An antinuclear antibody profile detection kit, a preparation method and a detection method

Abstract

The application belongs to the technical field of kits, and discloses an antinuclear antibody spectrum detection kit, a preparation method and a detection method. The detection kit comprises a detection film strip coated with a plurality of antigens in parallel, an enzyme combination liquid, a sample buffer and a substrate liquid. The antigens coated on the detection film strip include at least one of nRNP / Sm, Sm, SS-A, SS-B, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosome, histone, ribosome P protein, M2 and Ro52. The kit provided in the application can detect 15 kinds of antinuclear antibodies at the same time, has high accuracy, and is of great significance for assisting clinical diagnosis, clinical typing, disease observation, prognosis and treatment evaluation of autoimmune diseases.

Description

Technical Field

[0001] This application relates to the field of reagent kits, specifically to an antinuclear antibody spectrum detection kit, its preparation method, and its detection method. Background Technology

[0002] Antinuclear antibodies (ANA) are a collective term for autoantibodies that target various components of eukaryotic cells. ANA target antigens are mainly distributed in the cell nucleus, cytoplasm, cytoskeleton, and cell cycle proteins. Based on the different properties of various molecules within the cell nucleus, ANA can be classified into anti-DNA antibodies, anti-nucleosome antibodies, anti-histone antibodies, anti-nucleolar antibodies, and anti-centromere antibodies, etc. Each major class is further subdivided into many different types due to different antigenic characteristics. Therefore, ANA, broadly speaking, is a group of autoantibodies with different clinical significance; a more accurate name would be the antinuclear antibody spectrum. ANA is mainly found in IgG, but also in IgM, IgA, and even IgD and IgE.

[0003] Clinically, ANA is commonly seen in patients with systemic (non-organ-specific) AIDs such as systemic lupus erythematosus (SLE), and can also be found in patients with organ-specific AIDs, such as autoimmune liver diseases. In addition, ANA can also occur in chronic infectious diseases, tumors, and healthy individuals. Detecting ANA is of significant clinical importance for the diagnosis, differential diagnosis, classification, and monitoring of disease activity in AIDs.

[0004] The antibodies detected in the antinuclear antibody spectrum include:

[0005] (1) Anti-U1-nRNP antibody: High titers of this antibody are a marker of mixed connective tissue disease (MCTD, Sharp syndrome), with a positive rate of 95-100%. The antibody titer is correlated with disease activity. This antibody can also be detected in 30-40% of patients with systemic lupus erythematosus, but anti-Sm antibody is almost always present.

[0006] (2) Anti-Sm antibody: A specific antibody for systemic lupus erythematosus (SLE), which, along with anti-dsDNA antibody, is a diagnostic marker for SLE. However, the positive rate is only 5-10%.

[0007] (3) Anti-SS-A antibodies: These are associated with various autoimmune diseases, most commonly seen in Sjögren's syndrome (40–80%), and also in systemic lupus erythematosus (30–40%) and primary biliary cirrhosis (20%), and occasionally in chronic active hepatitis. Furthermore, anti-SS-A antibodies are positive in 100% of neonatal lupus erythematosus cases. This antibody can be transmitted to the fetus via the placenta, causing inflammatory responses and congenital heart block in newborns.

[0008] (4) Anti-SS-B antibody: It is almost exclusively found in female patients with Sjögren's syndrome (40-80%) and systemic lupus erythematosus (10-20%), with a male-to-female ratio of 1:29. In Sjögren's syndrome, anti-SS-A antibody and anti-SS-B antibody often appear simultaneously.

[0009] (5) Anti-Scl70 antibody (anti-topoisomerase I antibody): Found in 25-75% of patients with progressive sclerosis (diffuse type), depending on the experimental method and disease activity. Not found in localized sclerosis.

[0010] (6) Anti-PM-Scl antibody: It is commonly found in the polymyositis and sclerosis overlap syndrome. 50% of patients with positive anti-PM-Scl antibody have the polymyositis and sclerosis overlap syndrome. Anti-PM-Scl antibody can also be found in patients with polymyositis alone, with a positive rate of 8%. The positive rate in diffuse sclerosis is 2-5%.

[0011] (7) Anti-Jo-1 antibody: found in polymyositis, with a positive rate of 25-35%, often associated with pulmonary interstitial fibrosis.

[0012] (8) Anticentromere (CENP-B) antibody: This antibody is associated with localized progressive systemic sclerosis (CREST syndrome: calcification, Raynaud's disease, esophageal dysfunction, finger scleroderma, distal vasodilation), with a positive rate of 70-90%. It can also be detected in patients with primary biliary cirrhosis (positive rate 10-30%). It can also be found in Raynaud's syndrome.

[0013] (9) Anti-PCNA antibody: PCNA is a proliferating cell nuclear antigen, and its expression is related to the cell cycle. Anti-PCNA antibody is a specific antibody for systemic lupus erythematosus, but the positive rate is only 3%. Some literature reports that anti-PCNA antibody may be related to the development of diffuse proliferative glomerulonephritis in patients with systemic lupus erythematosus.

[0014] (10) Anti-dsDNA antibody: It has high specificity for systemic lupus erythematosus. In addition to anti-Sm antibody, anti-dsDNA antibody can also be used as a serological marker for the disease, with a positive rate of 40-90%. Moreover, the anti-dsDNA antibody titer is correlated with the activity of the disease and can be used for monitoring the treatment efficacy.

[0015] (11) Antinucleosome antibodies: The positive rate in the serum of patients with systemic lupus erythematosus (SLE) is 50-90%, with almost 100% for specific types. Nucleosomes are functional subunits of cellular chromosomes, composed of DNA and histones in a specific manner. Antinucleosome antibodies appear earlier in the early stages of SLE than anti-dsDNA antibodies and anti-histone antibodies, playing an important role in the induction and pathogenesis of SLE. Clinical data show that 15-19% of SLE patients who are negative for anti-dsDNA antibodies are positive for antinucleosome antibodies. Therefore, combined detection of anti-dsDNA and nucleosome antibodies can improve the serological detection rate of SLE.

[0016] (12) Anti-histone (His) antibodies: Anti-histone antibodies or anti-H2A-H2B complex antibodies are common in drug-induced lupus (procainamide, hydralazine, and other drugs) (positive rate 95%). In addition, anti-histone antibodies can also be detected in 30-70% of systemic lupus erythematosus patients and 15-50% of rheumatoid arthritis patients.

[0017] (13) Anti-ribosomal P protein antibody (P0): It is a specific marker for systemic lupus erythematosus (SLE), with a positive rate of 5-15%. It is generally believed that the titer of this antibody is related to the activity of SLE, as well as SLE complicated with central nervous system symptoms, kidney or liver involvement.

[0018] (14) Anti-M2 antibody: High titers of anti-mitochondrial type 2 antibody are a marker of primary biliary cirrhosis, with a positive rate of 85-95%. In addition, anti-M2 antibody can also be detected in other chronic liver diseases (30%) and progressive systemic sclerosis (7-25%), but at lower titers.

[0019] (15) Anti-Ro52 antibody: SS-A antigen is a small ribonucleoprotein composed of an RNA molecule and two different proteins (molecular weights of 52 kDa and 60 kDa, respectively). Antibodies against SS-A 60 kDa protein are more specific for Sjögren's syndrome and systemic lupus erythematosus, while antibodies against SS-A 52 kDa alone do not have disease specificity and are prone to cross-reaction with other antibodies.

[0020] The aforementioned antinuclear antibodies can appear before clinical symptoms appear. Some autoantibodies are also involved in the development of the disease. Furthermore, multiple antinuclear antibodies can be detected in a certain autoimmune disease, and one antinuclear antibody may also be involved in multiple related autoimmune diseases.

[0021] Common methods for clinical detection of antinuclear antibodies include indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and enzyme-catalyzed chemiluminescence assay. However, these methods all have shortcomings, as detailed below.

[0022] 1) Indirect immunofluorescence assay

[0023] The basic principle of this method is to use a specific antibody to bind to an antigen on a glass slide to form an antigen-antibody complex. Then, a fluorescent antibody binds to the antigen-antibody complex to form a fluorescent antigen-antibody complex. Under a fluorescence microscope, the luminescence of the complex is used to determine the antibody being detected. This method is complex to operate, requires an expensive fluorescence microscope, is difficult to implement in many primary hospitals, has high background noise, is prone to false positives, and its sensitivity and specificity need improvement. Furthermore, it is difficult to standardize and automate.

[0024] 2) Enzyme-linked immunosorbent assay (ELISA)

[0025] Enzyme-linked immunosorbent assay (ELISA) has been widely used, but the detection operation is mostly done manually. The amount of reagents and samples added is not very accurate. The operation process is cumbersome and prone to errors. It also has the disadvantages of limited combination of detection items and low positive detection rate.

[0026] 3) Enzyme-catalyzed chemiluminescence method

[0027] Enzymatic chemiluminescence assays mainly employ two methods: horseradish peroxidase (HRP) and alkaline phosphatase (ALP). Both methods have limitations. The main drawback of horseradish peroxidase is that luminol will be oxidized by H₂O₂ and emit its own light even in the absence of horseradish peroxidase, resulting in a relatively high background, affecting the signal-to-noise ratio. Furthermore, the reaction kinetics are complex, influenced by numerous factors, and the results are not stable enough. Obtaining substrates with high sensitivity and long plateau phases is also difficult. The main drawbacks of alkaline phosphatase are that the substrate takes a long time to reach its plateau phase, and the substrate cost is high, leading to high detection costs and a heavy burden on patients.

[0028] Currently, most tests for the aforementioned autoantibodies are individual tests for single autoantibodies. However, because the development mechanism of autoimmune diseases is closely related to immune function, and there are many types of symptoms, multiple symptoms may coexist. Therefore, the test results for a single autoantibody alone cannot accurately pinpoint the specific autoimmune disease. Since these autoantibody tests are individual tests, obtaining results for multiple autoantibodies requires separate testing, which takes a considerable amount of time. Summary of the Invention

[0029] The purpose of this application is to provide an antinuclear antibody (IgG) spectrum detection kit that can simultaneously perform semi-quantitative or quantitative detection of 15 antinuclear antibodies with high accuracy.

[0030] The objective of this application is achieved through the following technical solution:

[0031] An antinuclear antibody (IgG) detection kit, the kit comprising: 16 detection strips coated with 15 antigens (nRNP / Sm, Sm, SS-A, SS-B, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosome, histone, ribosomal P protein, M2, Ro52), 1 bottle of enzyme conjugation stock solution, 1 bottle of sample buffer, 1 bottle of washing buffer stock solution and substrate solution.

[0032] The sample buffer solution includes buffer, electrolyte, surfactant, reaction enhancer, preservative, stabilizer, and blocking agent;

[0033] The cleaning buffer solution includes a buffer solution, an electrolyte, a surfactant, and a preservative.

[0034] The enzyme conjugate solution includes a buffer solution, electrolytes, enzyme stabilizers and enhancers, and preservatives.

[0035] The substrate solution includes a buffer solution, electrolyte, stabilizer, colorimetric enhancer, and preservative.

[0036] The coating solution used in the coating process of the antigen-coated membrane strip described in this application includes three or more types of zwitterionic buffers, electrolytes, surfactants, preservatives, etc.

[0037] In one specific embodiment provided in this disclosure, the buffers used in the sample buffer, washing buffer stock solution, enzyme conjugation stock solution, and substrate solution are Tris buffer and MES buffer.

[0038] In one specific embodiment provided in this disclosure, the coating buffer is selected from Tris buffer, MOPS buffer, CBS buffer, Bis-Tris buffer, and PB buffer (for quality control band coating);

[0039] The electrolyte used in all the buffer solutions is an inorganic salt, selected from one or more of NaCl, MgCl2, KCl, and ZnCl2;

[0040] The surfactants used in all the buffer solutions are any one or more of Tween 20, Tween 80, Triton X-100, NP40, and SDS.

[0041] The stabilizers used in the sample buffer, enzyme conjugate, and substrate solution are one or more of BSA, gelatin, EDTA, Scavenger-ALP, and trehalose.

[0042] The enhancers used in the sample buffer, enzyme conjugate, and substrate solution are one or more selected from serine protease inhibitors, sodium carboxymethyl cellulose, L-arginine, PEG 6000, and sodium thiosulfate. The preservatives used in all the buffer solutions are one or more selected from Proclin 300, MB-1, sodium azide, and gentamicin sulfate.

[0043] In one specific embodiment provided in this disclosure, in order to improve reagent sensitivity and reaction specificity, sodium carboxymethyl cellulose and serine protease inhibitor are added to the sample buffer.

[0044] In one embodiment, the concentration of sodium carboxymethyl cellulose added is 10 g / L to 30 g / L, and the concentration of serine protease inhibitor added is 0.005 mg / mL to 0.05 mg / mL;

[0045] In one specific embodiment provided in this disclosure, in order to improve enzyme stability and reaction sensitivity, this application adds the enhancer L-arginine and PEG 6000 to the enzyme conjugation solution;

[0046] In one embodiment, the concentration of L-arginine added is 18 g / L to 25 g / L, and the amount of PEG 6000 added is 10 g / L to 25 g / L;

[0047] In one specific embodiment provided in this disclosure, in order to improve the coloring intensity of the substrate, sodium thiosulfate, an enhancing agent, is added to the substrate solution;

[0048] In one embodiment, the concentration of sodium thiosulfate added is 1 g / L to 5 g / L.

[0049] In one specific embodiment provided in this disclosure, in order to further improve the reaction specificity, two surfactants, Tween 20 and Tween 80, are added to the washing buffer stock solution; Tween 20 is selected in the coating buffer solution.

[0050] In addition, in order to improve the effective adhesion of dsDNA and nucleosome antigens to the solid-phase NC membrane in this application, a high-temperature short-time drying process is adopted after membrane scrubbing.

[0051] In one specific embodiment provided in this disclosure, the drying process for these two indicators is carried out at 60°C to 85°C for a time of 25 min to 50 min.

[0052] In one specific embodiment provided in this disclosure, the sample buffer of this kit contains: 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 0.5 g / L to 1.2 g / L EDTA, 2 g / L to 8 g / L Tween 20, 4 g / L to 10 g / L BSA, 5 g / L to 20 g / L goat serum, 10 g / L to 30 g / L sodium carboxymethyl cellulose, 0.005 mg / mL to 0.05 mg / mL serine protease inhibitor, and 0.5 g / L to 2 g / L Proclin 300.

[0053] The washing buffer stock solution includes 50 g / L to 120 g / L Tris, 60 g / L to 90 g / L NaCl, 10 g / L to 25 g / L Tween 20, 5 g / L to 20 g / L Tween 80, and 0.5 g / L to 2 g / L Proclin 300.

[0054] The enzyme conjugate solution includes 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 8 g / L to 25 g / L sucrose, 0.3 g / L to 2 g / L polyoxyethylene lauryl ether, 0.2 g / L to 1 g / L magnesium chloride, 0.01 g / L to 0.05 g / L zinc chloride, 10 g / L to 30 g / L BSA, 18 g / L to 25 g / L L-arginine, 10 g / L to 25 g / L PEG 6000, 0.02 g / L to 0.1 g / L gentamicin sulfate, and 0.5 g / L to 2 g / L Proclin 300.

[0055] The substrate solutions include 8 g / L to 30 g / L Tris, 3 g / L to 8 g / L NaCl, 0.1 g / L to 0.25 g / L NBT, 0.02 g / L to 0.15 g / L BCIP, 0.1 g / L to 0.5 g / L magnesium chloride, 1 g / L to 5 g / L sodium thiosulfate, and 0.5 g / L to 2 g / L Proclin 300.

[0056] The coating solution included 5 g / L–20 g / L NaCl, 10 g / L–50 g / L trehalose, 0.05 g / L–1 g / L Tween 20, and 0.5 g / L–2 g / L Proclin 300. The zwitterionic buffers added were 6 g / L–20 g / L Tris (Tris buffer), 15 g / L–40 g / L Bis-Tris (Bis-Tris buffer), 10 g / L–40 g / L MOPS (MOPS buffer), 1 g / L–10 g / L anhydrous sodium carbonate and 1 g / L–10 g / L sodium bicarbonate (CBS buffer), and 1 g / L–10 g / L disodium hydrogen phosphate (PB buffer).

[0057] The advantages of this application are: The kit for determining antinuclear antibody (IgG) profiles uses immunoblotting, a sensitive, specific, and rapid method that can be automated. It is simple to operate, improves reagent repeatability and sensitivity, and has a high positive / negative concordance rate, especially for histones, nucleosomes, and dsDNA. Compared to other existing methods, this product enables rapid, fully automated, and quantifiable ANA detection with high sensitivity. Furthermore, compared to commercially available immunoblotting products, it saves time while further improving accuracy. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the linear detection results of the reagent kit in this application. Detailed Implementation

[0059] The present application is further described below through embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of the present application in any way.

[0060] Example 1: Antinuclear Antibody Profile Combined Detection Kit

[0061] The antinuclear antibody spectrum combined detection kit includes detection membrane strips, enzyme conjugates, sample buffer, washing buffer, substrate solution, and coating solution for detecting membrane strip coating;

[0062] The antigen coated on the detection membrane strip is selected from at least one of the following antigens: nRNP / Sm, Sm, SS-A, SS-B, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosome, histone, ribosomal P protein, M2, Ro52.

[0063] The sample buffer solution includes buffer, electrolyte, surfactant, reaction enhancer, preservative, stabilizer, and blocking agent;

[0064] The cleaning buffer solution includes a buffer solution, an electrolyte, a surfactant, and a preservative.

[0065] The enzyme conjugate solution includes a buffer solution, electrolytes, enzyme stabilizers and enhancers, and preservatives;

[0066] The substrate solution includes buffer solution, electrolyte, stabilizer, colorimetric enhancer, and preservative;

[0067] The coating solution includes three or more zwitterionic buffers, electrolytes, surfactants, and preservatives.

[0068] In one embodiment, the buffers used in the sample buffer, washing buffer, enzyme conjugation solution, and substrate solution are at least one of Tris buffer and MES buffer.

[0069] The electrolyte is one or more of NaCl, MgCl2, KCl, and ZnCl2;

[0070] The surfactant is one or more of Tween 20, Tween 80, Triton X-100, NP40, and SDS;

[0071] The stabilizer is one or more of BSA, gelatin, EDTA, Scavenger-ALP, and trehalose;

[0072] The preservative is any one or more of Proclin 300, MB-1, sodium azide, and gentamicin sulfate;

[0073] In one embodiment, the enhancer is one or more of the following: serine protease inhibitor, sodium carboxymethyl cellulose, L-arginine, PEG 6000, and sodium thiosulfate.

[0074] In one embodiment, the zwitterionic buffer is selected from one or more of Tris buffer, MOPS buffer, CBS buffer, Bis-Tris buffer, and PB buffer; the PB buffer is used for quality control tape coating.

[0075] In one embodiment, the enhancer is sodium carboxymethyl cellulose and a serine protease inhibitor; the concentration of sodium carboxymethyl cellulose is 10 g / L to 30 g / L; and the concentration of the serine protease inhibitor is 0.005 mg / mL to 0.05 mg / mL.

[0076] In one embodiment, the enhancer is L-arginine or PEG 6000; the concentration of L-arginine is 18 g / L to 25 g / L; and the amount of PEG 6000 added is 10 g / L to 25 g / L.

[0077] In one embodiment, the color-enhancing agent is sodium thiosulfate; the concentration of sodium thiosulfate added is 1 g / L to 5 g / L.

[0078] In one embodiment, the sample buffer comprises: 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 0.5 g / L to 1.2 g / L EDTA, 2 g / L to 8 g / L Tween 20, 4 g / L to 10 g / L BSA, 5 g / L to 20 g / L goat serum, 10 g / L to 30 g / L sodium carboxymethyl cellulose, 0.005 mg / mL to 0.05 mg / mL serine protease inhibitor, and 0.5 g / L to 2 g / L Proclin 300.

[0079] In one embodiment, the washing buffer stock solution comprises 50 g / L to 120 g / L Tris, 60 g / L to 90 g / L NaCl, 10 g / L to 25 g / L Tween 20, 5 g / L to 20 g / L Tween 80, and 0.5 g / L to 2 g / L Proclin 300.

[0080] In one embodiment, the enzyme conjugation stock solution comprises 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 8 g / L to 25 g / L sucrose, 0.3 g / L to 2 g / L polyoxyethylene lauryl ether, 0.2 g / L to 1 g / L magnesium chloride, 0.01 g / L to 0.05 g / L zinc chloride, 10 g / L to 30 g / L BSA, 18 g / L to 25 g / L L-arginine, 10 g / L to 25 g / L PEG 6000, 0.02 g / L to 0.1 g / L gentamicin sulfate, and 0.5 g / L to 2 g / L Proclin 300.

[0081] In one embodiment, the substrate solution comprises 8 g / L to 30 g / L Tris, 3 g / L to 8 g / L NaCl, 0.1 g / L to 0.25 g / L NBT, 0.02 g / L to 0.15 g / L BCIP, 0.1 g / L to 0.5 g / L magnesium chloride, 1 g / L to 5 g / L sodium thiosulfate, and 0.5 g / L to 2 g / L Proclin 300.

[0082] In one embodiment, the coating solution comprises 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, 0.5 g / L to 2 g / L Proclin 300, and zwitterionic buffer.

[0083] In one embodiment, the zwitterionic buffer comprises 6 g / L to 20 g / L Tris buffer, 15 g / L to 40 g / L Bis-Tris buffer, 10 g / L to 40 g / L MOPS buffer, 1 g / L to 10 g / L anhydrous sodium carbonate, 1 g / L to 10 g / L CBS buffer, and 1 g / L to 10 g / L PB buffer.

[0084] Example 2: Preparation of the reagent kit of this application

[0085] (1) Preparation of sample buffer

[0086] Add 6 g / L to 9 g / L NaCl, 0.5 g / L to 1.2 g / L EDTA, 2 g / L to 8 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to a suitable container in the following order to a Tris buffer solution (5 g / L to 12 g / L). Stir slowly until completely dissolved. Adjust the pH to 7.4. Add 4 g / L to 10 g / L BSA and dilute to volume with water. Filter through a 0.22 μm filter. Then add 5 g / L to 20 g / L goat serum, 10 g / L to 30 g / L sodium carboxymethyl cellulose, and 0.005 mg / mL to 0.05 mg / mL serine protease inhibitor. Stir slowly until completely dissolved and store in a cold storage at 2–8 °C.

[0087] In this embodiment, the concentration of sodium carboxymethyl cellulose added was 10 g / L to 30 g / L, and the concentration of serine protease inhibitor added was 0.005 mg / mL to 0.05 mg / mL.

[0088] The sample buffer in this application has the following two advantages compared with ordinary sample buffer: (1) Sodium carboxymethyl cellulose can significantly enhance specific reactions and reduce the incidence of non-specific reactions; (2) The addition of serine protease inhibitors can significantly improve the stability of proteins in the sample buffer.

[0089] The differences between the sample buffer and the ordinary buffer in this embodiment are compared. Taking the positive and negative concordance rate of histone indicators as an example, the results are shown in Table 1. The positive and negative concordance rate of the sample buffer in this application is significantly higher than that of the ordinary sample buffer, and the average value is higher than the detection value of the marketed products.

[0090] Table 1. Results of positive and negative concordance rates for histone indicators.

[0091]

[0092] (2) Preparation of the washing buffer stock solution:

[0093] Add 60 g / L to 90 g / L NaCl, 10 g / L to 25 g / L Tween 20, 5 g / L to 20 g / L Tween 80, and 0.5 g / L to 2 g / L Proclin 300 to a 50 g / L to 120 g / L Tris buffer solution in that order. Stir slowly until completely dissolved, adjust the pH to 7.4, add water to bring the volume to a final depth, filter through a 0.22 μm filter, and store in a cold storage at 2 to 8 °C.

[0094] (3) Preparation of enzyme conjugate stock solution:

[0095] Add the following in sequence to a Tris buffer solution (5 g / L–12 g / L): 6 g / L–9 g / L NaCl, 8 g / L–25 g / L sucrose, 0.3 g / L–2 g / L polyoxyethylene lauryl ether, 0.2 g / L–1 g / L magnesium chloride, 0.01 g / L–0.05 g / L zinc chloride, 0.02 g / L–0.1 g / L gentamicin sulfate, and 0.5 g / L–2 g / L Proclin 300. Stir slowly until completely dissolved. Adjust the pH to 8.0, add 10 g / L–30 g / L BSA, and dilute to volume with water. Then add 18 g / L–25 g / L L-arginine and 10 g / L–25 g / L PEG 6000. Stir slowly until completely dissolved, filter through a 0.22 μm filter, and store in a cold storage at 2–8 °C.

[0096] (4) Preparation of substrate solution:

[0097] Add the following in sequence to a Tris buffer solution (8 g / L–30 g / L): 3 g / L–8 g / L NaCl, 0.1 g / L–0.25 g / L NBT, 0.02 g / L–0.15 g / L BCIP, 0.1 g / L–0.5 g / L magnesium chloride, and 0.5 g / L–2 g / L Proclin 300. Stir slowly until completely dissolved. Adjust the pH to 9.6, add water to make up the volume, then add 1 g / L–5 g / L sodium thiosulfate. Stir slowly until completely dissolved, then filter through a 0.22 μm filter and store in a cold storage at 2–8 °C.

[0098] (5) Preparation of membrane strips

[0099] 1) Preparation of antigen coating solution and quality control band coating solution

[0100] Tris antigen coating solution: Add 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to 6 g / L to 20 g / L Tris buffer in sequence. Stir at low speed until completely dissolved, adjust the pH to 9.58, add water to make up to volume, filter at 0.22 μm, and store in a cold storage at 2 to 8 °C.

[0101] CBS antigen coating solution: Add 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to 1 g / L anhydrous sodium carbonate and 1 g / L to 10 g / L sodium bicarbonate buffer, respectively, and stir at low speed until completely dissolved. Adjust the pH to 9.6, add water to make up to the final volume, filter at 0.22 μm, and store in a cold storage at 2–8 °C.

[0102] MOPS antigen coating solution: Add 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to 10 g / L to 40 g / L MOPS buffer in sequence, stir at low speed until completely dissolved, adjust the pH to 7.5, add water to make up to volume, filter at 0.22 μm, and store in a cold storage at 2 to 8 °C.

[0103] Bis-Tris antigen coating solution: Add 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to 15 g / L to 40 g / L Bis-Tris buffer in sequence, stirring at low speed until completely dissolved. Adjust the pH to 7.5, add water to make up to the final volume, filter at 0.22 μm, and store in a cold storage at 2–8 °C.

[0104] PB quality control tape coating solution: Add 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, and 0.5 g / L to 2 g / L Proclin 300 to 1 g / L disodium hydrogen phosphate buffer in sequence, stirring at low speed until completely dissolved. Adjust the pH to 7.5, add water to make up to volume, filter at 0.22 μm, and store in a cold storage at 2–8 °C.

[0105] 2) Scraping of 15 antigens and quality control bands

[0106] Based on the amount of membrane strips prepared, calculate the required amount of each antigen and the amount of quality control strips, add the corresponding coating solution, stir evenly at low speed, and then perform membrane etching at a humidity of 45% to 60% and a temperature of 18℃ to 28℃.

[0107] 3) Drying and film application

[0108] Except for nucleosomes and dsDNA, which are placed at 60℃~85℃ for 25min~50min, all other antigens are placed at 37℃ for 12h~24h. After drying, they are placed in a resealable bag containing desiccant and stored at 2~8℃. Before lamination, the resealable bag is removed and lamination is performed at a humidity of 20%~30%.

[0109] 4) Membrane cutting and inner wrapping

[0110] Example 3: Detection method of the reagent kit of this application

[0111] Using the Bangqi Fully Automated Immunoblot Analyzer (model: BQ100) as the detection tool, the specific steps are as follows:

[0112] (1) Preparation for testing

[0113] Equilibrate to room temperature: Remove the test kit and equilibrate to 18–26°C, then remove the membrane strip;

[0114] Washing buffer stock solution, diluted 10 times: 30mL washing buffer stock solution + 270mL distilled water;

[0115] Enzyme conjugate stock solution, 10-fold dilution: 2 mL enzyme conjugate stock solution + 18 mL sample diluent.

[0116] (2) Instrument Operation

[0117] 1) Pre-filling: After cleaning and emptying the instrument tubing, pre-fill the tubing with sample buffer, diluted washing buffer, diluted enzyme conjugate, and substrate solution to fill the entire tubing. Place the membrane tray in the designated position on the instrument, remove the membrane strip with tweezers, and place it into the tray with the numbers facing up. Add 1.5 mL of sample buffer to each tray and incubate at room temperature for 3 minutes.

[0118] 2) Antigen and antibody incubation for 30 min: After aspirating the liquid from the tank with the pipette tip, add 15 μL of quality control sample and 1.5 mL of sample buffer respectively, and shake for 25 min;

[0119] 3) Cleaning for 7.5 min: After removing the liquid from the tank with a pipette tip, clean the membrane strip three times with 1.5 mL of diluted cleaning buffer on a shaking table, each time for 2.5 min;

[0120] 4) Add diluted enzyme conjugate (secondary antibody): Add 1.5 mL of diluted enzyme conjugate and incubate for 20 min to form antigen-antibody (1) st Antibody) - Goat anti-human IgG (2 nd Antibody complex;

[0121] 5) Cleaning for 7.5 min: After removing the liquid from the tank with a pipette tip, clean the membrane strip three times with 1.5 mL of diluted cleaning buffer on a shaking table, each time for 2.5 min;

[0122] 6) Add substrate solution: Aspirate the liquid from the tank and add 1.5 mL of substrate solution to each tank. Shake at room temperature for 8 minutes;

[0123] 7) Terminate the reaction with distilled water: After removing the liquid from the tank, rinse once with 1.5 mL of distilled water, and then let the instrument air dry automatically for 30 minutes.

[0124] 8) Automatic instrument interpretation

[0125] Comparison of manufacturers: Detection methods for commercially available reagent kits:

[0126] Using the commercially available fully automated immunoblotting instrument (model: EUROLiNineMasterPlus) as the testing tool, the specific steps are as follows:

[0127] (1) Preparation for testing

[0128] Equilibrate to room temperature: Remove the test kit and equilibrate to 18–25°C, then remove the membrane strip;

[0129] Washing buffer stock solution, diluted 10 times: 30mL washing buffer stock solution + 270mL distilled water;

[0130] Enzyme conjugate stock solution, 10-fold dilution: 2 mL enzyme conjugate stock solution + 18 mL sample diluent.

[0131] (2) Instrument Operation

[0132] 1) Pre-filling: After cleaning and emptying the instrument tubing, pre-fill the tubing with sample buffer, diluted wash buffer, diluted enzyme conjugate, and substrate solution to fill the entire tubing. Place the membrane tray in the designated position on the instrument, remove the membrane strip with tweezers, and place it into the tray with the numbers facing up. Add 1.5 mL of sample buffer to each tray and incubate at room temperature for 5 minutes.

[0133] 2) Antigen and antibody incubation for 30 min: After aspirating the liquid from the tank with the pipette tip, add 15 μL of quality control sample and 1.5 mL of sample buffer respectively, and shake for 30 min;

[0134] 3) Cleaning for 15 min: After removing the liquid from the tank with a pipette tip, clean the membrane strip three times with 1.5 mL of diluted cleaning buffer on a shaking table, each time for 5 min;

[0135] 4) Add diluted enzyme conjugate (secondary antibody): Add 1.5 mL of diluted enzyme conjugate and incubate for 30 min to form antigen-antibody (1) st Antibody) - Goat anti-human IgG (2 nd Antibody complex;

[0136] 5) Cleaning for 15 min: After removing the liquid from the tank with a pipette tip, clean the membrane strip three times with 1.5 mL of diluted cleaning buffer on a shaking table, each time for 5 min;

[0137] 6) Add substrate solution: Remove the liquid from the tank and add 1.5 mL of substrate solution to each tank. Shake at room temperature for 10 min;

[0138] 7) Terminate the reaction with distilled water: After removing the liquid from the tank, rinse with 1.5 mL of distilled water 3 times, 1 minute each time, and then let the instrument air dry automatically for 30 minutes.

[0139] 8) Automatic instrument interpretation

[0140] Example 4: Performance evaluation of the reagent kit of this application

[0141] Clinical samples were tested using the method described in Example 3.

[0142] (1) LOD detection

[0143] Taking histone (His) index as an example, the LOD was detected. The results are shown in Table 2. After the mixed serum was diluted to 1 / 128, both this kit and the commercially available reagents reached the gray zone. Therefore, it can be concluded that the LOD detection limit is at a dilution rate of 1 / 64. Thus, the detection limits of this kit and the commercially available reagents are consistent.

[0144] The LOD values ​​for the other 14 indicators are also consistent with those of commercially available test kits.

[0145] Table 2 LOD Detection Results

[0146]

[0147] 2) Sensitivity detection

[0148] Experimental methods: Positive samples for mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE), polymyositis (PM), dermatomyositis (DM), systemic sclerosis (SSc), Sjögren's syndrome (SS), and primary biliary cirrhosis (PBC) were confirmed using commercially available reagents. Positive samples for different diseases were then tested using Bangqi products. The ratio of the number of positive samples detected by Bangqi reagents to the total number of positive samples (i.e., sensitivity) was used.

[0149] The experimental results are shown in Table 3. The sensitivity for primary biliary cirrhosis is as high as 100%, and the sensitivity for mixed connective tissue disease, systemic lupus erythematosus, and Sjögren's syndrome is all above 90%, with an average value close to 90%.

[0150] Table 3 Sensitivity Detection

[0151] sample Sample size Positive Sensitivity / % Mixed connective tissue disease (MCTD) 37 34 91.89％ Systemic lupus erythematosus (SLE) 129 120 93.02％ Polymyositis (PM) 28 20 71.43％ Dermatomyositis (DM) 10 6 60％ Systemic sclerosis (Ssc) 65 52 80％ Sjögren's syndrome (SS) 44 43 97.73％ Primary biliary cirrhosis (PBC) 30 30 100％ total 343 305 88.92％

[0152] 3) Specific detection

[0153] Experimental methods: Negative samples for rheumatoid arthritis, gluten intolerance, and Wegener's granulomatosis (PBC) were confirmed using commercially available reagents. Negative samples for each disease were tested using Bangqi products. The ratio of the number of negative samples detected by Bangqi reagents to the total number of negative samples (i.e., specificity) was calculated.

[0154] The experimental results are shown in Table 4. The specificity for Wegener's granulomatosis and gluten intolerance is as high as 100%, with an average value of 94.71%.

[0155] Table 4 Specificity Detection

[0156] sample Sample size Negative Specificity / % rheumatoid arthritis 126 119 94.44％ gluten allergy 14 14 100％ Wegener's granulomatosis 10 10 100％ Other autoimmune diseases 77 72 93.51％ total 227 215 94.71％

[0157] 4) Precision testing

[0158] Intra-batch precision test method: Repeat the test 10 times using the enterprise precision reference, and control the CV within 15%.

[0159] Inter-batch precision test method: Take 3 batches of reagents and test the same precision reference sample. Repeat each batch 10 times and control the CV within 15%.

[0160] The experimental results are shown in Tables 5 and 6. The intra-batch precision was between 7% and 13%, and the inter-batch precision was between 9% and 14%.

[0161] Table 5 Precision Testing

[0162] Value of products already on the market nucleosomes dsDNA Centrifuge point M2 P0 PCNA Jo-1 Serum 1(+) 14 15 13 12 12 13 12 Intra-batch precision 9％ 12％ 13％ 7％ 12％ 10％ 11％ Inter-batch precision 13％ 12％ 14％ 11％ 10％ 13％ 12％ Serum 2(++) 28 29 27 26 28 28 32 Intra-batch precision 10％ 8％ 9％ 11％ 12％ 11％ 8％ Inter-batch precision 14％ 12％ 10％ 13％ 11％ 11％ 13％

[0163] Table 6 Precision Testing

[0164]

[0165]

[0166] 5) Linear detection

[0167] Experimental Method: Dilute the high-concentration sample (close to the upper limit of linearity) to at least five concentrations (Xi), one of which should be close to the lower limit of linearity. Test each kit separately, performing three tests at each dilution, and calculate the mean (Yi) of the results. Using the theoretical dilution concentration (Xi) as the independent variable and the mean (Yi) of the results as the dependent variable, derive the linear regression equation and calculate the correlation coefficient (r) according to the formula, as shown in the table below.

[0168] The results are shown in Table 7 and... Figure 1 As shown, the sample values ​​range from 0.56 to 143.51, with a relative deviation of ≤±5%, and the linear correlation coefficient R is 0.9434, which is consistent with the measured value R of the marketed product of 0.9288.

[0169] Table 7 Linearity Detection Results

[0170]

[0171] 6) Interference detection

[0172] Mixed serum samples were prepared and various interfering agents were added, including bilirubin, hemoglobin, ascorbic acid, triglycerides, and RF. Taking histone (His) as an example, the values ​​of the mixed serum and the mixed serum with each interfering agent were measured separately, and the deviation between the two was calculated, with ±10% considered acceptable. The results are shown in Table 8. All 15 indicators met the NCCLS document standards under interfering agent assays and can be used for accurate assessment of antinuclear antibody status in clinical laboratories.

[0173] Table 8 Interference Detection Results

[0174]

[0175] 7) Methodological Comparison Experiment

[0176] Using this kit and commercially available kits, 21 clinical samples were tested. Taking histones, nucleosomes, and dsDNA as examples, Table 9-11 shows that the results of this kit are 100% consistent with those of commercially available products.

[0177] Table 9. Results of the comparative experiment on the methodology of histone indicators.

[0178]

[0179]

[0180] Table 10 Results of the comparative experiment on nucleosome index methodologies

[0181]

[0182]

[0183] Table 11 Comparative Experiment Results of dsDNA Index Methodology

[0184]

[0185]

[0186] This kit has the following advantages: firstly, the total reaction time of this kit is relatively short; secondly, it has high accuracy, especially for dsDNA detection.

[0187] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any non-substantial modifications made to this application using this concept shall be considered an infringement of the scope of protection of this application.

Claims

1. A combined detection kit for antinuclear antibody profiles, characterized in that, This includes detection membrane strips, enzyme conjugation solution, sample buffer, washing buffer, substrate solution, and coating solution for coating the detection membrane strips; The antigens coated on the detection membrane strips are: nRNP / Sm, Sm, SS-A, SS-B, Scl-70, PM-Scl, Jo-1, CENP-B, PCNA, dsDNA, nucleosomes, histones, ribosomal P protein, M2, and Ro52. The sample buffer solution consisted of: 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 0.5 g / L to 1.2 g / L EDTA, 2 g / L to 8 g / L Tween 20, 4 g / L to 10 g / L BSA, 5 g / L to 20 g / L goat serum, 10 g / L to 30 g / L sodium carboxymethyl cellulose, 0.005 mg / mL to 0.05 mg / mL serine protease inhibitor, and 0.5 g / L to 2 g / L Proclin 300. The cleaning buffer solution includes a buffer solution, an electrolyte, a surfactant, and a preservative. The enzyme conjugate solution includes a buffer solution, electrolytes, enzyme stabilizers and enhancers, and preservatives; The substrate solution includes a buffer solution, electrolyte, stabilizer, colorimetric enhancer, and preservative. The coating solution includes three or more zwitterionic buffers, electrolytes, surfactants, and preservatives.

2. The detection kit according to claim 1, characterized in that, The electrolyte is NaCl or MgCl₂. 2、 One or more of KCl and ZnCl2; The surfactant is one or more of Tween 20, Tween 80, Triton X-100, NP40, and SDS; The stabilizer is one or more of BSA, gelatin, EDTA, Scavenger-ALP, and trehalose; The preservative is any one or more of Proclin 300, MB-1, sodium azide, and gentamicin sulfate.

3. The detection kit according to claim 1, characterized in that, The enhancing agent is L-arginine and PEG6000; The concentration of L-arginine added is 18 g / L to 25 g / L; the amount of PEG 6000 added is 10 g / L to 25 g / L.

4. The detection kit according to claim 1, characterized in that, The color-enhancing agent is sodium thiosulfate; the concentration of sodium thiosulfate added is 1 g / L to 5 g / L.

5. The detection kit according to claim 1, characterized in that, The washing buffer consists of 50 g / L to 120 g / L Tris, 60 g / L to 90 g / L NaCl, 10 g / L to 25 g / L Tween 20, 5 g / L to 20 g / L Tween 80, and 0.5 g / L to 2 g / L Proclin 300. The enzyme conjugate solution includes 5 g / L to 12 g / L Tris, 6 g / L to 9 g / L NaCl, 8 g / L to 25 g / L sucrose, 0.3 g / L to 2 g / L polyoxyethylene lauryl ether, 0.2 g / L to 1 g / L magnesium chloride, 0.01 g / L to 0.05 g / L zinc chloride, 10 g / L to 30 g / L L-BSA, 18 g / L to 25 g / L L-arginine, 10 g / L to 25 g / L PEG 6000, 0.02 g / L to 0.1 g / L gentamicin sulfate, and 0.5 g / L to 2 g / L Proclin 300. The substrate solution includes 8 g / L to 30 g / L Tris, 3 g / L to 8 g / L NaCl, 0.1 g / L to 0.25 g / L NBT, 0.02 g / L to 0.15 g / L BCIP, 0.1 g / L to 0.5 g / L magnesium chloride, 1 g / L to 5 g / L sodium thiosulfate, and 0.5 g / L to 2 g / L Proclin 300; The coating solution includes 5 g / L to 20 g / L NaCl, 10 g / L to 50 g / L trehalose, 0.05 g / L to 1 g / L Tween 20, 0.5 g / L to 2 g / L Proclin 300, and zwitterionic buffer.

6. A method for preparing an antinuclear antibody spectrum combined detection kit according to any one of claims 1 to 5, characterized in that, The preparation steps include the following: (1) Prepare enzyme conjugate solution, sample buffer, washing buffer, and substrate solution respectively; (2) Prepare antigen coating solution and quality control band coating solution respectively; (3) Antigen and quality control strips were etched onto the membrane; (4) Drying and film application; (5) Cutting the membrane and wrapping the inner membrane.

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