CN113917136A - Detection method of anti-double-stranded deoxyribonucleic acid antibody immunoglobulin G - Google Patents
Detection method of anti-double-stranded deoxyribonucleic acid antibody immunoglobulin G Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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Abstract
A method for detecting immunoglobulin G (IgG) of an anti-double-strand deoxyribonucleic acid antibody comprises the following steps: adding the serum of a patient sample containing the anti-double-stranded deoxyribonucleic acid antibody into a double-stranded deoxyribonucleic acid antigen sheet double-stranded deoxyribonucleic acid reaction area, reacting with double-stranded deoxyribonucleic acid on a short membrane worm on the antigen sheet double-stranded deoxyribonucleic acid reaction area, combining the anti-double-stranded deoxyribonucleic acid antibody with the double-stranded deoxyribonucleic acid, and washing away the unbound substances; secondly, adding a fluorescein isothiocyanate labeled goat anti-human immunoglobulin G solution into the antigen tablet reaction area for reaction to generate an immune complex, and washing to remove unreacted substances; thirdly, sealing the tablet in the reaction area of the reacted antigen tablet, covering a cover glass, and observing a homogeneous or annular double-chain deoxyribonucleic acid antibody fluorescent substance between the cell nucleus of the short membrane worm and the moving matrix by fluorescence; high sensitivity, strong specificity, good accuracy, low cost, simple operation and wide applicability, and is used for monitoring clinical course of disease and treatment effect and judging prognosis.
Description
Technical Field
The invention relates to an in vitro diagnosis and detection technology, in particular to a detection method of an anti-double-strand deoxyribonucleic acid antibody immunoglobulin G.
Background
The detection method of the anti-double-chain deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) is used for qualitatively detecting the anti-double-chain DNA antibody in human serum in vitro and assisting in diagnosing the systemic lupus erythematosus.
Systemic lupus erythematosus is an autoimmune disease involving multiple systems and multiple organs. Lupus nephritis is a major complication of systemic lupus erythematosus, and has a high incidence rate in systemic lupus erythematosus patients. The autoantibody plays an important role in the development process of lupus nephritis, can form a circulating immune complex to be deposited on the kidney, is combined with the inherent antigen of the kidney, activates autoimmune reaction and causes the occurrence of lupus nephritis.
The anti-double-stranded DNA antibody is a specific antibody of the systemic lupus erythematosus, is also an important basis for diagnosing the systemic lupus erythematosus, and is also a marker of disease activity, particularly kidney injury.
The anti-dsDNA antibody is closely related to systemic lupus erythematosus and rises and falls with the activity of the disease, and the titer of the patient with better disease decreases or even turns negative. It is now believed that anti-double-stranded DNA antibodies that bind complement play an important role in the pathogenesis of systemic lupus erythematosus, particularly in those with complicated nephritis (lupus nephritis). Systemic lupus erythematosus complicated with nephritis is an immune complex disease mediated by anti-double-stranded DNA. Because of the higher sensitivity of anti-double-stranded DNA to anti-systemic lupus erythematosus, currently, in the standard for diagnosing systemic lupus erythematosus in the field, anti-double-stranded DNA has been used as a diagnostic index. Besides being used for diagnosing the systemic lupus erythematosus, the kit can also be used for monitoring the clinical course of disease and the treatment effect, and has certain value for judging prognosis. The short membrane worm body is used as a coating substrate, only a single and complete dsDNA molecule is contained on the short membrane worm body, no other substance is contained, and the specificity is good. Therefore, a method for detecting immunoglobulin G (IgG) as an anti-double-stranded deoxyribonucleic acid (DNA) antibody is to be further perfected.
Disclosure of Invention
The invention mainly aims to provide a method for detecting an anti-double-stranded deoxyribonucleic acid antibody immunoglobulin G, which is suitable for detecting double-stranded deoxyribonucleic acid (DNA) antibodies, can be used for diagnosing systemic lupus erythematosus, can also be used for monitoring clinical course and treatment effect, and has certain value for judging prognosis.
The purpose of the invention is realized by the following technical scheme.
The invention relates to a method for detecting immunoglobulin G (IgG) of an anti-double-strand deoxyribonucleic acid (DNA) antibody, which comprises the following steps:
the first step is as follows: adding a patient sample serum containing anti-double-stranded deoxyribonucleic acid (DNA) antibody to a double-stranded deoxyribonucleic acid (DNA) reaction area of an antigen sheet, carrying out a first reaction with double-stranded deoxyribonucleic acid (DNA) on a short membrane worm on the double-stranded deoxyribonucleic acid (DNA) reaction area of the antigen sheet, wherein specific anti-double-stranded deoxyribonucleic acid (DNA) antibody is combined with the double-stranded deoxyribonucleic acid (DNA), and the rest of substances which are not combined are washed and removed by a washing solution;
secondly, adding a fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG) solution into the antigen sheet reaction area for secondary reaction to generate an immune complex; the immune complex is double-stranded deoxyribonucleic acid (DNA) -specific anti-double-stranded deoxyribonucleic acid (DNA) antibody-fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG); washing with a washing solution to remove substances which do not undergo the reaction;
and thirdly, adding a sealing tablet in the antigen tablet reaction area after the first reaction and the second reaction, then covering a cover glass, observing the result under a fluorescence microscope, and observing a homogeneous or circular double-chain deoxyribonucleic acid antibody fluorescent substance between the cell nucleus and the moving matrix of the short membrane worm to finish the detection.
The method for detecting immunoglobulin G (IgG) of anti-double-stranded deoxyribonucleic acid (DNA) antibody, wherein the double-stranded DNA antigen sheet is a solution of attaching short membrane worms on a glass slide;
adding a mixed liquid consisting of fluorescein isothiocyanate labeled goat anti-human immunoglobulin G and 5 to 10 percent of bovine serum albumin with the volume concentration of 0.01 to 0.05 percent into 0.01 to 0.05mol/L of phosphate buffer solution;
the blocking tablet is a mixed solution of 0.01 to 0.05mol/L phosphate buffer solution and 50 to 80 percent of glycerol and 1 to 5 percent of 4', 6-diamidino-2-phenylindole by volume concentration;
the washing solution used in the first step is prepared by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate buffer solution and mixing;
the washing liquid used in the second step is formed by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate solution and mixing.
The method for detecting an immunoglobulin G as an anti-dsdna antibody described above, wherein: the short membrane worm solution is cultured by adopting a short membrane worm culture medium, washed and precipitated by phosphate buffer solution and then resuspended by using a fixing solution, and the resuspended short membrane worm solution is smeared on a glass slide;
the glass slide is soaked in a poly-L-lysine solution with the molecular weight of 50000 and the volume concentration of 0.1-0.5%, and is dried at 40 ℃ for later use after soaking.
The method for detecting an immunoglobulin G as an anti-dsdna antibody described above, wherein: the short membrane worm culture medium adopted by the short membrane worm solution is a mixed liquid prepared from 1 +/-0.2% of tryptone, 0.1 +/-0.5% of yeast extract, 0.01 +/-0.05% of liver extract, 1 to 2% of sucrose, 1 +/-0.5% of triethanolamine, 10 +/-5% of hemin, 1 +/-0.5% of fetal calf serum and the balance of deionized water according to volume concentration;
the resuspension fixing solution is phosphate buffer solution containing 20 to 60 volume percent of formaldehyde, the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.4.
The method for detecting the anti-double-chain deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) has the beneficial effects that the method is suitable for detecting the double-chain deoxyribonucleic acid (DNA) antibody, can be used for diagnosing the systemic lupus erythematosus, can also be used for monitoring the clinical course and the treatment effect, and has certain value for judging prognosis. The short membrane worm body is a coating substrate, only contains a single and complete dsDNA molecule on the short membrane worm body, does not contain any other substance, has good specificity, can be used for diagnosing systemic lupus erythematosus, can also be used for monitoring clinical course and treatment effect, and has certain value for judging prognosis. The method has the advantages of high sensitivity, strong specificity, good accuracy, low cost, simple operation and wide applicability.
Detailed Description
The features and resulting efficacy of the method for detecting immunoglobulin G (IgG) as an anti-double-stranded deoxyribonucleic acid (DNA) antibody of the present invention are further understood and appreciated, and the following detailed description of preferred embodiments is provided.
The invention relates to a method for detecting immunoglobulin G (IgG) of an anti-double-strand deoxyribonucleic acid (DNA) antibody, which comprises the following steps:
the first step is as follows: adding a patient sample serum containing anti-double-stranded deoxyribonucleic acid (DNA) antibody to a double-stranded deoxyribonucleic acid (DNA) reaction area of an antigen sheet, carrying out a first reaction with double-stranded deoxyribonucleic acid (DNA) on a short membrane worm on the double-stranded deoxyribonucleic acid (DNA) reaction area of the antigen sheet, wherein specific anti-double-stranded deoxyribonucleic acid (DNA) antibody is combined with the double-stranded deoxyribonucleic acid (DNA), and the rest of substances which are not combined are washed and removed by a washing solution;
secondly, adding a fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG) solution into the antigen sheet reaction area for secondary reaction to generate an immune complex; the immune complex is double-stranded deoxyribonucleic acid (DNA) -specific anti-double-stranded deoxyribonucleic acid (DNA) antibody-fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG); washing with a washing solution to remove substances which do not undergo the reaction;
and thirdly, adding a sealing tablet in the antigen tablet reaction area after the first reaction and the second reaction, then covering a cover glass, observing the result under a fluorescence microscope, and observing a homogeneous or circular double-chain deoxyribonucleic acid antibody fluorescent substance between the cell nucleus and the moving matrix of the short membrane worm to finish the detection.
The invention relates to a method for detecting immunoglobulin G (IgG) of an anti-double-strand deoxyribonucleic acid (DNA) antibody, wherein:
the double-stranded deoxyribonucleic acid antigen sheet is formed by adhering a short membrane worm solution to a glass slide; adding a mixed liquid consisting of fluorescein isothiocyanate labeled goat anti-human immunoglobulin G and 5 to 10 percent of bovine serum albumin with the volume concentration of 0.01 to 0.05 percent into 0.01 to 0.05mol/L of phosphate buffer solution; the blocking tablet is a mixed solution of 0.01 to 0.05mol/L phosphate buffer solution and 50 to 80 percent of glycerol and 1 to 5 percent of 4', 6-diamidino-2-phenylindole by volume concentration; the washing solution used in the first step is prepared by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate buffer solution and mixing; the washing liquid used in the second step is formed by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate solution and mixing.
The short membrane worm solution is cultured by adopting a short membrane worm culture medium, washed and precipitated by phosphate buffer solution and then resuspended by using a fixing solution, and the resuspended short membrane worm solution is smeared on a glass slide; the glass slide is soaked in a poly-L-lysine solution with the molecular weight of 50000 and the volume concentration of 0.1-0.5%, and is dried at 40 ℃ for later use after soaking.
The short membrane worm culture medium adopted by the short membrane worm solution is a mixed liquid prepared from 1 +/-0.2% of tryptone, 0.1 +/-0.5% of yeast extract, 0.01 +/-0.05% of liver extract, 1 to 2% of sucrose, 1 +/-0.5% of triethanolamine, 10 +/-5% of hemin, 1 +/-0.5% of fetal calf serum and the balance of deionized water according to volume concentration; the resuspension fixing solution is phosphate buffer solution containing 20 to 60 volume percent of formaldehyde, the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.4.
The first embodiment is as follows:
1. preparing double-stranded deoxyribonucleic acid (DNA) antigen tablets:
the short membrane worms in the double-stranded deoxyribonucleic acid (DNA) antigen sheet are cultured for 2 days by using a short membrane worm culture medium (formed by mixing 0.8% of tryptone, 0.1% of yeast extract, 0.02% of liver extract, 1.5% of cane sugar, 0.5% of triethanolamine, 5% of hemocrytine, 0.5% of fetal bovine serum and the balance of deionized water), the cultured short membrane worms are centrifuged to remove supernatant, then washed for 5 times by using 0.01mol/L phosphate buffer solution with pH7.4, the supernatant is removed after precipitation, the short membrane worm precipitate is resuspended by using a stationary liquid (0.01 mol/L phosphate liquid with pH7.4 and containing 40% of formaldehyde by volume concentration), and the resuspended short membrane worm solution is smeared on a glass slide, dried, sealed and stored.
2. Preparation of fluorescein isothiocyanate-labeled goat anti-human IgG:
0.02 vol% fluorescein isothiocyanate-labeled goat anti-human immunoglobulin G (IgG) and 10% bovine serum albumin were added to 0.05mol/L phosphate buffer.
3. Preparing a sealing tablet: adding 70% of glycerol and 5% of 4', 6-diamidino-2-phenylindole into 0.05mol/L of phosphate buffer solution, and mixing.
4. Preparation of a washing solution: adding 0.1% polysorbate into 0.02mol/L phosphate solution, and mixing.
Example two:
the detection method of the invention for the anti-double-strand deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) is used for detecting the sample of the systemic lupus erythematosus patient.
1. Sample serum: 96 cases of serum of systemic lupus erythematosus patients, wherein the diagnosis standard of systemic lupus erythematosus meets the classification standard of systemic lupus erythematosus of American college of rheumatology, and 100 cases of serum of healthy examiners.
The first step is as follows: adding a patient sample serum containing anti-double-stranded deoxyribonucleic acid (DNA) antibody to a double-stranded deoxyribonucleic acid (DNA) reaction area of an antigen sheet, carrying out a first reaction with double-stranded deoxyribonucleic acid (DNA) on a short membrane worm on the double-stranded deoxyribonucleic acid (DNA) reaction area of the antigen sheet, wherein specific anti-double-stranded deoxyribonucleic acid (DNA) antibody is combined with the double-stranded deoxyribonucleic acid (DNA), and the rest of substances which are not combined are washed and removed by a washing solution;
secondly, adding a fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG) solution into the antigen sheet reaction area for secondary reaction to generate an immune complex; the immune complex is double-stranded deoxyribonucleic acid (DNA) -specific anti-double-stranded deoxyribonucleic acid (DNA) antibody-fluorescein isothiocyanate labeled goat anti-human immunoglobulin G (IgG); washing with a washing solution to remove substances which do not undergo the reaction;
2. and thirdly, adding a sealing tablet in the antigen tablet reaction area after the first reaction and the second reaction, then covering a cover glass, observing the result under a fluorescence microscope, and observing a homogeneous or circular double-chain deoxyribonucleic acid antibody fluorescent substance between the cell nucleus and the moving matrix of the short membrane worm to finish the detection.
3. And (3) detection results: experiments show that the specificity of the detection method of the anti-double-stranded deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) is 97 percent, and the sensitivity is 89.6 percent. The double-chain deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) is detected by the detection method of the double-chain deoxyribonucleic acid (DNA) antibody, so that the diagnosis and the treatment of the systemic lupus erythematosus are facilitated.
The content that will not be described in this embodiment is the prior art, and therefore, the description thereof is omitted.
The method for detecting the anti-double-strand deoxyribonucleic acid (DNA) antibody immunoglobulin G (IgG) has the advantages that: the short membrane worm body is used as a coating substrate, and has extremely high specificity because the short membrane worm body has a large mitochondrion (moving matrix) containing pure circular dsDNA, the antigen concentration of the short membrane worm body is 10 times higher than that of the antigen concentration of human laryngeal epidermoid carcinoma cells and liver cell nucleuses of other systemic lupus erythematosus detection substrates, and more importantly, the short membrane worm body does not contain any other antigens. The short membrane worms have simple structure, obvious negative and positive difference, clear fluorescence result and easy observation of the result. Clinically, the kit is combined with other systemic lupus erythematosus diagnosis methods, can improve the detection rate of the systemic lupus erythematosus and reduce false positive.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiment according to the technical spirit of the present invention still fall within the scope of the technical solution of the present invention.
Claims (4)
1. A method for detecting an immunoglobulin G (IgG) as an anti-double-strand deoxyribonucleic acid antibody, which comprises the following steps:
the first step is as follows: adding the serum of a patient sample containing anti-double-stranded deoxyribonucleic acid antibody into a double-stranded deoxyribonucleic acid reaction area of a double-stranded deoxyribonucleic acid antigen sheet, carrying out a first reaction with double-stranded deoxyribonucleic acid on a short membrane worm on the antigen sheet double-stranded deoxyribonucleic acid reaction area, combining the specific anti-double-stranded deoxyribonucleic acid antibody with the double-stranded deoxyribonucleic acid, and washing and removing the rest substances which are not combined by a washing solution;
secondly, adding a fluorescein isothiocyanate labeled goat anti-human immunoglobulin G solution into the antigen tablet reaction area subjected to the reaction in the first step for secondary reaction to generate an immune complex; the immune complex is a double-chain deoxyribonucleic acid-specific anti-double-chain deoxyribonucleic acid antibody-fluorescein isothiocyanate-labeled goat anti-human immunoglobulin G; washing with a washing solution to remove substances which do not undergo the reaction;
and thirdly, adding a sealing tablet in the antigen tablet reaction area after the first reaction and the second reaction, then covering a cover glass, observing the result under a fluorescence microscope, and observing a homogeneous or circular double-chain deoxyribonucleic acid antibody fluorescent substance between the cell nucleus and the moving matrix of the short membrane worm to finish the detection.
2. The method for detecting immunoglobulin G as an anti-dsDNA antibody according to claim 1, wherein the dsDNA antigen sheet is a slide glass to which a solution of Bremia is attached;
adding a mixed liquid consisting of fluorescein isothiocyanate labeled goat anti-human immunoglobulin G and 5 to 10 percent of bovine serum albumin with the volume concentration of 0.01 to 0.05 percent into 0.01 to 0.05mol/L of phosphate buffer solution;
the blocking tablet is a mixed solution of 0.01 to 0.05mol/L phosphate buffer solution and 50 to 80 percent of glycerol and 1 to 5 percent of 4', 6-diamidino-2-phenylindole by volume concentration;
the washing solution used in the first step is prepared by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate buffer solution and mixing;
the washing liquid used in the second step is formed by adding polysorbate with the volume concentration of 0.1-0.5% into 0.01-0.05 mol/L phosphate solution and mixing.
3. The method for detecting immunoglobulin G as an anti-dideoxyribonucleic acid antibody according to claim 2, wherein: the short membrane worm solution is cultured by adopting a short membrane worm culture medium, washed and precipitated by phosphate buffer solution and then resuspended by using a fixing solution, and the resuspended short membrane worm solution is smeared on a glass slide;
the glass slide is soaked in a poly-L-lysine solution with the molecular weight of 50000 and the volume concentration of 0.1-0.5%, and is dried at 40 ℃ for later use after soaking.
4. The method for detecting an immunoglobulin G as an anti-double-stranded deoxyribonucleic acid antibody according to claim 3, wherein: the short membrane worm culture medium adopted by the short membrane worm solution is a mixed liquid prepared from 1 +/-0.2% of tryptone, 0.1 +/-0.5% of yeast extract, 0.01 +/-0.05% of liver extract, 1 to 2% of sucrose, 1 +/-0.5% of triethanolamine, 10 +/-5% of hemin, 1 +/-0.5% of fetal calf serum and the balance of deionized water according to volume concentration;
the resuspension fixing solution is phosphate buffer solution containing 20 to 60 volume percent of formaldehyde, the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.4.
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| CN115819604A (en) * | 2022-09-09 | 2023-03-21 | 季华实验室 | Anti-double-stranded DNA antibody, nucleotide fragment, magnetic beads and method for extracting free DNA from peripheral blood |
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