A detection kit for detecting anti-aconitate hydratase-IgG antibodies

Abstract

The present invention provides a kind of detection kit for detecting anti-aconitate hydratase IgG antibodies, by aconitate hydratase (aconitate hydratase) antigen protein, labeled antibody liquid, the solid phase carrier coated with aconitate hydratase antigen, sample diluent, antibody and antigen diluent, substrate color developing solution, washing solution, stop solution, standard substance, positive and negative quality control product composition. The present invention is through the contact of the serum sample being tested with the target antigen aconitate hydratase polypeptide or its binding fragment, an immune reaction occurs, an antigen-antibody complex is formed, and the antigen-antibody complex formed is tested for anti-aconitate hydratase IgG autoantibodies. The present invention identifies for anti-aconitate hydratase IgG antibodies for the first time, and provides a better detection method for the identification of autoimmune nephrotic syndrome related to anti-aconitate hydratase IgG antibodies at home and abroad.

Description

[0001] This application is a divisional application of application number 202110742938.0, application date July 1, 2021, and invention name: A detection kit for detecting anti-aconitate hydratase-IgG antibodies. Technical Field

[0002] The invention belongs to the technical field of biomedicine and relates to a detection kit for detecting anti-aconitate hydratase-IgG antibodies. Background Art

[0003] Primary nephrotic syndrome (PNS) is a clinical syndrome characterized by multiple factors leading to increased glomerular basement membrane permeability, which in turn increases plasma protein filtration and loss in the urine, resulting in a series of pathological changes. PNS is a common glomerular disease in children, with a reported annual incidence of 1.15 to 16.9 per 100,000 children. Minimal change nephrotic syndrome (MCNS) is the most common pathological type. PNS is classified into steroid-sensitive (SSNS), steroid-dependent (SDNS), and steroid-resistant (SRNS) based on its response to steroid therapy. Although the vast majority of children respond to steroid therapy and have a good prognosis, 10% to 20% of patients are either dependent or resistant to steroid therapy. Without additional pharmacological intervention, these children may develop glomerulosclerosis and ultimately end-stage renal disease, severely impacting their health and placing a heavy economic burden on their families and society. Furthermore, the pathogenesis of this disease remains unclear. The clinical symptoms of PNS are usually edema, heavy proteinuria, hypoproteinemia, and hyperlipidemia. However, the causes of these clinical manifestations are very complex, making it difficult to diagnose PNS. Therefore, if the exact cause of PNS can be found, then PNS can be diagnosed as early as possible, allowing for early intervention and treatment, which will be of great clinical significance.

[0004] Currently, the vast majority of children with PNS who receive treatment with steroids and immunosuppressants experience improvement, indirectly suggesting a close link between PNS and autoimmunity. B lymphocytes are known to play a crucial role in humoral immunity. However, the lack of localized antibody deposition in the kidneys of children with PNS has led to a neglected role for B lymphocyte-mediated humoral immunity in PNS. Recent studies have shown a significant increase in the number of B cells in the peripheral blood of patients with relapsed steroid-sensitive nephrotic syndrome, and a significant increase in activated B cells in patients with steroid-dependent nephrotic syndrome. However, B cell numbers are significantly decreased in patients with steroid-dependent nephrotic syndrome in remission, suggesting that B lymphocyte dysfunction plays a key role in PNS. In recent years, studies worldwide have demonstrated that rituximab, which specifically targets CD20+ B cells, has achieved significant therapeutic efficacy in the treatment of refractory nephrotic syndrome in children. However, during the treatment of SDNS with rituximab, it was found that its B cell depletion effect lasted for about 5 months, but at 6 or 7 months, the patient's condition relapsed due to a rebound in B cell count. This suggests that pathological B cell clones occur in PNS patients. If these pathological B cell clones can be identified and accurately eliminated, it will be beneficial for the recovery of PNS and also reduce the risk of humoral immune deficiency caused by indiscriminate B cell depletion using methods such as rituximab.

[0005] However, the target antigens of pathological B cells in children with PNS remain unclear. Pathologically, minimal change disease or focal segmental glomerulosclerosis is believed to be a podocytopathy caused by loss or alteration of podocyte function, leading to excessive proteinuria. Podocytes are renal glomerular epithelial cells that are attached to the outer side of the glomerular basement membrane. They serve as the final barrier to protein loss, and podocyte damage often results in excessive proteinuria.

[0006] Aconitase (aconitate hydratase) is an essential enzyme located in mitochondria that catalyzes the interconversion of citrate and isocitrate in the tricarboxylic acid cycle. Existing studies have shown that the expression of aconitate hydratase may be altered in certain types of cancer. Wang et al. performed real-time quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemistry staining on gastric cancer specimens to measure the expression of aconitate hydratase in tumor tissues and matched adjacent non-tumor tissues. The results showed that compared with matched adjacent non-tumor tissues, the expression of aconitate hydratase in gastric cancer tissues was significantly downregulated and correlated with the TNM stage of gastric cancer. Aconitate hydratase may play an important role in gastric cancer and may serve as a prognostic biomarker (Wang, et al. Decreased expression of the mitochondrial metabolic enzyme aconitase (aconitate hydratase) is associated with poor prognosis in gastric cancer. Med Oncol, 2013 Jun; 30(2): 552.). Aconitate hydratase activity is increased in prostate cancer and decreased in rectal cancer. A recent study on breast cancer found that decreased aconitate hydratase expression may inhibit cell proliferation by increasing the level of enzyme in cells, which may be related to enhanced oxidative metabolism (Fabio Ciccarone, et al. Aconitase 2 inhibits the proliferation of MCF-7 cells promoting mitochondrial oxidative metabolism and ROS / FoxO1-mediated autophagic response. Br J Cancer, 2020 Jan; 122(2): 182-193.).

[0007] However, there are currently no reports on the relationship between aconitate hydratase and nephrotic syndrome. In addition, in the prior art, there is currently no research on identifying autoimmune nephrotic syndrome by detecting serum anti-aconitate hydratase-IgG antibodies. Summary of the Invention

[0008] The present invention aims to provide a detection kit for detecting serum anti-aconitate hydratase-IgG autoantibodies, wherein the kit detects antibodies in a sample through an immune reaction with an aconitate hydratase antigen protein (particularly as shown by the sequence identification number SEQ ID NO.1).

[0009] The kit includes: aconitate hydratase antigen protein, labeled antibody solution (enzyme-labeled or chemiluminescent anti-human IgG solution), a solid phase carrier coated with aconitate hydratase antigen, sample diluent, antibody diluent, antigen diluent, substrate color development solution, washing solution, stop solution, standard substance, positive quality control substance, and positive quality control substance.

[0010] The sequence of the aconitate hydratase antigen protein is shown in SEQ ID NO: 1:

[0011] MAPYSLLVTRLQKALGVRQYHVASVLCQRAKVAMSHFEPNEYIHYDLLEKNINIVRKRLNRPLTLSEKIVYGHLDDPASQEIERGKSYLRLRPDRVAMQDATAQMAMLQFISSGLSKVAVPSTIHCDHLIEAQVGGEKDLRRAKDINQEVYNFLATAGAKY GVGFWKPGSGIIHQIILENYAYPGVLLIGTDSHTPNGGLGLGICIGVGGADAVDVMAGIPWELKCPKVIGVKLTGSLSGWSSPKDVILKVAGILTVKGGTGAIVEYHGPGVDSISCTGMATICNMGAEIGATTSVFPYNHRMKKYLSKTGREDIANLADEF.

[0012] According to the present invention, the aconitate hydratase antigen protein is purified by molecular sieve, gel filtration chromatography, affinity chromatography, ion exchange column and hydrophobic column.

[0013] The aconitate hydratase antigen protein of the present invention can be a fusion protein, using a tag with biological or physical function, particularly an N-terminal or C-terminal tag, preferably a C-terminal tag. These tags are conducive to antigen protein purification, fixation, and precipitation. In a preferred embodiment, the tag is a sequence or domain that can specifically bind to the ligand, and the tag peptide is selected from: His tag, GST tag, maltose binding protein, thioredoxin and fluorescent tag or biotin tag.

[0014] According to the present invention, the antigenic protein aconitate hydratase can be expressed in bacteria such as Escherichia coli, fungi, yeast, and mammalian cells.

[0015] In a preferred embodiment, the aconitate hydratase antigen protein of the present invention is presented in an immobilized form. The term "immobilized" refers to binding to an insoluble solid support in an aqueous solution. Preferably, the binding occurs through electrostatic interactions, hydrophobic interactions, or covalent bonds. Preferred solid supports include polystyrene, microplates, nitrocellulose membranes, and magnetic beads.

[0016] Described aconitate hydratase antigen protein immobilization mode comprises reversible immobilization mode or irreversible immobilization mode.For example, molecule combines by cleavable covalent bond (as can add the disulfide bond that the reagent containing thiol cracks), and this fixing is reversible.In addition, as molecule is fixed on carrier by the covalent bond (key formed by the reaction of epoxide group and the amine group that lysine side chain is coupled to affinity column) that can not crack in aqueous solution, then fixing is irreversible.Fixing can also be indirect mode fixed protein: as fixing the antibody that described molecule has specific affinity, then form complex to reach the effect of fixed molecule-antibody complex.

[0017] The aconitate hydratase antigen protein immobilization method described in the present invention is a direct coating method: (1) the antigen is bound to a nitrocellulose membrane or a polystyrene microplate by physical adsorption or non-covalent bonding; (2) magnetic microparticles with carboxyl functional groups are bound to the amino groups of the protein, and the antigen is bound to the magnetic microparticles by chemical coupling.

[0018] The substrate color developing solution is TMB, luminol, hydrogen peroxide, and acridinium ester; the antigen diluent is: 1xPBS, pH 7.40; the antibody diluent is: 0.15% BSA + 0.01M PBS (pH 7.40); the sample diluent is: 6% fetal bovine serum + 0.01M PBS (pH 7.40); the washing solution is: 1xPBS (pH 7.40) + 0.1Tween-20; the stop solution is: 2M sulfuric acid.

[0019] In one embodiment of the present invention, the standard and positive control are preferably prepared from antigens homologous to humans, such as recombinant human anti-tag peptide immunoglobulin G or its fragment, or anti-aconitatehydratase-IgG antibodies extracted from patient serum as positive control and standard products, and the negative control is the serum of healthy subjects.

[0020] The labeled antibody solution of the present invention can be horseradish peroxidase (HRP)-labeled anti-human IgG, biotin-labeled anti-human IgG, or acridinium ester-labeled anti-human IgG.

[0021] According to the present invention, the sample to be tested is a liquid sample containing antibodies, preferably pleural effusion, ascites, urine, whole blood, plasma, and most preferably serum. The serum is mammalian serum, preferably human serum. Prior to testing, the sample to be tested may be further processed, including fractionation, centrifugation, or enrichment.

[0022] The present invention relates to a detection kit which uses a recombinant protein aconitate hydratase expressed and purified by a gene recombination method as an antigen protein in a kit, which is then coated on a solid phase carrier, and is then incubated with a positive quality control product, a standard product, or a serum to be tested. A labeled secondary antibody is then added for reaction, and the secondary antibody combines with an anti-aconitate hydratase-IgG antibody in the serum to form a coating antigen aconitate hydratase-anti-aconitate hydratase-IgG antibody in the serum to be tested-labeled anti-human IgG antibody complex. The light signal is detected by an optical method such as a photochromic method, a chemiluminescence method, or a fluorescence method to qualitatively or quantitatively analyze the concentration of the anti-aconitate hydratase-IgG antibody in the serum.

[0023] Using the kit of the present invention, an anti-aconitate hydratase-IgG autoantibody was detected for the first time in some patients with autoimmune nephrotic syndrome, and the target antigen of the autoantibody was determined to be aconitase on glomerular podocytes. Therefore, the kit of the present invention can be used to detect anti-aconitate hydratase-IgG autoantibodies, providing a basis for the study of autoimmune nephrotic syndrome.

[0024] Compared with the prior art, the advantages of the kit of the present invention are as follows:

[0025] (1) This invention identifies for the first time the presence of anti-aconitate hydratase-IgG autoantibodies in patients with autoimmune nephrotic syndrome and develops a detection kit for these antibodies. Currently, there is no research related to aconitate hydratase-IgG antibodies in China or abroad. This invention fills a gap in the detection of aconitate hydratase-IgG antibodies both domestically and internationally.

[0026] (2) The kit of the present invention relates to the qualitative detection of anti-aconitate hydratase-IgG antibodies in serum. The solid-phase membrane immunoassay is simple to operate, requires a small amount of reagent, and can absorb trace amounts of antigens due to the strong adsorption capacity of the NC membrane. The adsorbed NC membrane can be stored for a long time (at -20°C for half a year) without affecting its activity. It is suitable for large-scale screening.

[0027] (3) The present invention relates to a magnetic microparticle chemiluminescent immunoassay quantitative detection kit. Magnetic microparticles with a diameter of only 1.0 μm are used as solid-phase carriers. This greatly increases the coating surface area, increases the amount of antigen adsorbed, improves the reaction rate, and simplifies cleaning and separation, thereby reducing contamination and the probability of cross-infection. Furthermore, anti-human IgG is directly labeled with an acridinium ester luminescent agent. The chemical reaction is simple, rapid, and catalyst-free. Acridinium ester chemiluminescence is a flash type. Its emission intensity reaches its maximum 0.4 seconds after the luminescent reagent (H2O2, NaOH) is activated. The half-life is 0.9 seconds, and the emission essentially ends within 2 seconds, facilitating rapid detection.

[0028] (4) Currently, there are no reports on the relationship between aconitate hydratase and nephrotic syndrome. This invention is the first to discover the presence of aconitate hydratase-IgG antibodies in nephrotic syndrome, clarifying the cause of autoimmunity. In the existing technology, there is no research on identifying autoimmune nephrotic syndrome by detecting serum anti-aconitate hydratase-IgG antibodies. This invention provides a non-invasive detection method for this disease. Compared with the existing renal biopsy detection method, the use of the kit of this invention will reduce the pain and financial burden of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :Aconitate hydratase protein on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Figure 1 A: Two-dimensional electrophoresis protein spots with the primary antibody being healthy human serum; Figure 1 B: Two-dimensional electrophoresis protein spots of serum from patients with autoimmune nephrotic syndrome in which the primary antibody was used; Figure 1 C: Mass spectrometry identification of the target antigen aconitate hydratase protein.

[0030] Figure 2 : SDS-PAGE identification of the expressed recombinant aconitate hydratase antigen protein.

[0031] Figure 3:Solid phase membrane immunoassay kit for detecting anti-aconitatehydratase-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome.

[0032] Figure 4 : Schematic diagram of the principle of detecting anti-aconitate hydratase-IgG antibody using magnetic microparticle chemiluminescence kit.

[0033] Figure 5 : Schematic diagram of carboxyl magnetic microparticles coated with the antigen protein aconitate hydratase.

[0034] Figure 6 : Detection of anti-aconitate hydratase-IgG antibodies in patients with different kidney diseases, including NC: healthy subjects; HP: Henoch-Schonlein purpura; HPN: purpuric nephritis; IgAN: IgA nephropathy; NS: autoimmune nephrotic syndrome.

[0035] Figure 7 : Receiver operating characteristic (ROC) curve was used to evaluate the application value of anti-aconitate hydratase-IgG antibody in the detection of autoimmune nephrotic syndrome. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0037] Example 1: Aconitate hydratase on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0038] (1) Extraction of total protein from glomerular podocytes: Culture the podocyte cell line (MPC5), wash with PBS 2-3 times, and then use a focused ultrasound device (Covaris S220, Gene) to fully lyse on ice in a lysis buffer containing 30 mm Tris-HCl, 8 m urea, 4% CHAPS and protease inhibitors (#ab65621; Abcam, 1:200 dilution). Then place the sample in a centrifuge, 12000 g, 4 ° C, and centrifuge for 30 minutes. Collect the supernatant, which is the collected total protein from glomerular podocytes. The concentration of the collected total protein from glomerular podocytes was determined using a BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: The total protein from glomerular podocytes was extracted and transferred to a nitrocellulose membrane for two-dimensional electrophoresis. The membrane was incubated with the serum of patients with autoimmune nephrotic syndrome and healthy people as the primary antibody, and then a secondary antibody was added for development. Figure 1A, 1B. (3) Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis: After developing step (2), differential analysis of positive spots was performed. Protein spots that were strongly positive in patients with nephrotic syndrome and negative or weakly positive in healthy people on the two-dimensional electrophoresis gel were selected. The selected protein spots were cut out from the gel. The dried gel was digested with trypsin (0.1 μg / μL). Then, 10 μL of 25 mM ammonium bicarbonate was added to the reaction mixture and incubated at 37°C overnight. Then, peptides were extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptides were analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass spectrum, which was identified as aconitate hydratase protein. Figure 1 C.

[0039] Example 2 Expression and purification of recombinant aconitate hydratase antigen protein

[0040] The gene encoding aconitate hydratase protein was used as a template for PCR amplification by genetic engineering, and then an expression vector was constructed for protein expression. The antigen protein expressed by the present invention contained a His-tagged peptide. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc. Finally, SDS-PAGE was used to identify the molecular weight of the recombinant protein aconitate hydratase to be 37KDa. Figure 2 .

[0041] Example 3 The present invention uses orthogonal experimental design to optimize the reaction conditions of the kit

[0042] An orthogonal table was selected based on four factors: the antigen aconitate hydratase coating concentration (50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL), reaction time (30 min, 45 min), temperature (25°C, 35°C), and the optimal dilution of the enzyme-labeled secondary antibody (1:100, 1:500, 1:1000, and 1:1500). For each factor, replicate measurements were performed on standard positive and negative sera at two levels. The ratio (P / N) of the highest luminescence value (P) of the positive serum to the lowest luminescence value (N) of the negative serum was selected. The average P / N value of the replicates was statistically analyzed to determine the optimal coating conditions and secondary antibody dilution for orthogonal optimization, which significantly improved the positive detection rate of the standard positive serum. Through orthogonal design, we obtained that the optimal antigen coating concentration of this kit is 100 μg / mL, the optimal antigen-antibody reaction temperature is 25°C, the optimal antigen-antibody reaction time is 45 minutes, and the optimal working dilution of the best labeled anti-human IgG antibody is 1:500.

[0043] Example 4 Preparation of a Solid-Phase Membrane Immunoassay Kit for Detecting Anti-aconitate Hydratase-IgG Antibodies

[0044] 4.1 Composition of the solid-phase membrane immunoassay kit for detecting anti-aconitate hydratase-IgG:

[0045] 1. Nitrocellulose membrane coated with aconitate hydratase antigen protein;

[0046] 2. Standard: human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang),

[0047] 3. Antibody diluent,

[0048] 4. Antigen diluent,

[0049] 5. Horseradish peroxidase-labeled anti-human IgG antibody,

[0050] 6. Detergent,

[0051] 7. TMB color developer,

[0052] 8. Stop solution.

[0053] 4.2 The detection steps are as follows:

[0054] 4.2.1 Coating and blocking: Dilute the antigen with 0.01M PBS (pH 7.4) and spot 10 μl on a nitrocellulose membrane. Place the membrane in a 37°C incubator for 30 minutes. Place the nitrocellulose membrane in a plate holder and add 150 μl of 3% BSA. Block the membrane in a 37°C incubator for 15 minutes. Aspirate the blocking solution and wash the membrane twice with washing solution.

[0055] 4.2.2 Serum Incubation (First Incubation): 100 μl of the standard diluted with the antigen release solution and the serum sample to be tested are added to the reaction chamber. Simultaneously, negative and positive controls are performed. Do not allow the pipette tip to touch the membrane surface. Change the pipette tip after adding each serum. Place the reaction chamber with the added samples on a shaker and incubate at room temperature (20-25°C) for 45 minutes.

[0056] 4.2.3 Cleaning (First Cleaning): Pour out the liquid in the reaction tank and rinse with diluted detergent for 10 seconds. During rinsing, ensure that the detergent flows thoroughly through the reaction tank. Repeat this process five times. When pouring and rinsing, ensure that the liquid flows down the reaction tank to avoid cross contamination. After cleaning, spin dry the reaction tank.

[0057] 4.2.4 Incubation with secondary antibody working solution (second incubation): Dilute horseradish peroxidase-labeled anti-human IgG antibody with antibody diluent, then add 6 drops (300 μl) of secondary antibody working solution to the reaction chamber and incubate on a shaker at room temperature (20-25°C) for 45 minutes.

[0058] 4.2.5 Cleaning (second cleaning): The process is the same as step 3.

[0059] 4.2.6. Color development incubation (third incubation): Add 6 drops (300 μl) of color development solution to the reagent strip and incubate on a shaker at room temperature (20-25°C) for 20 minutes.

[0060] 4.2.7. Termination of reaction: Rinse the reaction tank with running water to terminate the reaction.

[0061] 4.2.8. Interpretation of results: Take out the test strip and blow dry it with a hair dryer (about 5 minutes) or place it in a 37℃-50℃ oven to dry for more than 20 minutes. Make a qualitative judgment with the naked eye. If obvious brown spots appear, it is positive (see Figure 3 ) or place the membrane strip on a developer for scanning. The developer's built-in analysis software uses the reference standard concentration as the ordinate and the grayscale value read by the instrument as the abscissa to draw a standard curve for semi-quantitative analysis of the anti-aconitate hydratase-IgG level in the serum.

[0062] Example 5 Preparation of a Chemiluminescent Immunoassay Kit for Detecting Anti-aconitate Hydratase-IgG Antibodies

[0063] 5.1 Composition of the chemiluminescent immunoassay kit for detecting anti-aconitate hydratase-IgG:

[0064] 1. Magnetic microparticle solution coated with aconitate hydratase antigen protein,

[0065] 2. Standard: human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang),

[0066] 3. Sample diluent,

[0067] 4. Acridinium ester labeled anti-human IgG solution,

[0068] 5. Quality control products,

[0069] 6. Pre-excitation solution,

[0070] 7. Exciting fluid,

[0071] 8. Detergent.

[0072] 5.2 Detection Principle: This kit uses an indirect method to detect anti-aconitate hydratase-IgG antibodies in human serum. The entire process consists of two steps: First, the magnetic bead solution is mixed with the diluted sample, and the specific anti-aconitate hydratase-IgG antibody is bound to the magnetic beads. The residual liquid is then washed away. Second, acridinium ester-labeled anti-human IgG antibody is added to form a magnetic bead-antigen-anti-aconitate hydratase-IgG antibody-acridinium ester antibody complex. After washing away the residual liquid, a pre-excitation solution (H2O2) and an excitation solution (NaOH) are added to carry out a luminescent reaction. The luminescence value is recorded. The antibody concentration is proportional to the luminescence value, and the concentration measurement value is calculated using the calibration curve (see Figure 4 ).

[0073] 5.3 The solid phase carrier of this kit is magnetic particles containing carboxyl functional groups.

[0074] 5.4 The antigen coating method of this kit is to activate the carboxyl magnetic particles with EDC / Sulfo-NHS, and then covalently bind to the antigen (amino residue) to form a magnetic particle solution. The coating steps are as follows:

[0075] a) Add 40 μl of magnetic bead stock solution to 400 μl of 0.05 M phosphate buffer, mix for 8 minutes, separate on a magnet, and discard the supernatant;

[0076] b) Take 200 μL of 2% dextran solution and add 200 μL of 10 mg / mL sodium periodate to react;

[0077] c) After the reaction, add 10 mg / mL EDC solution and 10 mg / mL Sulfo-NHS solution prepared in phosphate buffer and mix for 60 minutes;

[0078] d) Separate the beads by magnet, discard the supernatant, wash the beads with 400 μl of 0.05 M phosphate buffer, and add 400 μl of sealing solution to the fixed volume for storage.

[0079] The supernatant of the activated magnetic beads was discarded, and 1 ml of pre-cooled 20 mM MES was added to continue washing the magnetic beads twice; 200 μL of 2 mg / mL antigen protein aconitate hydratase was added to the activated magnetic beads, mixed thoroughly, and allowed to react at room temperature for 16 hours; after the reaction, pH 7.4 PBS buffer containing 0.2% Tween20 was added, and the magnetic beads were washed twice; then pH 7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA was added to a final concentration of 10 mg / mL magnetic beads, mixed thoroughly, and allowed to react at room temperature for 30 minutes; after the reaction, the supernatant was discarded, and the magnetic beads were resuspended with pH 7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA to complete the cross-linking of the activated magnetic beads and the antigen protein aconitate hydratase (see Figure 5 ).

[0080] 5.5 Acridinium ester labeled anti-human IgG solution, steps are:

[0081] a) preparing a 2 mg / mL acridinium ester solution in dimethylformamide;

[0082] b) preparing 1 mg / mL anti-human IgG antibody using 0.2 M (pH 8.0) carbonate buffer;

[0083] c) mixing acridinium ester and anti-human IgG antibody at a molar ratio of 4:1, stirring, and reacting for 40 minutes;

[0084] d) adding 20 μl of carbonate buffer containing 5% lysine for 30 minutes to terminate the reaction;

[0085] e) removing impurities by desalting to obtain an acridinium ester-labeled anti-human IgG solution.

[0086] 5.6 The detection steps are as follows:

[0087] 5.6.1 Dilute the sample in a certain ratio;

[0088] 5.6.2 Take the diluted sample, add the magnetic microparticle solution and sample diluent, and incubate at 37°C for 12 minutes;

[0089] 5.6.3 Wash 3 times with detergent;

[0090] 5.6.4 Add acridinium ester-labeled anti-human IgG solution and react at 37°C for 12 minutes;

[0091] 5.6.5 Wash 3 times with detergent;

[0092] 5.6.6 Add pre-excitation solution (H2O2) and excitation solution (NaOH) to react and collect luminescence measurements;

[0093] 5.6.7 Calculate the concentration value using the calibration curve.

[0094] Example 6 Detection of anti-aconitate hydratase-IgG antibodies in various types of kidney disease patients

[0095] 6.1 Subjects enrolled: Patients diagnosed with nephrotic syndrome between June 2018 and June 2020; healthy controls were selected from healthy individuals undergoing physical examinations during the same period. Serum samples were obtained from patients with nephrotic syndrome and healthy controls. All subjects underwent the first serum sample collection before receiving immunosuppressive therapy.

[0096] 6.2 Detection of anti-aconitate hydratase-IgG antibodies in the serum of patients with different kidney diseases The kit of the present invention was used to detect the levels of anti-aconitate hydratase-IgG antibodies in the serum of patients diagnosed with various kidney diseases from June 2018 to June 2020, including 466 cases of nephrotic syndrome, 168 cases of Henoch-Schönlein purpura, 137 cases of purpuric nephritis, 133 cases of IgA nephropathy, and 195 healthy children of the same period. The results showed that some patients with autoimmune nephrotic syndrome were positive for anti-aconitate hydratase-IgG antibodies, while patients with purpuric nephritis, Henoch-Schönlein purpura, IgA nephropathy, and healthy children were negative for anti-aconitate hydratase-IgG antibodies (see Figure 6 ).

[0097] Example 7 ROC Curve Evaluation of the Value of Anti-aconitate Hydrate-IgG Antibody as a Serological Marker for Detecting Patients with Autoimmune Nephrotic Syndrome The results of anti-aconitate hydratase-IgG antibody detection in patients with autoimmune nephrotic syndrome in 6.2 of Example 6 were analyzed using an ROC curve to determine the value of the antibody in detecting autoimmune nephrotic syndrome. The results showed that anti-aconitate hydratase-IgG antibody as a serum marker has a good value in diagnosing patients with autoimmune nephrotic syndrome. When the diagnostic definition is greater than 53.4, the diagnostic sensitivity is 56.8%, the specificity is 87.2%, and the area under the curve is 0.773 (see Figure 7 ). SEQUENCE LISTING <110> Zhejiang University <120> A detection kit for detecting anti-aconitate hydratase-IgG antibodies <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 322 <212> PRT <213> Artificial sequence (Unknow) <400> 1 Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu Gly 1 5 10 15 Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala Lys Val 20 25 30 Ala Met Ser His Phe Glu Pro Asn Glu Tyr Ile His Tyr Asp Leu Leu 35 40 45 Glu Lys Asn Ile Asn Ile Val Arg Lys Arg Leu Asn Arg Pro Leu Thr 50 55 60 Leu Ser Glu Lys Ile Val Tyr Gly His Leu Asp Asp Pro Ala Ser Gln 65 70 75 80 Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg Leu Arg Pro Asp Arg Val 85 90 95 Ala Met Gln Asp Ala Thr Ala Gln Met Ala Met Leu Gln Phe Ile Ser 100 105 110 Ser Gly Leu Ser Lys Val Ala Val Pro Ser Thr Ile His Cys Asp His 115 120 125 Leu Ile Glu Ala Gln Val Gly Gly Glu Lys Asp Leu Arg Arg Ala Lys 130 135 140 Asp Ile Asn Gln Glu Val Tyr Asn Phe Leu Ala Thr Ala Gly Ala Lys 145 150 155 160 Tyr Gly Val Gly Phe Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile 165 170 175 Ile Leu Glu Asn Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp 180 185 190 Ser His Thr Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val 195 200 205 Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu 210 215 220 Lys Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser Gly 225 230 235 240 Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala Gly Ile Leu Thr 245 250 255 Val Lys Gly Gly Thr Gly Ala Ile Val Glu Tyr His Gly Pro Gly Val 260 265 270 Asp Ser Ile Ser Cys Thr Gly Met Ala Thr Ile Cys Asn Met Gly Ala 275 280 285 Glu Ile Gly Ala Thr Thr Ser Val Phe Pro Tyr Asn His Arg Met Lys 290 295 300 Lys Tyr Leu Ser Lys Thr Gly Arg Glu Asp Ile Ala Asn Leu Ala Asp 305 310 315 320 Glu Phe

Claims

1. Use of an aconitate hydratase polypeptide capable of forming any antigen-antibody complex in contact with a sample obtained from a patient in the preparation of a nephrotic syndrome detection reagent or kit; wherein: The antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The aconitate hydratase polypeptide sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the sample is serum.

3. The use according to claim 1, wherein the sample is a sample from a patient before immunotherapy.

4. Use of an aconitate hydratase polypeptide capable of forming any antigen-antibody complex in contact with a sample obtained from a patient in the preparation of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy; wherein: The antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The aconitate hydratase polypeptide sequence is shown in SEQ ID NO.

1.

5. A kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy, comprising: An aconitate hydratase polypeptide capable of forming any antigen-antibody complex in contact with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody (anti-aconitate hydratase-IgG antibody) complex; and A magnetic particle solution coated with aconitate hydratase antigen protein; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The aconitate hydratase polypeptide sequence is shown in SEQ ID NO.1.

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