence analysis, indirect immunofluorescence and the like. The indirect immunofluorescence method is the most widely applied in clinic, the method takes the body of the short membrane worm of the green fly as a coating substrate, only contains a simple and complete dsDNA molecule on the body, does not contain any other substance, and has good specificity; sensitivity is limited due to binding of only high affinity antibodies in serum, and the procedure involves manual manipulation and fluorescence microscopy, while requiring extensive experience, subject to a variety of factors. The immunoblotting method combines the high resolution of sodium dodecyl sulfate polyacrylamide gel electrophoresis and the high specificity of enzyme immunoadsorption technology, and is widely used for group antigen component analysis, but the method has lower sensitivity in detecting anti-ds-DNA antibodies.

CN101776682A discloses a preparation method of double-stranded DNA antigen and a kit for detecting anti-double-stranded DNA antibody of human serum. The method comprises (1) treating plasmid DNA with buffer solution P1 containing Tris, EDTA salt and RNase A, centrifuging to remove supernatant, and adding buffer solution P1 for suspension; (2) treating the suspension obtained in the step (1) with an alkaline buffer solution P2 containing SDS and alkali metal ions; and (3) treating the product obtained in the step (2) by using a buffer solution P3 containing potassium acetate, centrifuging to obtain a supernatant, filtering the supernatant, treating the supernatant by using isopropanol, and centrifuging to obtain a precipitate. The antigen provided by the patent is low in cost and has stable and reliable antigen performance, but the sensitivity of the antigen to double-stranded DNA antibodies is not high enough. CN109444404A provides a method for rapidly detecting an anti-double-stranded DNA antibody, and the DNA extraction step comprises the following steps: labeling double-stranded DNA antigen with biotin, labeling mouse anti-human immunoglobulin G with fluorescence, preparing a test strip and a sample diluent, and measuring, wherein the detection is carried out quickly and accurately by utilizing a centrifugal tube, a centrifugal machine, a water bath box, an electronic pH meter, a fluorescence immunoassay analyzer, a connection point membrane gold spraying all-in-one machine and corresponding reagents and materials; when the test strip provided by the patent is used for detection, the detection time is short, but the sensitivity is not high enough.

Therefore, it is desirable to provide a novel detection reagent to improve the detection sensitivity against double-stranded DNA antibodies.

Disclosure of Invention

The invention aims to provide an anti-double-chain DNA antibody detection reagent. The anti-double-chain DNA antibody detection reagent provided by the invention has the advantages of higher detection sensitivity, lower detection error and shorter detection time, and can be used for quickly and accurately detecting.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an anti-double-stranded DNA antibody detection reagent, which comprises magnetic particles coated with double-stranded DNA antigens, an alkaline phosphatase labeled antibody and a chemiluminescent substrate;

the magnetic particles are silicon dioxide magnetic composite particles.

According to the invention, the double-stranded DNA antigen is coated by the specific selected silicon dioxide magnetic composite particles, so that the coating performance is good, and the detection error of the detection reagent is small; meanwhile, the invention selects the chemiluminescence label as the judgment standard of quantitative analysis, and has the characteristics of long luminescence time, high intensity and small change in luminescence time.

The coating performance is good, and the subsequent detection of the sample to be detected is facilitated, so that the detection error cannot be increased due to the coating problem.

In the invention, the silicon dioxide magnetic composite particles comprise a silicon dioxide shell layer and a magnetic ferric oxide nanometer inner core.

Preferably, the magnetic fine particles have a particle size of 0.5 to 2 μm, for example, 0.8 μm, 1.0 μm, 1.2 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.8 μm, etc., more preferably 1 to 1.5 μm, for example, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, etc.

The silica magnetic composite particles of the present invention are magnetic microspheres available in the prior art.

The magnetic particles with the particle size of 0.5-2 mu m are preferably selected, if the particle size is too large, the coated double-stranded DNA antigen can be too dense, so that part of the antigen can not be combined with the object to be detected when being combined with the object to be detected subsequently, waste is caused, and the sensitivity of the antigen can be influenced; if the particle size is too small, the amount of double-stranded DNA antigen coated is too small, and measurement accuracy is impaired.

In the present invention, the chemiluminescent substrate is 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt.

In the present invention, the luminescent substrate further comprises an enhancer.

Preferably, the enhancer is selected from polyquaterniums, further preferably bis-decyl dimethyl ammonium bromide and/or bis-decyl dimethyl ammonium chloride.

In the invention, the reinforcing agent can enhance the luminous intensity of the substrate of alkaline phosphatase enzymolysis chemiluminescence, polyquaternium is specifically selected as the reinforcing agent, particularly, bis-decyl dimethyl ammonium bromide and/or bis-decyl dimethyl ammonium chloride are/is selected as the reinforcing agent, and the reinforcing factor can reach more than 10000 times, so that the sensitivity of the reagent for detecting the anti-double-chain DNA antibody provided by the invention can be obviously improved.

In the present invention, the double-stranded DNA antigen is selected from a plasmid DNA antigen and/or an Escherichia coli double-stranded DNA antigen, and a plasmid DNA antigen is more preferable.

Preferably, the antibody is selected from any one of anti-human IgG, IgA or IgM or a combination of at least two thereof, further preferably anti-human IgG.

In the present invention, the magnetic particles are coupled with streptavidin.

Preferably, the double stranded DNA antigen is coated on the magnetic particle by streptavidin.

In the present invention, the preparation method of the magnetic particle coated with the double-stranded DNA antigen is as follows:

and adding the antigen solution into the magnetic particles, and incubating and coating to obtain the magnetic particles coated with the double-stranded DNA antigen.

Preferably, the preparation method is as follows:

(1) adding a streptavidin solution into an enzyme label plate for coupling reaction to obtain streptavidin coupled magnetic particles;

(2) and adding the antigen solution into streptavidin coupled magnetic particles for incubation coating to obtain the magnetic particles coated with the double-stranded DNA antigen.

In the present invention, the preparation method of the alkaline phosphatase-labeled antibody is as follows:

and mixing the activated antibody with the activated alkaline phosphatase, and purifying to obtain the alkaline phosphatase labeled antibody.

The preparation methods of the magnetic particles coated with the double-stranded DNA antigen and the alkaline phosphatase labeled antibody can be prepared by adopting the common preparation method in the prior art.

Illustratively, the preparation method of the activated anti-human IgG antibody is as follows:

adding the anti-human IgG antibody into a coupling agent 2-iminothiolane hydrochloride, standing, adding a glycine solution, standing again, and desalting by using a gel column to obtain the activated anti-human IgG antibody.

The activated alkaline phosphatase was prepared as follows:

adding the alkaline phosphatase solution into the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution, standing, and desalting with gel column to obtain activated alkaline phosphatase.

In the present invention, in the anti-double-stranded DNA antibody detection reagent, the alkaline phosphatase-labeled antibody is present in the form of a solution.

Preferably, the concentration of the solution is 0.1-2. mu.g/mL, such as 0.4. mu.g/mL, 0.6. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL, 1.2. mu.g/mL, 1.5. mu.g/mL, 1.8. mu.g/mL, and the like.

In the present invention, in the anti-double-stranded DNA antibody detection reagent, the magnetic fine particles coating the double-stranded DNA antigen are present in the form of a dispersion.

Preferably, the concentration of the dispersion is 1-4. mu.g/mL, such as 1.2. mu.g/mL, 1.5. mu.g/mL, 1.8. mu.g/mL, 2.0. mu.g/mL, 2.5. mu.g/mL, 3.0. mu.g/mL, 3.5. mu.g/mL, and the like.

In the present invention, the detection method of the anti-dsDNA antibody is a detection method commonly used in the prior art, and exemplarily, the use method of the anti-dsDNA antibody detection reagent is as follows:

(1) adding a dispersion liquid of magnetic particles coated with double-stranded DNA antigens and a sample to be detected into a detection tube, mixing, incubating, separating and cleaning;

(2) adding alkaline phosphatase labeled antibody solution into the system obtained in the step (1), uniformly mixing, performing secondary incubation, separation and washing;

(3) adding chemiluminescence substrate and enhancer, mixing thoroughly, incubating for 12-15min, and detecting relative luminescence intensity to obtain the amount of anti-double-stranded DNA antibody.

In the present invention, the separation may be performed by applying a magnetic field.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the double-stranded DNA antigen is coated by the specific selected silicon dioxide magnetic composite particles, so that the coating performance is good, and the detection error of the detection reagent is small; meanwhile, the invention selects the chemiluminescence label as the judgment standard of quantitative analysis, and has the characteristics of long luminescence time, high intensity and small change in luminescence time.

(2) In the invention, the reinforcing agent can enhance the luminous intensity of the substrate of alkaline phosphatase enzymolysis chemiluminescence, polyquaternium is specifically selected as the reinforcing agent, particularly, bis-decyl dimethyl ammonium bromide and/or bis-decyl dimethyl ammonium chloride are/is selected as the reinforcing agent, and the reinforcing factor can reach more than 10000 times, so that the sensitivity of the reagent for detecting the anti-double-chain DNA antibody provided by the invention can be obviously improved.

(3) The detection reagent for the double-chain DNA antibody has the detection advantages of high sensitivity and small error value, wherein the LOD of the detection reagent is below 0.05IU/mL and can be below 0.005IU/mL at the lowest; the detection error is below 2 percent, and the lowest detection error can reach below 1 percent.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.