A method for preparing a highly immunogenic native dsDNA antigen

Abstract

The present application relates to the technical field of biology, and in particular to a method for preparing natural dsDNA antigen with high immunogenicity. The method for separating and purifying natural dsDNA antigen from biological tissues provided by the present application comprises the following steps: firstly, preparing a crude dsDNA antigen through lysis digestion and phenol chloroform extraction; and then, further removing impurities through CM cation exchange chromatography and dextran-agarose composite gel filtration medium 200 molecular sieve chromatography. The prepared dsDNA antigen has high yield, strong immunogenicity, good sensitivity and specificity, and is more suitable for a diagnostic method with high requirements for the quality of diagnostic raw materials, such as chemiluminescence.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and specifically to a method for preparing a highly immunogenic natural dsDNA antigen. Background Technology

[0002] Autoimmune diseases are illnesses caused by damage to the body's own tissues due to an immune response to autoantibodies. First proposed by Donath and Landsteiner, they are characterized by high titers of autoantibodies and the presence of sensitized lymphocytes, encompassing dozens of diseases. Common autoimmune diseases are generally divided into three categories: the first category primarily manifests as joint pain, such as rheumatoid arthritis and ankylosing spondylitis; the second category is connective tissue diseases represented by systemic lupus erythematosus and Sjögren's syndrome; and the third category is systemic vasculitis primarily manifested as vascular involvement. Currently, the pathogenesis of autoimmune diseases is not fully understood. It is generally believed that in genetically susceptible individuals, the combined effects of multiple environmental factors such as infection, ultraviolet radiation, tumors, and drugs lead to abnormal autoimmune responses, resulting in pathological immune responses against self-antigens and causing organ or system damage.

[0003] Systemic lupus erythematosus (SLE) is a relatively common systemic autoimmune disease that primarily affects connective tissue and multiple organ systems. It commonly affects young and middle-aged women and presents with diverse clinical manifestations, including various autoantibodies, primarily antinuclear antibodies (ANA), widespread small artery lesions, and multi-system involvement. Renal failure, brain damage, and heart failure are common causes of death in SLE patients. Antinuclear antibodies (ANA), also known as anti-nucleic acid antigen antibodies, are a group of autoantibodies against DNA, RNA, proteins, or molecular complexes of these substances within the cell nucleus. ANA shows no species or organ-specific differences; most are IgG antibodies, with only a small portion belonging to IgM and IgA. The target antigens of ANA, such as intracellular nucleic acids and nucleoprotein components, play important roles in cellular physiological activities; therefore, specific detection of ANA is of significant reference value for the definitive diagnosis of the disease. Currently, more than 20 types of antinuclear antibodies (ANAs) with different clinical significance have been discovered, forming a preliminary systematic spectrum of antinuclear antibodies (ANAs). These ANAs have important clinical value in the diagnosis, differential diagnosis, assessment of treatment efficacy, and prognosis of diseases such as systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS), systemic sclerosis (SSc), polymyositis / dermatomyositis, and primary biliary cirrhosis (PBC).

[0004] Anti-double-stranded DNA antibodies (dsDNA antibodies) are a type of anti-DNA antibody. Arana discovered them in the serum of SLE patients in 1967. Because they are autoantibodies that can bind to natural DNA, they are called natural DNA antibodies or dsDNA antibodies. Anti-dsDNA antibodies are mainly found in the serum of SLE patients, and the antibody titer is correlated with SLE activity. In the blood circulation of SLE patients, the concentration of large DNA molecules is significantly higher than in normal individuals. DNA can also bind to microvascular structures in various organs, including heparan sulfate on the glomerular basement membrane. Anti-dsDNA antibodies can react with these circulating and in-situ organ DNA molecules to form antigen-antibody complexes, activating inflammatory systems such as the complement activation pathway, leading to tissue damage. Therefore, anti-double-stranded DNA antibodies are a serological marker for systemic lupus erythematosus (SLE).

[0005] Currently, methods for detecting dsDNA antibodies include radioimmunoassay, indirect immunofluorescence, colloidal gold assay, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Radioimmunoassay is increasingly being phased out due to risks such as radioactive contamination. Indirect immunofluorescence results are heavily influenced by subjective human factors and require specialized equipment such as fluorescence microscopes, making large-scale implementation difficult. Colloidal gold assays are only qualitative, with poor sensitivity and susceptibility to interference, insufficient for clinical diagnostic needs. Western blotting can result in antigen loss or denaturation during the transfer process, leading to undeveloped or unclear bands, making results difficult to observe and prone to false positives or missed detections, thus failing to meet diagnostic requirements. ELISA offers quantitative detection, but due to the difficulty in obtaining stable and highly immunoreactive dsDNA antigens, the detection of anti-dsDNA antibodies often suffers from poor specificity, relatively low sensitivity and accuracy, making it impossible to guarantee detection sensitivity and accuracy.

[0006] Therefore, obtaining double-stranded DNA antigens with high purity and immunoreactivity is of great significance for improving the sensitivity and accuracy of detection methods with high diagnostic quality requirements, such as chemiluminescence immunoassay. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide a method for preparing highly immunogenic natural dsDNA antigens. The antigens prepared by this method can meet the requirements of diagnostic raw materials in terms of purity, activity, specificity and stability, and the immunogenicity is far higher than that of commercially available similar products.

[0008] This invention achieves the above-mentioned technical objective through the following technical solution: a method for preparing highly immunogenic natural dsDNA antigen, comprising the following steps:

[0009] Add lysis buffer, proteinase K and RNase A to the tissue sample, homogenize and place in a water bath at 45-60℃ for digestion, centrifuge and collect the supernatant;

[0010] Extraction reagent was added to the supernatant according to the volume ratio of sample: saturated phenol: chloroform: isopropanol 50:(25~40):(32~40):(1~2), and the supernatant was collected. After precipitation with ethanol, the crude dsDNA antigen was obtained.

[0011] The crude dsDNA antigen was purified by sequentially passing it through CM cation exchange chromatography and dextran-agarose composite gel filtration medium 200 molecular sieve chromatography.

[0012] The method for isolating and purifying natural dsDNA antigens from biological tissues provided by this invention first prepares crude dsDNA antigens through lysis digestion and phenol-chloroform extraction. Then, it is further purified by CM chromatography and dextran-agarose composite gel filtration medium 200 molecular sieve chromatography to remove impurities. The prepared dsDNA antigens have high yield, strong immunogenicity, good sensitivity and specificity, and small batch-to-batch variability, making them more suitable for diagnostic methods such as chemiluminescence, which have high requirements for the quality of diagnostic raw materials.

[0013] In a preferred embodiment, the buffer solution used when packing the CM cation exchange chromatography column is 10–50 mM Hepes, 0.5–5 mM EDTA, 0.05–0.5 M NaCl, and pH 5.0–10.0.

[0014] As a preferred embodiment, the chromatography column used for CM cation exchange chromatography is CM-6FF, and the molecular sieve chromatography using dextran-agarose composite gel filter medium 200 is Prepack-GL26 / 600Finedex 200.

[0015] As a preferred embodiment, the buffer solution used when packing the 200 molecular sieve chromatography column with dextran-agarose composite gel filter media is 5-50 mM Tris-HCl, 0.5-5 mM EDTA, pH 4.5-9.0.

[0016] As a preferred embodiment, the lysis buffer is: 5-50 mM Tris-HCl, 0.5-5% SDS, 0.1-1.0% Triton, and 0.1-1.0 M NaCl.

[0017] The tissue samples include, but are not limited to, calf thymus tissue, frog fish essence, and HeLa cells.

[0018] As a preferred embodiment, when the tissue sample contains connective tissue, it is necessary to remove the connective tissue through pretreatment. The specific steps are: homogenization and filtration, lysis and digestion, chloroform extraction, CM cation exchange chromatography, and dextran-agarose composite gel filtration medium 200 molecular sieve chromatography.

[0019] As a preferred embodiment, the pretreatment method is as follows: add homogenization buffer and filter with gauze; wherein the homogenization buffer is: 10-100mM Tris-HCl, 0-0.1M NaCl, pH 5.0-8.5.

[0020] Another object of this invention is to protect the application of the dsDNA prepared by the above method in the preparation of clinical auxiliary diagnostic kits for systemic lupus erythematosus (SLE), an autoimmune disease. The dsDNA antigen prepared by this invention is suitable for detection by ELISA, chemiluminescence, immunoturbidimetry, etc., including but not limited to the clinical auxiliary diagnosis of autoimmune diseases such as SLE. Attached Figure Description

[0021] Figure 1 This is the DEAE tomography spectrum in Comparative Example 1 of the present invention;

[0022] Figure 2 This is a comparison of the detection results of the antigen prepared in Comparative Example 1 of this invention before and after purification and the Sigma antigen on different serums.

[0023] Figure 3 The results of SDS-PAGE analysis of the dsDNA antigens prepared in Examples 1 and 2 and commercially available products are shown.

[0024] Figure 4 The results of agarose gel electrophoresis detection of the dsDNA antigens prepared in Examples 1 and 2 and commercially available products are shown.

[0025] Figure 5 The results show the ELISA titers of the dsDNA antigens prepared in Examples 1 and 2 and commercially available products at different coating concentrations. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0027] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0028] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0029] Double-stranded DNA is a double helix structure formed by two antiparallel chains of deoxyribonucleotides. The outer side of the DNA structure consists of a basic backbone of alternating deoxyribose and phosphate groups, while the inner side consists of base pairs linked by hydrogen bonds. Base pairing follows the complementary base pairing rule (AT, CG). In normal cells, the hydrogen bonds between base pairs are broken, requiring the action of helicases. The higher the proportion of G and C base pairs in DNA, the more stable the structure, and the higher the temperature required for complete unwinding into single strands.

[0030] For the preparation of dsDNA antigens, the base ratio in animal DNA is closer to that in human DNA. Calf thymus cells have a high proportion of nuclei and high DNA content, and low deoxyribonuclease activity. Therefore, calf thymus tissue is easy to extract, has low raw material cost, and is suitable for large-scale production. The method provided by this invention is particularly suitable for the large-scale extraction and production of this tissue material.

[0031] Example 1

[0032] This embodiment provides a method for extracting natural dsDNA antigen from calf thymus. The animal tissue is homogenized, lysed, and extracted with phenol-chloroform. After dissolution with dsDNA dissolution buffer and centrifugation, the precipitate is removed by CM cation exchange column purification, and finally purified by molecular sieve chromatography to obtain 20.4 mg of dsDNA antigen. Detailed steps are as follows:

[0033] (1) After removing connective tissue and other impurities from fresh calf thymus, weigh 25.0g and add 37.5mL of homogenization buffer at a mass-to-volume ratio of 1:1.5. Homogenize multiple times using a homogenizer (5s each time, 5s pause) until no lumps remain. Then, filter the homogenized thymus through a 50-mesh filter bag to remove excess tissue. The homogenization buffer consisted of 50mM Tris-HCl, 30mM NaCl, and pH 7.5.

[0034] (2) Add the filtered sample to a 50mL centrifuge tube, balance it, centrifuge at 5000rpm for 20min, carefully discard the supernatant, collect the centrifuged precipitate, and weigh the precipitate.

[0035] (3) Add tissue lysis buffer to the precipitate at a mass-to-volume ratio of 1:1.5, followed by proteinase K and RNase A to a final concentration of 100 μg / mL. Homogenize the mixture using a hand homogenizer and place it in a 60℃ water bath for 1.5 h, stirring every 15 min during this time. After the water bath, transfer the sample to a 50 mL centrifuge tube, balance the solution, and centrifuge at 5000 rpm for 20 min. Carefully collect the supernatant. The lysis buffer consisted of 10 mM Tris-HCl, 1% SDS, 0.5% Triton, and 0.5 M NaCl.

[0036] (4) Weigh the volume of the supernatant and add the sample, Tris saturated phenol, chloroform and isopropanol in the following order according to the volume ratio of 50:37.5:36:1.5. Mix by inversion and dispense into 50mL high-speed centrifuge tubes. After balancing, centrifuge at 22000rpm and 4℃ for 15min. After centrifugation, the sample can be seen to be divided into three layers: the upper layer is a clear and transparent aqueous phase, the middle layer is a milky white protein layer and the lower layer is pale yellow. Carefully transfer the upper aqueous phase to a graduated cylinder and weigh the volume.

[0037] (5) Add 2.5 times the volume of 95% ethanol to a graduated cylinder, mix well, and dispense into 50mL high-speed centrifuge tubes. Balance the mixture and centrifuge at 22000rpm at 4℃ for 15min. Carefully discard the supernatant after centrifugation. Add 20mL of 75% ethanol to each tube and pipette the precipitate. Centrifuge again at 22000rpm at 4℃ for 15min. Repeat this operation, collect the precipitate after centrifugation, add an appropriate volume of dsDNA dissolution buffer, and gently shake to dissolve. The dsDNA dissolution buffer is: 20mM HEPES, 1mM EDTA, pH=8.0.

[0038] (6) After dissolution, the sample was filtered through a 0.45 μm filter and then loaded onto a CM-6FF cation exchange column (Wuhan Huiyan Biotechnology Co., Ltd.). During loading, the sample eluent was collected when the absorbance A260 rose to 0.01. After loading, the sample was washed with chromatography initiation buffer until the absorbance dropped to 0.01, at which point collection was stopped; this eluent, approximately 25 mL in volume, was obtained. The buffer used during column packing consisted of 20 mM HEPES, 1 mM EDTA, 0.15 M NaCl, and pH = 8.0.

[0039] (7) The eluent was further purified in batches by Prepack-GL 26 / 600 Finedex 200 (Jiangsu Qianchun Biotechnology Co., Ltd.) molecular sieve chromatography, and 5 mL of eluent was collected per tube. The molecular sieve buffer consisted of 10 mM Tris-HCl, 1 mM EDTA, and pH 7.5. The samples after molecular sieve chromatography were then subjected to protein electrophoresis and nucleic acid agarose gel electrophoresis for detection.

[0040] In this embodiment, 25g of fresh calf thymus was used as raw material. The purified dsDNA antigen was obtained by homogenization, lysis, extraction and chromatography. The A260 was measured to be 408.0 by ultraviolet light detector. Based on the calculation that 20 corresponds to 1mg of dsDNA antigen, a total of 20.4mg of dsDNA antigen was obtained.

[0041] The dsDNA antigen prepared in Example 1 was tested for stability. Multiple samples were placed in an environment of 37 degrees Celsius for 72 hours and then detected by ELISA. The results showed no statistically significant difference compared with those before stabilization. The dsDNA antigen was prepared multiple times according to the method of Example 1, and the coefficient of variation (CV) between batches was less than 15%.

[0042] Example 2

[0043] This embodiment provides a method for extracting natural dsDNA antigens from frog-fish sperm biological tissue. The biological tissue used is frog-fish sperm. Because frog-fish sperm lacks connective tissue, no sample pretreatment such as homogenization is required. The specific steps are as follows:

[0044] (1) Add 100 mL of tissue lysis buffer to 25.0 g of frog-fish sperm tissue, then add proteinase K and RNase A to a final concentration of 100 μg / mL. Homogenize the mixture using a hand homogenizer and place it in a 60℃ water bath for 1.5 h, stirring once every 15 min. After the water bath, transfer the sample to a 50 mL centrifuge tube, balance the solution, and centrifuge at 5000 rpm for 20 min. Carefully collect the supernatant. The lysis buffer consisted of 20 mM Tris-HCl, 5% SDS, 0.5% Triton, and 0.5 M NaCl.

[0045] (2) Weigh the volume of the supernatant and add the sample, Tris saturated phenol, chloroform and isopropanol in the following order according to the volume ratio of 50:37.5:36:1.5. Mix by inversion and dispense into 50mL high-speed centrifuge tubes. After balancing, centrifuge at 22000rpm and 4℃ for 15min. After centrifugation, the sample can be seen to be divided into three layers: the upper layer is a clear and transparent aqueous phase, the middle layer is a milky white protein layer and the lower layer is pale yellow. Carefully transfer the upper aqueous phase into a graduated cylinder and weigh the volume.

[0046] (3) Add 2.5 times the volume of 95% ethanol to a graduated cylinder, mix well, and dispense into 50mL high-speed centrifuge tubes. Balance the mixture and centrifuge at 22000rpm for 4℃ for 15min. Carefully discard the supernatant after centrifugation. Add 20mL of 75% ethanol to each tube and gently pipette to remove the precipitate. Centrifuge again at 22000rpm for 4℃ for 15min. Repeat this operation, collect the precipitate after centrifugation, add an appropriate volume of dsDNA dissolution buffer, and gently shake to dissolve. The dsDNA dissolution buffer is: 10mM HEPES, 1mM EDTA, pH=7.5.

[0047] (4) After dissolution, the sample was filtered through a 0.45 μm filter and then loaded onto a CM-6FF cation exchange column (Wuhan Huiyan Biotechnology Co., Ltd.). During loading, the sample eluent was collected when the A260 absorbance rose to 0.01. After loading, the sample was washed with chromatography initiation buffer until the absorbance dropped to 0.01, at which point collection was stopped; this eluent, approximately 25 mL in volume, was obtained. The buffer used during column packing consisted of 20 mM HEPES, 1 mM EDTA, 0.15 M NaCl, and pH = 8.0.

[0048] (5) The eluent was further purified in batches by Prepack-GL 26 / 600 Finedex 200 molecular sieve chromatography (Jiangsu Qianchun Biotechnology Co., Ltd.), and 5 mL of eluent was collected per tube. The molecular sieve buffer was: 10 mM Tris-HCl, 1 mM EDTA, pH = 7.5.

[0049] In this embodiment, 25g of fresh frogfish extract was used as raw material. The purified dsDNA antigen was obtained by lysis, extraction and chromatography. The A260 was measured to be 232.00 by ultraviolet light detector. Based on the calculation that 20 corresponds to 1mg of dsDNA antigen, a total of 11.6mg of dsDNA antigen was obtained.

[0050] Comparative Example 1

[0051] This comparative example provides a method for extracting natural dsDNA antigens from calf thymus, which is one of the improved extraction methods in the experiment. The specific steps are as follows:

[0052] (1) 25.0g of fresh calf thymus, after removing connective tissue and other impurities, was added to PBS buffer at a mass-to-volume ratio of 1:5 and homogenized multiple times using a homogenizer (5s each time, 5s pause) until no lumps remained. The homogenized thymus was then placed in a 50-mesh filter bag and squeezed to remove excess tissue. The PBS buffer consisted of 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, and pH 7.4.

[0053] (2) Add the filtered sample to a 50mL centrifuge tube, balance it, centrifuge at 5000rpm for 20min, carefully discard the supernatant, collect the centrifuged precipitate, and weigh the precipitate.

[0054] (3) Add tissue lysis buffer to the precipitate at a mass-to-volume ratio of 1:5, followed by proteinase K and RNase A to a final concentration of 100 μg / mL. Homogenize the mixture using a hand homogenizer and incubate at 55°C for 2 hours, stirring every 15 minutes. After the water bath, transfer the sample to a 50 mL centrifuge tube, balance the solution, and centrifuge at 5000 rpm for 20 minutes. Carefully collect the supernatant. The lysis buffer consisted of 10 mM Tris-HCl, 1% SDS, 0.5% Triton, and 0.5 M NaCl.

[0055] (4) Weigh the volume of the supernatant and add the sample, Tris saturated phenol, chloroform and isopropanol in the following order according to the volume ratio of 50:25:24:1. Mix by inverting and dispense into 50mL high-speed centrifuge tubes. After balancing, centrifuge at 18000rpm and 4℃ for 15min. After centrifugation, the sample can be seen to be divided into three layers: the upper layer is a clear and transparent aqueous phase, the middle layer is a milky white protein layer and the lower layer is pale yellow. Carefully transfer the upper aqueous phase to a graduated cylinder and weigh the volume.

[0056] (5) Add 2.5 times the volume of 95% ethanol to a graduated cylinder, mix well, and dispense into 50mL high-speed centrifuge tubes. Balance the mixture and centrifuge at 18000rpm for 15min at 4℃. Carefully discard the supernatant after centrifugation. Add 20mL of 75% ethanol to each tube and gently pipette to remove the precipitate. Centrifuge again at 18000rpm for 15min at 4℃. Repeat this operation, collect the precipitate after centrifugation, add an appropriate volume of TE buffer, and gently shake to dissolve and obtain the unpurified dsDNA antigen. The TE buffer is: 10mM Tris-HCl, 1mM EDTA, pH=7.5.

[0057] (6) The dissolved sample was filtered through a 0.45 μm filter and then loaded onto a DEAE anion exchange chromatography column (purchased from Wuhan Huiyan Biotechnology Co., Ltd.) for purification. The buffer used during column packing was 20 mM Tris-HCl, 1 mM EDTA, 0.15 M NaCl, pH 8.0. During elution, elution buffer 1 consisted of 20 mM Tris-HCl, 1 mM EDTA, 0.5 M NaCl, pH 8.0, and elution buffer 2 consisted of 20 mM Tris-HCl, 1 mM EDTA, 4 M NaCl, pH 8.0. After loading, elution with elution buffer 1 showed a distinct elution peak. Elution buffer 1 was collected, and elution with elution buffer 2 was continued. Subsequent elution peaks were not obvious. The DEAE chromatogram is shown below. Figure 1 As shown. By Figure 1 It can be seen that the target product appears in elution peak 1, the yield of the obtained sample is low, the volume is significantly increased, and the concentration is low.

[0058] Enzyme-linked immunosorbent assay (ELISA) was used to verify the reaction titers of natural dsDNA antigen with Sigma antigens against control samples 1, 2, and 3 containing human anti-double-stranded DNA antibodies at different coating antigen concentrations (0.50 and 2.00 ug / ml). The results are shown in Table 2 and... Figure 1 As shown.

[0059] Table 1 compares the detection results of the antigen prepared in Comparative Example 1 before and after purification and the Sigma antigen against different quality control products.

[0060]

[0061] From Table 1 and Figure 2 It can be seen that after further purification by anion exchange chromatography, the purified dsDNA antigen showed significant differences in detection results against the three screening positive sera (quality control 1, quality control 2, and quality control 3). The purified antigen titers against quality control 1 and quality control 3 were higher than those against the unpurified antigen, but quality control 2 did not change with the increase of antigen coating concentration at different coating concentrations. This indicates that the dsDNA antigen prepared by this method cannot meet the requirements of diagnostic raw materials.

[0062] Detection and Analysis

[0063] (1) Purity analysis

[0064] Simultaneously, the purity of purified natural dsDNA prepared in Examples 1 and 2 and purified dsDNA antigen (Product No: D4522) purchased from Sigma was compared in parallel. The experimental methods included polyacrylamide gel electrophoresis (SDS-PAGE, Coomassie blue staining), nucleic acid agarose gel electrophoresis (EB substitute Golden view), and then densitometry analysis of the electrophoretic gel results was performed using a BioRad ChemiDocMP imaging system.

[0065] SDS-PAGE test results are as follows: Figure 3 As shown, neither the dsDNA antigen purified by Sigma nor the antigen prepared in the examples of this application showed any protein bands, indicating that there was no obvious protein residue. The agarose gel electrophoresis results are as follows: Figure 4 As shown, the purified dsDNA antigen in the examples has the same band position as the product from Sigma, and the band is single with no obvious tailing degradation, indicating that the integrity of the dsDNA prepared by the present invention is comparable to that of commercially available products.

[0066] (2) Verification of sensitivity and specificity

[0067] Simultaneously, the purified natural dsDNA prepared in Examples 1 and 2 and the purified dsDNA antigen (Product No: D4522) purchased from Sigma were used for parallel comparison of sensitivity and specificity. The experimental method was enzyme-linked immunosorbent assay (ELISA). The reaction titers of quality control samples containing corresponding antibodies were detected at different coated antigen concentrations (0.25, 0.50, 1.00, 2.00 μg / mL), and standard curves were plotted. The results are shown in Table 2 and... Figure 5 As shown in the titer comparison, it can be seen that the detection results of Examples 1 and 2 are much higher than those of the control antigen at all coating concentrations.

[0068] Table 2. ELISA titers of dsDNA antigen at different coating concentrations.

[0069]

[0070] The antigens prepared in Examples 1 and 2 were used to detect 214 SLE samples. The detection results are shown in Table 3 below. The detection results showed high sensitivity, all above 92%.

[0071] Table 3. Results of serum sensitivity testing for dsDNA antigen in 214 samples

[0072]

[0073]

[0074] The antigens prepared in Examples 1 and 2 were used to test 401 samples (124 healthy samples from physical examinations and 277 samples from related diseases). The test results are shown in Table 4. All samples showed high clinical specificity, with a specificity of 88.03%.

[0075] Table 4. Serum-specific detection results of dsDNA antigen in 401 samples

[0076]

[0077] The natural dsDNA antigen extracted and purified by this invention has passed a series of high-standard quality tests within the company, proving that it has significantly improved in terms of yield, purity, activity, specificity, and stability. The test results from downstream customers have also shown good feedback, all of which prove that the natural dsDNA antigen prepared by the method provided by this invention has higher immunogenicity and higher potency.

[0078] The specific method of ELISA is as follows: The antigen is conjugated to a solid-phase carrier to form a solid-phase antigen. After washing, unbound antigen and impurities are removed. Diluted test serum is added, and the reaction is incubated. Specific antibodies in the serum bind to the solid-phase antigen, forming a solid-phase antigen-antibody complex. After washing, only the specific antibody remains on the solid-phase carrier; other components in the serum are washed away. After adding HRP-labeled antibody, the antibody in the solid-phase immune complex binds to the enzyme-labeled antibody, thus indirectly labeling the enzyme. After washing, the amount of enzyme on the solid-phase carrier is positively correlated with the amount of the test antibody. Enzyme substrate is added to induce enzyme color development. The absorbance of the ELISA test plate is measured using a microplate reader, and the content of the colored final product can be used to measure the content of the test antibody. In this invention, the coating buffer used in the ELISA detection is a pH 7.5 PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4). First, prepare the required sample concentration using coating buffer and add 100 μL / well to a 96-well ELISA plate. Incubate at 4°C for 24 h. Then, discard the liquid in the wells, wash three times with PBST buffer, and add 200 μL / well of blocking buffer containing 5% BSA. Block at 37°C for 40 min and discard. Add 100 μL of diluted serum (primary antibody serum dilution ratio 1:300), mix well, incubate at 37±0.5°C for 5–20 min, discard the supernatant, wash three times with PBST buffer, add 100 μL of diluted secondary antibody serum (secondary antibody serum dilution ratio 1:3000), mix well, incubate at 37±0.5°C for 5–20 min, discard the supernatant, wash three times with PBST buffer, add 100 μL of luminescent substrate to the detection tube, mix well, and detect the luminescence intensity using a microplate reader.

[0079] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing highly immunogenic natural dsDNA antigens, characterized in that, Includes the following steps: Add lysis buffer, proteinase K and RNase A to the tissue sample, homogenize and place in a water bath at 45~60℃ for digestion, centrifuge and collect the supernatant; Extraction reagent was added to the supernatant according to the volume ratio of sample: saturated phenol: chloroform: isopropanol 50: (25~40): (32~40): (1~2). The supernatant was collected, precipitated with ethanol, and then dissolved to obtain crude dsDNA antigen. The crude dsDNA antigen was purified by sequentially passing it through CM cation exchange chromatography and dextran-agarose composite gel filtration medium 200 molecular sieve chromatography. The chromatography column used for the CM cation exchange chromatography is CM-6FF, and the dextran-agarose composite gel filtration medium 200 molecular sieve chromatography is Prepack-GL 26 / 600 Finedex 200. The buffer solution used when packing the CM cation exchange chromatography column is 20 mM HEPES, 1 mM EDTA, 0.15 M NaCl, pH 8.

0. The buffer solution used when packing the 200 molecular sieve chromatography column with the dextran-agarose composite gel filter medium was 10 mM Tris-HCl, 1 mM EDTA, pH 7.

5.

2. The method for preparing highly immunogenic natural dsDNA antigen according to claim 1, characterized in that, The lysis buffer is: 5~50mM Tris-HCl, 0.5~5% SDS, 0.1~1.0% Triton, and 0.1~1.0M NaCl.

3. The method for preparing highly immunogenic natural dsDNA antigen according to claim 1, characterized in that, Tissue samples include, but are not limited to, calf thymus tissue, frog fish essence, and HeLa cells.

4. The method for preparing highly immunogenic natural dsDNA antigen according to claim 1, characterized in that, When tissue samples contain connective tissue, pretreatment is required to remove the connective tissue.

5. The method for preparing highly immunogenic natural dsDNA antigen according to claim 4, characterized in that, The pretreatment method is as follows: add homogenization buffer and filter with gauze; wherein the homogenization buffer is: 10~100mM Tris-HCl, 0~0.1M NaCl, pH 5.0~8.

5.

6. The use of the dsDNA prepared by the method according to any one of claims 1 to 5 in the preparation of a clinical auxiliary diagnostic kit for systemic lupus erythematosus, an autoimmune disease.

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