CN114878810A - Rheumatism autoantibody detection kit and detection method - Google Patents
Rheumatism autoantibody detection kit and detection method Download PDFInfo
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- CN114878810A CN114878810A CN202210516942.XA CN202210516942A CN114878810A CN 114878810 A CN114878810 A CN 114878810A CN 202210516942 A CN202210516942 A CN 202210516942A CN 114878810 A CN114878810 A CN 114878810A
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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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention discloses a rheumatism autoantibody detection kit and a detection method thereof. Antigens include U1-RNP, Sm, RO60, SSA, SS-B, Scl-70, Jo-1 and Rib 52. The enzyme conjugate comprises one or more of alkaline phosphatase-labeled goat anti-human IgG, IgM and IgA. The detection method and principle is to adopt an immunoblotting method for in vitro diagnosis. The detection kit provided by the invention can conveniently and rapidly carry out in-vitro detection, and is simple to operate and high in sensitivity; provides a reliable basis for clinical diagnosis, typing, disease observation, prognosis and treatment evaluation of rheumatism.
Description
Technical Field
The invention relates to the field of kits, in particular to a kit and a method for detecting a rheumatism autoantibody.
Background
Autoimmune diseases (Autoimmune diseases) refer to diseases caused by the body's immune response to autoantigens resulting in damage to the tissues of the body. Rheumatism is a group of diseases mainly attacking joints, bones, muscles, blood vessels and related soft tissues or connective tissues, and most of them are autoimmune diseases. The disease is usually more hidden and slower, the disease course is longer, and most of the diseases have hereditary tendency. Both diagnosis and treatment are difficult; different autoantibodies can be detected in blood, and may be associated with different HLA subtypes; has good short-term or long-term remission response to nonsteroidal anti-inflammatory drugs (NSAID), glucocorticoids and immunosuppressants. In a broad sense, diseases causing pain in bones and joints and muscles are classified as rheumatism. In the classification of rheumatism, there are over 100 kinds of diseases in broad sense, including infectious, immune, metabolic, endocrine, hereditary, degenerative, neoplastic, endemic, toxic and other diseases. In a narrow sense, it should be limited to a few dozen diseases in the medical and immune-related categories. Some of these diseases are also interdisciplinary, such as gout, osteoarthritis, infectious arthritis, and the like. Common diagnosis methods of the rheumatism autoantibody are an immunoblotting method, a chemiluminescence method and the like, and detection of the rheumatism autoantibody has important significance for assisting clinical diagnosis, clinical typing, disease observation, prognosis and treatment evaluation.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a rheumatism autoantibody detection kit and a detection method, the detection kit can rapidly carry out in-vitro qualitative detection on the detection problem of the rheumatism autoantibody in human serum or plasma by an immunoblotting method, and provides a reliable basis for clinical diagnosis, typing, disease observation, prognosis and treatment evaluation.
The technical scheme is as follows: the kit for detecting the autoantibody of the rheumatism comprises a detection membrane strip coated with an antigen in parallel, an enzyme conjugate, a sample buffer solution and a substrate solution.
Wherein the antigens coated in parallel on the detection membrane strip include at least 7 of U1-RNP (anti-ribonucleoprotein U1 fragment antibody), Sm (anti-micronuclein antibody), RO60 (anti-cytoplasmic soluble substance A-60kd antibody), SSA (anti-cytoplasmic soluble substance A antibody), SS-B (anti-nuclear soluble substance B antibody), Scl-70 (anti-deoxyribonuclease topoisomerase antibody), Jo-1 (anti-cytoplasmic histone acyl-transport ribonuclease synthetase antibody), and Rib 52 (anti-ribosomal P protein antibody).
The antigens on the test membrane strip are preferably combined into U1-RNP, Sm, RO60, SSA, SS-B, Scl-70, Jo-1 and Rib 52.
The enzyme conjugate comprises one or more of alkaline phosphatase-labeled goat anti-human IgG, IgM and IgA.
Wherein the pH value of the enzyme conjugate is 7-7.1, and the enzyme conjugate also comprises Tris (hydroxymethyl) aminomethane (Tris), sodium chloride, BSA (bovine serum albumin), sucrose, preservative (Proclin 300), Brij 35 (polyoxyethylene lauryl ether), acetaminophen, gentamicin sulfate and Tween 20.
The content of each liter of enzyme conjugate is as follows: 5.451-6.663g of Tris (hydroxymethyl) aminomethane (Tris), 8.1-9.9g of sodium chloride, 22.5-27.5g of BSA (bovine serum albumin), 13.5-16.5g of sucrose, 0.9-1.1ml of preservative (Proclin 300), 0.9-1.1g of Brij 35 (polyoxyethylene lauryl ether), 0.45-0.55g of acetaminophen, 0.036-0.044g of gentamycin sulfate, 200.45-0.55 ml of Tween and 0.72-0.88mg/L of AP enzyme goat antibody.
The pH value of the enzyme conjugate is preferably 7.05, and the content of each component in each liter of the enzyme conjugate is preferably as follows: 6.057g of Tris (hydroxymethyl) aminomethane (Tris), 9g of sodium chloride, 25g of BSA (bovine serum albumin), 15g of sucrose, 1ml of preservative (Proclin 300), 1g of Brij 35 (polyoxyethylene lauryl ether), 0.5g of acetaminophen, 0.04g of gentamicin sulfate, 200.5 ml of Tween and 0.8mg/L of AP enzyme goat antibody.
The enzyme conjugate was concentrated 10-fold, diluted with a sample buffer at the time of use, and used within one day after dilution.
The detection kit also comprises a washing buffer solution.
The wash buffer comprises TBST buffer (Tris-sodium chloride-tween 20). Wherein the pH value of the washing buffer solution is 7-8, and the washing buffer solution also comprises Proclin 300.
The content of each component in each liter of washing buffer solution is as follows: tris 54.513-66.627g, sodium chloride 81-99g, Proclin 3000.45-0.55 ml and Tween 2018-22 ml.
The pH value of the washing buffer is preferably 7.4, and the content of each component in each liter of the washing buffer is preferably as follows: tris 60.57g, sodium chloride 90g, Proclin 3000.5 ml and Tween 2020 ml.
The washing buffer was concentrated 10 times, and diluted with distilled water before use, and used within one day after dilution.
The sample buffer comprises TBST buffer/skimmed milk powder (Tris-sodium chloride-Tween 20/skimmed milk powder), and can be directly used.
Wherein the pH value of the sample buffer solution is 7-8, and the sample buffer solution also comprises EDTA and Proclin 300.
The content of each component in each liter of sample buffer solution is as follows: tris 5.451-6.663g, sodium chloride 7.893-9.647g, EDTA 0.9-1.1g, Proclin 3000.9-1.1 ml, Tween 204.5-5.5 ml and skimmed milk powder 9-11 g.
The pH value of the sample buffer is preferably 7.4, and the content of each component in each liter of the sample buffer is preferably as follows: tris 6.057g, sodium chloride 8.77g, EDTA 1g, Proclin 3001 ml, Tween 205 ml and skimmed milk powder 10 g.
The substrate solution comprises tetrazole nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate (NBT/BCIP).
Wherein the pH value of the substrate solution is 9-10, and the substrate solution also comprises Tris, NaCl, Proclin 300, N-dimethylformamide and MgCl 2 。
The content of each liter of substrate liquid is as follows: tris 10.903-13.325g, NaCl 5.256-6.424g, Proclin 3000.45-0.55 ml, NBT 0.149-0.182g, BCIP 0.0747-0.0913g, N-dimethylformamide 4.95-6.05ml, MgCl 2 0.086-0.105g。
The pH value of the substrate liquid is preferably 9.6, and the content of each component in each liter of the substrate liquid is preferably as follows: tris 12.114g, NaCl 5.84g, Proclin 3000.5 ml, NBT 0.165g, BCIP 0.083g, N-dimethylformamide 5.5ml, MgCl 2 0.095g。
The substrate liquid can be directly used, but the bottle cap is covered immediately after use due to the sensitivity of the substrate liquid to light.
The detection membrane strip is provided with a quality control band which can detect whether the detection experiment operation is correct or not, and the width of the quality control band is not less than 2 mm.
The detection method and the principle for detecting the antibody by using the kit of the invention are as follows:
the in vitro diagnosis is carried out by adopting an immunoblotting method, and highly purified antigens are coated on a detection membrane strip of the kit in parallel. In the first incubation step, the diluted patient sample is incubated with a test membrane strip. If the sample is positive, specific IgG antibodies (including IgA and IgM) bind to the corresponding antigenic sites.
To detect the bound antibody, an enzyme-labeled anti-human IgG (enzyme conjugate) was added for a second incubation; then enzyme substrate is added and a third incubation step is performed to generate a color reaction that is observable.
Has the advantages that: 1. the detection kit provided by the invention can conveniently and rapidly carry out in-vitro detection, and is simple to operate and high in sensitivity; 2. provides a reliable foundation for clinical diagnosis, typing, disease observation, prognosis and treatment evaluation of rheumatism; 3. the anti-U1-nRNP antibody had a specificity of 90% for MCTD (mixed connective tissue disease), the anti-SSA, anti-SSB and anti-Ro-52 antibodies had a specificity of 80%, 95%, 62% for SS (Sjogren's syndrome), the anti-Scl-70 antibody had a specificity of 100% for SSC (systemic sclerosis), and the anti-Jo-1 antibody had a specificity of 100% for PM/DM (myositis/dermatomyositis); the anti-Sm antibody has high specificity to SLE (systemic lupus erythematosus), is frequently accompanied with nervous system damage when being singly found in SLE, is related to lupus nephritis, has no correlation with the activity of SLE, can still be positive in SLE patients after being treated and relieved, and has great help for early atypical SLE or retrospective diagnosis after treatment; anti-SSA and anti-SSB antibodies are mainly related to SS, and the sensitivity in SLE varies with the detection method. anti-SSA antibody, anti-SSB antibody, and anti-Ro-52 antibody are associated with SLE disease, and SLE patients with anti-SSA antibody, anti-SSB antibody, and anti-Ro-52 antibody are more common with photosensitivity, skin lesions, and purpura. anti-SSA antibodies and anti-SSB antibodies are particularly found in neonatal SLE, the former occurring at 95% -100% and the latter at 75%, and can cause congenital heart diseases such as neonatal SLE and infantile heart transmission block; anti-Scl-70 antibodies can be found in 25% -75% SSC, about 40% -65% diffuse, and 5% -15% confined. The anti-Jo-1 antibody is found in PM/DM, the positive rate of PM patients can reach 40% -50%, the positive rate of anti-Jo-1 antibody of patients with scleroderma superposition can reach 85%, and the positive rate of PM/DM patients is 25%. anti-Rib antibodies are SLE-specific autoantibodies, but the incidence is only about 10%. The sensitivity and specificity of the single detection of various antibodies are different, the limitation of case statistics and the non-uniformity of each laboratory method exist, certain results are different from each other, but the sensitivity and specificity of the multiple combined detection of the autoantibodies can be improved. Clinical manifestations of various rheumatism are various, and combined detection of autoantibodies provides strong evidence for early correct diagnosis and treatment of rheumatism.
Drawings
FIG. 1 is a schematic view of the structure of a detection membrane strip in the detection kit of the present invention;
FIG. 2 is a schematic view showing the detection procedure when the detection kit of the present invention is used.
Detailed Description
Referring to fig. 1, the kit for detecting an autoantibody in rheumatism according to an embodiment of the present invention includes a detection membrane strip coated with an antigen in parallel, an enzyme conjugate, a sample buffer, a washing buffer, and a substrate solution.
Wherein the antigens coated on the detection membrane strip in parallel comprise U1-RNP, Sm, RO60, SSA, SS-B, Scl-70, Jo-1 and Rib 52.
The detection membrane strip is provided with a quality control band which can detect whether the detection experiment operation is correct. The width of the film strip is 2.5 mm.
The pH value of the enzyme conjugate is 7.05, and each liter of the enzyme conjugate comprises the following components in percentage by weight: tris 6.057g, sodium chloride 9g, BSA 25g, sucrose 15g, Proclin 3001 ml, Brij 351 g, acetaminophen 0.5g, gentamicin sulfate 0.04g, Tween 200.5 ml and AP enzyme goat anti-human 0.8 mg/L.
The enzyme conjugate was concentrated 10-fold, diluted with a sample buffer at the time of use, and used within one day after dilution.
The pH value of the sample buffer solution is 7.4, and each liter of the sample buffer solution comprises the following components in percentage by weight: tris 6.057g, sodium chloride 8.77g, EDTA 1g, Proclin 3001 ml, Tween 205 ml and skimmed milk powder 10 g. The sample buffer can be used directly.
The pH value of the washing buffer solution is 7.4, and each liter of the washing buffer solution comprises the following components in percentage by weight: tris 60.57g, sodium chloride 90g, Proclin 3000.5 ml and Tween 2020 ml.
The washing buffer was concentrated 10 times, and diluted with distilled water before use, and used within one day after dilution.
The pH value of the substrate liquid is 9.6, and each liter of the substrate liquid comprises the following components in percentage by weight: tris 12.114g, NaCl 5.84g, Proclin 3000.5 ml, NBT 0.165g, BCIP 0.083g, N-dimethylformamide 5.5ml, MgCl 2 0.095g。
The substrate liquid can be directly used, but the bottle cap is covered immediately after use due to the sensitivity of the substrate liquid to light.
Incubation trays are required to complete the detection assays of the present invention. Meanwhile, the detection kit is also suitable for a full-automatic immunoblotting instrument.
Referring to fig. 2, the detection kit of the present invention is used for detecting related antibodies by immunoblotting, and the principle and specific steps are as follows:
first, sample requirement
Sample preparation: a human serum sample. Avoid the use of severely lipemic, jaundice, hemolysis and contaminated samples.
Sample stability: the sample of the patient to be detected can be stable for 14 days at the temperature of 2-8 ℃, and the diluted sample can be detected in the same working day.
Sample dilution: patient samples were diluted 1:101 in sample buffer, e.g., 15. mu.L serum was diluted 1.5mL sample buffer and mixed well by vortex mixer during manual operation, but not by a sample injector. When the instrument is automatically detected, the dilution process is completed by the instrument.
Second, inspection method
1. Preparation and stabilization of reagents
Note that: all reagents had to be equilibrated at room temperature (18-25 ℃) for 30 minutes before use. From the first use, the kit is, for example, stored at 2-8 ℃ and, without contamination, can be stabilized to the indicated expiration date.
(1) Antigen-coated test membrane strip: can be used directly. To prevent condensation of the film strip, the package can be opened only after the film strip has equilibrated to room temperature. The strips should be removed and the original package sealed immediately and stored at 2-8 ℃.
(2) Enzyme conjugate: concentrating by 10 times. In use, the desired enzyme conjugate is pipetted from the vial using a clean pipette and diluted 1:10 with sample buffer. If necessary, incubate one test membrane strip and dilute 0.15mL of enzyme conjugate with 1.35mL of sample buffer. The diluted enzyme conjugate should be used up on the same working day.
(3) Sample buffer: can be used directly.
(4) Washing buffer solution: concentrating by 10 times. When in use, a clean suction pipe is used for sucking required amount from the bottle and diluting the bottle with distilled water at a ratio of 1: 10. If necessary, incubate one strip and dilute 1mL of concentrated buffer with 9mL of distilled water. The diluted buffer should be used up on the same working day.
(5) Substrate solution: the product can be directly used and is sensitive to light, and the bottle cap should be closed immediately after use.
2. Operation process
(1) Pretreatment: the desired membrane strip is removed and placed in an incubation well. The side of the film strip with the number faces upwards. 1.5mL of each sample buffer was added to each of the incubation tanks, and after incubation for 5 minutes on a rocking shaker at room temperature, the liquid in the incubation tanks was aspirated.
(2) Incubation with serum:
s1: 1.5mL of each diluted serum sample was added to the incubation tank and incubated for 30 minutes at room temperature (18-25 ℃) on a rocking shaker.
Cleaning: the tank was aspirated and the membrane strips were washed 3 times 5 minutes each time on a rocking shaker with 1.5mL working concentration of wash buffer.
(3) Incubation of enzyme conjugate:
s2: 1.5mL of diluted enzyme conjugate (alkaline phosphatase-labeled anti-human IgG) was added to each of the incubation tanks, and incubated on a rocking shaker at room temperature for 30 minutes.
Cleaning: the tank was aspirated and the membrane strips were washed 3 times 5 minutes each time on a rocking shaker with 1.5mL working concentration of wash buffer.
(4) Substrate incubation:
s3: 1.5mL of the substrate solution was added to each of the incubation tanks, and incubated for 10 minutes at room temperature (18-25 ℃) on a rocking shaker.
And (4) terminating: the liquid in the tank was aspirated off, and the membrane strip was washed with distilled water 3 times for 1 minute each time.
(5) And (4) judging a result: and (5) placing the detection membrane strip in a result judgment template, and judging the result after air drying.
Quality control procedure: the detection membrane strip is provided with a quality control band, and if the quality control band has strong color reaction, the experimental operation is correct. The white band appearing at the position where the membrane strip coats the antigen should be judged negative.
The results can be classified as negative, suspicious, positive and strongly positive according to the criteria in Table 1 and then according to the shade of the staining of the antigen bands.
TABLE 1 judgment standards for test results
Claims (10)
1. A kit for detecting autoantibodies against rheumatism, which is characterized in that: comprises a detection membrane strip coated with antigen in parallel, an enzyme conjugate, a sample buffer solution and a substrate solution,
wherein the antigens coated on the detection membrane strip in parallel comprise at least 7 of U1-RNP, Sm, RO60, SSA, SS-B, Scl-70, Jo-1 and Rib 52.
2. The detection kit according to claim 1, characterized in that: the enzyme conjugate comprises one or more of alkaline phosphatase-labeled goat anti-human IgG, IgM and IgA.
3. The detection kit according to claim 1 or 2, characterized in that: the enzyme conjugate was concentrated and used diluted with sample buffer.
4. The detection kit according to claim 1, characterized in that: the washing buffer solution comprises TBST buffer solution.
5. The detection kit according to claim 4, characterized in that: the wash buffer was concentrated.
6. The detection kit according to claim 1, characterized in that: the sample buffer comprises TBST buffer/skimmed milk powder.
7. The detection kit according to claim 1, characterized in that: the substrate solution comprises tetrazole nitroaniline blue/5-bromo-4-chloropyridine-3-indole-phosphate.
8. The detection kit according to claim 1, characterized in that: the detection membrane strip is provided with a quality control band which can detect whether the detection experiment operation is correct.
9. The detection kit according to claim 1, characterized in that: the width of the detection membrane strip is not less than 2 mm.
10. A method for detecting an autoantibody against rheumatism using the detection kit according to any one of claims 1 to 9, wherein the detection is carried out by immunoblotting, comprising the steps of:
(1) combining and reacting the diluted serum sample with a detection membrane strip, and carrying out a first-step incubation, wherein if the sample is positive, specific IgG, IgA or IgM antibodies are combined with corresponding antigen sites;
(2) adding diluted enzyme conjugate for second incubation in order to detect the antibody bound in step (1);
(3) in order to enable the detection result in the step (2) to be visually observed through color reaction, adding substrate solution to carry out third-step incubation;
(4) and (4) placing the detection membrane strip in the result judgment template and judging the detection result, namely finishing the detection of the related antibody.
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