Kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies

Abstract

The present invention provides a kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies, which consists of an antigen protein rich in serine / arginine splicing factor 9, a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color developer, a washing solution, a standard substance, a positive quality control product, and a negative quality control product. Anti-serine / arginine-rich splicing factor 9-IgG autoantibodies are detected by a specific kit. The present invention identifies autoantibodies against the target antigen rich in serine / arginine splicing factor 9 for the first time, and provides a detection kit for the autoantibodies. The kit provided by the present invention provides a basis for the molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to serine / arginine-rich splicing factor 9 and serine / arginine-rich splicing factor 9-IgG autoantibodies at home and abroad.

Description

[0001] This application is a divisional application of application number 202110743511.2, application date July 1, 2021, and invention name: Kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies. Technical Field

[0002] The present invention belongs to the field of biomedical technology and relates to a kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies. The kit is used to detect anti-serine / arginine-rich splicing factor 9-IgG antibodies in patient samples. Background Art

[0003] Autoimmune diseases are conditions caused by an abnormal immune response to substances and tissues normally present in the body. These diseases may be limited to certain organs, involve specific tissues in different locations, or be systemic. Treatment for autoimmune diseases often involves immunosuppression, which involves medications that reduce the immune response.

[0004] Primary nephrotic syndrome (PNS) is characterized by edema, increased protein in the urine, decreased protein in the blood, and increased fat in the blood (Roth KS, Amaker BH, Chan JC: Nephrotic syndrome: pathogenesis and management. Pediatr Rev (2002) 23(7): 237-48). In more than 90% of children with PNS, the pathological type is minimal change disease or focal segmental glomerulosclerosis, characterized by podocyte damage. Podocyte inflammation increases permeability to protein, leading to increased protein secretion. When the amount of protein excreted in the urine exceeds the liver's ability to compensate, less protein is detected in the blood, especially albumin, which accounts for the majority of circulating protein. As the amount of protein in the blood decreases, the osmotic pressure in the blood decreases. Since the osmotic pressure in the tissues remains unchanged, edema can result. This condition is exacerbated by the secretion of aldosterone by the adrenal glands, which is secreted in response to the decrease in circulating blood, leading to sodium and water retention. Hyperlipidemia is believed to be the result of increased liver activity (Brinkkoetter PT, Ising C, Benzing T: The role of the podocyte in albuminfiltration. Nat Rev Nephrol (2013) 9 (6): 328-36). Evidence has shown that the majority of steroid-sensitive nephrotic syndrome in children and two-thirds of steroid-resistant nephrotic syndrome are associated with autoimmune dysfunction (Li J, Wang L, Wan L, Lin T, Zhao W, Cui H, et al: Mutational spectrum and novel candidate genes in Chinese children with sporadic steroid-resistant nephrotic syndrome. Pediatr Res (2019) 85 (6): 816-21). We screened for autoantibodies against serine / arginine-rich splicing factor 9 in some patients with primary nephrotic syndrome.Serine / arginine-rich splicing factor 9 is a splicing factor involved in splicing site selection in alternative splicing (Matsumoto E, Matsumoto Y, Inoue J, Yamamoto Y, Suzuki T. AMP-activated protein kinase regulates beta-catenin protein synthesis by phosphorylatingserine / arginine-rich splicing factor 9. Biochem Biophys Res Commun (2021) 534: 347-52).

[0005] However, there are currently no reports of Ser / Arg-Rich Splicing Factor 9 (SRSF9) or autoantibodies against Ser / Arg-Rich Splicing Factor 9 (SRSF9) in urological diseases, particularly nephrotic syndrome. Furthermore, existing technologies do not address the use of Ser / Arg-Rich Splicing Factor 9 (SRSF9) or its autoantibodies as serological markers for autoimmune nephrotic syndrome. Research on the identification of autoimmune nephrotic syndrome by detecting serum anti-SRSF9 IgG antibodies is lacking. Summary of the Invention

[0006] The present invention provides a kit for detecting anti-serine / arginine-rich splicing factor 9 IgG antibodies. The kit is based on the target serine / arginine-rich splicing factor 9 and its corresponding autoantibodies. The kit can detect autoantibodies in tissues (renal biopsy tissue) or body fluids (particularly blood, plasma, and serum) through an antigen-antibody reaction with the antigenic protein serine / arginine-rich splicing factor 9 (particularly as represented by SEQ ID NO. 1).

[0007] The present invention relates to a detection kit for detecting anti-serine / arginine-rich splicing factor 9 autoantibodies. The kit comprises an antigen protein, serine / arginine-rich splicing factor 9, a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color developer, a washing solution, a stop solution, a standard, a positive quality control product, and a negative quality control product.

[0008] According to the present invention, the antigenic protein serine / arginine-rich splicing factor 9 can be expressed in bacteria such as Escherichia coli, yeast, and mammalian cells.

[0009] According to the present invention, the antigen protein can be a fusion protein, using a tag having certain biological or physical functions, the presence of which is conducive to the purification of the antigen protein, immobilization, precipitation or identification of other sequence motifs or polypeptides of the polypeptide of the present invention. In a more preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, for example, selected from: a GST tag, a c-Myc tag, a His tag, a Flag tag or a biotin tag.

[0010] According to the present invention, the antigen protein serine / arginine-rich splicing factor 9 is fixed on a solid phase carrier, and preferred solid phase carriers include: nitrocellulose membrane, magnetic beads, and enzyme-labeled microplate.

[0011] In one embodiment of the present invention, the standard and positive quality control products are recombinant human anti-tag peptide IgG or its fragments, or anti-serine / arginine-rich splicing factor 9 antibodies extracted from patient serum as positive quality control products and standard products, and the serum of healthy subjects is used as negative quality control product.

[0012] According to the present invention, the antigen protein rich in serine / arginine splicing factor 9 is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, and hydrophobic column.

[0013] Sample herein is the sample of body fluid to be tested or tissue.Preferred test sample comprises blood, serum, blood plasma, cerebrospinal fluid, urine, saliva, sputum and pleural effusion.In addition, after separation or purification procedure, for example whole blood is separated into serum or blood plasma component, will be more easily analyzed some test sample.Therefore, in a preferred embodiment of the present invention, sample is selected from the extract of blood sample, serum sample, blood plasma sample, cerebrospinal fluid sample, saliva sample and urine sample or any above-mentioned sample.Preferably, sample is a blood sample, more preferably a serum sample or blood plasma sample.

[0014] The detection kit also includes a substrate color developer, an antigen diluent, a sample dilution buffer, an antibody diluent, a washing solution, and a stop solution. The substrate color developer includes TMB, BCIP, AMPPD, and 4-MUP; the antigen dilution buffer includes 1xPBS (pH 7.4), 163mM NaCL, and 1% TritonX-100; the sample dilution buffer includes 0.01M PBS (pH 7.4) containing 10% BSA; the antibody dilution solution includes 1M D-glucose (19.82g) and 2% glycerol (2ml) added to 0.01M PBS containing 0.2% Tween20 to 100ml; the washing solution includes 1x PBS (pH 7.4), 163mM NaCL, 1% TritonX-100, and 10% glycerol; and the stop solution includes 2M sulfuric acid.

[0015] The antigen protein serine / arginine-rich splicing factor 9 fixation 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 bond; 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.

[0016] The labeled antibody selected in the present invention can be horseradish peroxidase (HRP)-labeled anti-human IgG, acridinium ester-labeled anti-human IgG, or biotin-labeled anti-human IgG.

[0017] The present invention successfully expressed and purified the recombinant protein serine / arginine-rich splicing factor 9 (SRS9) using a prokaryotic gene recombination method. This protein serves as the antigen in a test kit to develop a kit suitable for detecting anti-SRS9-IgG antibodies in patients with autoimmune nephrotic syndrome. The kit includes a detection kit for qualitative or quantitative analysis of anti-SRS9-IgG antibodies.

[0018] The principle of a kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies in serum is to use an antigen adsorbed on a solid phase carrier as a coating antigen, then add a positive quality control product or a standard product or a serum sample to be tested for incubation, and then add a labeled secondary antibody to react to form a ternary complex of coating antigen-serine / arginine-rich splicing factor 9-serine-rich splicing factor 9-IgG antibody in the serum to be tested-labeled anti-human IgG antibody. Finally, light signals are detected by photochromic method, chemiluminescence method, or fluorescence method to achieve the purpose of qualitative or quantitative analysis of anti-serine / arginine-rich splicing factor 9-IgG antibodies in human serum.

[0019] The sequence of the antigen protein rich in serine / arginine splicing factor 9 is shown in SEQ ID NO.1:

[0020] MSGWADERGGEGDGRIYVGNLPTDVREKDLEDLFYKYGRIREIELKNRHGLVPFAFVRFEDPRDAEDAIYGRNGYDYGQCRLRVEFPRTYGGRGGWPRGGRNGPPTRRSDF RVLVSGLPPSGSWQDLKDHMREAGDVCYADVQKDGVGMVEYLRKEDMEYALRKLDDTKFRSHEGETSYIRVYPERSTSYGYSRSRSGSRGRDSPYQSRGSPHYFSPFRPY.

[0021] The specific innovations can be summarized as follows:

[0022] (1) The present invention identifies for the first time an autoantibody against serine / arginine-rich splicing factor 9 and invents a detection kit for the autoantibody.

[0023] (2) At present, there are no studies related to Ser / Arg-Rich Splicing Factor 9 and anti-Ser / Arg-Rich Splicing Factor 9 autoantibodies in nephrotic syndrome at home and abroad, and there is no kit for detecting the level of anti-Ser / Arg-Rich Splicing Factor 9 autoantibodies. This invention fills the gap in the detection kit for anti-Ser / Arg-Rich Splicing Factor 9 autoantibodies at home and abroad.

[0024] (3) The advantage of the chemiluminescence method lies in the simplicity of the assay. Using a magnetic separation system, the advantages of acridinium ester-labeled chemiluminescence technology are that acridinium ester can be used as a luminescent agent in the absence of a catalyst and can also emit light in a dilute alkaline solution containing hydrogen peroxide; its small molecular weight avoids shielding the antibody binding site, improves the overall sensitivity of the system, and reacts quickly; it has a low background and a high signal-to-noise ratio, making it an effective chemiluminescent label.

[0025] (4) Solid-phase membrane immunoassays significantly shorten the detection time and can achieve rapid detection. The kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies in human serum introduces a biotin-avidin amplification system, which greatly improves the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Serine / arginine-rich splicing factor 9 protein on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Figure 1 A: Two-dimensional electrophoresis protein spots of healthy human serum; Figure 1 B: Two-dimensional electrophoretic protein spots of serum from patients with autoimmune nephrotic syndrome; Figure 1 C: Mass spectrometry identification of the target antigen serine / arginine-rich splicing factor 9 protein.

[0027] Figure 2 : SDS-PAGE identification of recombinant protein serine / arginine-rich splicing factor 9.

[0028] Figure 3 : Solid phase membrane immunoassay kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome.

[0029] Figure 4 : Schematic diagram of the detection principle of the chemiluminescence kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibody.

[0030] Figure 5: Schematic diagram of carboxyl magnetic microparticles coated with antigen protein serine / arginine-rich splicing factor 9.

[0031] Figure 6 :Detection of anti-serine / arginine-rich splicing factor 9-IgG antibodies in patients with various kidney diseases, including PNS: autoimmune nephrotic syndrome, HSP: Henoch-Schonlein purpura, HSPN: purpuric nephritis, IgAN: IgA nephropathy, NC: healthy children.

[0032] Figure 7 : Receiver operating characteristic (ROC) curve evaluation of anti-serine / arginine-rich splicing factor 9 autoantibodies as serological markers for the differential diagnosis of autoimmune nephrotic syndrome. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1: Serine / Arginine-Rich Splicing Factor 9 (SRSF9) on podocytes is a target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. This study, based on extensive clinical and molecular mechanisms, first discovered elevated serum IgG levels in patients with nephrotic syndrome and confirmed that SRSF9 on podocytes is one of the target antigens for autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative levels of anti-SRSF9 IgG antibodies in serum can aid in the early identification of autoimmune nephrotic syndrome, particularly for screening patients with related symptoms.

[0035] The specific implementation is as follows (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: After extracting the total protein from glomerular podocytes and performing two-dimensional electrophoresis, transfer it to a nitrocellulose membrane and incubate it with the serum of healthy people and patients with autoimmune nephrotic syndrome as the primary antibody, and then add a secondary antibody for development, see. 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, and 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, and 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, incubated at 37°C overnight, and 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 serine / arginine-rich splicing factor 9 protein, see Figure 1 C.

[0036] Example 2 Expression and purification of serine / arginine-rich splicing factor 9 antigen

[0037] The gene encoding the serine / arginine-rich splicing factor 9 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 contains a His-tagged peptide. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc., and finally the molecular weight of the recombinant protein serine / arginine-rich splicing factor 9 was identified by SDS-PAGE. The results are shown in FIG. Figure 2 .

[0038] Example 3 The present invention uses orthogonal experimental design to optimize the reaction conditions of the kit

[0039] An orthogonal table was selected based on four factors: the coating concentration of the antigen serine / arginine-rich splicing factor 9 (50 μg / ml, 90 μg / ml, 120 μg / ml, and 150 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, two replicates were performed on standard positive and negative sera at each level. 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 the optimal dilution of the secondary antibody 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 90μg / ml, the optimal antigen-antibody reaction time is 30min, and the optimal working dilution of the acridinium ester-labeled anti-human IgG is 1:1000.

[0040] Example 4 Anti-serine / arginine-rich splicing factor 9-IgG antibody solid phase membrane immunoassay kit

[0041] 4.1 Composition of the solid-phase membrane immunoassay kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies:

[0042] Table 1

[0043] Component name describe antigen Recombinant protein serine / arginine-rich splicing factor 9 solid phase carrier Satourius CN140 nitrocellulose membrane Positive quality control (standard) Human anti-His tag IgG Negative control Healthy physical examination serum 10 Labeled secondary antibody Biotinylated anti-human IgG antibody substrate Alkaline phosphatase-streptavidin Color development solution BCIP colorimetric solution

[0044] 4.2 The detection steps of the solid phase membrane immunoassay kit for detecting anti-serine / arginine-rich splicing factor 9-IgG are as follows:

[0045] (1) Coating and blocking: 8 μl of 90 μg / ml serine / arginine-rich splicing factor 9 antigen was directly spotted on the nitrocellulose membrane and incubated at 37°C for 30 min to dry. The nitrocellulose membrane was placed in the detection plate and blocked with 200 μl of 5% BSA in a 37°C incubator for 30 min. The blocking solution was discarded and the membrane was washed twice with washing solution.

[0046] (2) Antigen incubation: Add 10 μl of antibody standard or serum to be tested diluted with diluent to the test plate, and perform negative and positive controls at the same time. Incubate at room temperature for 30 min. Set up three parallel wells for each sample.

[0047] (3) Secondary antibody incubation: discard the liquid in the test plate, wash with washing solution 5 times × 1 min, add 20 μl 1:500 biotin-labeled anti-human IgG antibody, and incubate at room temperature for 30 min;

[0048] (4) Color development: discard the liquid in the test plate, wash with washing solution 5 times for 1 min, add 500 μl alkaline phosphatase-streptavidin, incubate at room temperature for 20 min, discard the liquid in the test plate, wash with washing solution 5 times for 1 min, then add BCIP color development solution, react at room temperature for 20 min, rinse the test plate with running water to terminate the enzyme reaction;

[0049] (5) Take out the test NC membrane strip and blow dry it with a hair dryer. Use the colorimetric card to make a qualitative judgment with the naked eye. If obvious brown spots appear, it is a positive result. 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 level of anti-serine / arginine-rich splicing factor 9-IgG in the serum.

[0050] Example 5 Anti-serine / arginine-rich splicing factor 9-IgG antibody chemiluminescence detection kit

[0051] 5.1 Anti-serine / arginine-rich splicing factor 9-IgG antibody chemiluminescent detection kit, including the following components:

[0052] (1) Acridinium ester-labeled anti-human IgG;

[0053] (2) carboxyl magnetic beads coupled to serine / arginine-rich splicing factor 9 antigen;

[0054] (3) Chemiluminescent pre-excitation solution A and chemiluminescent excitation solution B;

[0055] (4) anti-serine / arginine-rich splicing factor 9-IgG antibody series standard solutions, standard concentrations: 0 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, 20.0 μg / ml, buffer: 0.5-Tris-HCl 5.0% BSA and 0.1-0.5% PC300;

[0056] (5) Cleaning solution, in particular, a 25 mmol / L Tris-HCl solution at pH 7.2 containing 0.15 mol / L NaCL and 0.05% Tween-20.

[0057] 5.2 Preparation of magnetic bead-coupled antigens ( Figure 4 )

[0058] (1) Place 1 mg of carboxyl magnetic particles in a 0.5 mL centrifuge tube, add a certain amount of 0.1 mol / L MES buffer, vortex to mix, place on a magnetic stand, let it stand for 5 minutes to allow the magnetic particles to separate from the liquid, and discard the supernatant. Wash three times, then add a certain amount of MES (pH 5.0) buffer and vortex;

[0059] (2) Add 18 μL (18 μg) of serine / arginine-rich splicing factor 9 antigen, vortex, rotate the reaction tube, and incubate at room temperature for 30 min;

[0060] (3) Add 10 μL of 10 mg / mL coupling reagent EDC, vortex, rotate the reaction tube, and incubate at room temperature for 2 h;

[0061] (4) Remove the supernatant and add 200 μL of washing buffer (TBS + 0.05% Tween-20) and wash three times;

[0062] (5) Block with a buffer solution containing 1% BSA, repeat 4 times, 10 min each time. Store the magnetic particle suspension at 2-8°C.

[0063] 5.3 Preparation of Acridinium Ester-labeled Antibodies

[0064] (1) Place a certain amount of anti-human IgG antibody in a dialysis bag, and then place the dialysis bag in no less than 1 L of labeling buffer for dialysis. During the dialysis, the buffer was changed at least three times, and the last dialysis was performed overnight. The labeling buffer was Na2CO3-NaHCO3 buffer with a pH of 10.1 and a concentration of 0.1 mol / L.

[0065] (2) Weigh 1.7 mg of acridinium ester NSP-DMAE-NHS and dissolve it in 447 μL of anhydrous dimethylformamide (DMF) to form a 6.5 mmol / L NSP-DMAE-NHS DMF solution;

[0066] (3) The dialyzed antibody solution was placed in a 500 μL centrifuge tube, and a certain amount of 6.5 mmol / L NSP-DMAE-NHS DMF solution was added, with a molar ratio of acridinium ester to antibody of 7.4:1. 200 μL of labeling buffer was added, and the reaction was allowed to proceed at room temperature for 45 min. 10 μL of lysine was added, and the reaction was continued for 15 min to terminate the labeling reaction.

[0067] (4) The labeled NSP-DMAE-NHS-Ab was separated from the free NSP-DMAE-NHS by Sephadex G-50 column (1 × 25 cm) using a purification buffer containing pH 6.3 and a concentration of 0.1 mol / L;

[0068] (5) During the separation process, the protein peak was detected by chromatograph, and the chemiluminescence intensity and absorbance at 430 nm of the effluent were measured respectively;

[0069] (6) Collect the eluate with high brightness and high absorbance, add 1% BSA (volume), and store on ice.

[0070] 5.4 Sample preparation: dilute the sample in a certain ratio

[0071] 5.5 The detection steps of the chemiluminescence kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibody are as follows:

[0072] (1) 100 μL of the sample to be tested, 150 μL of the coupled magnetic particle suspension, and 150 μL of the acridinium ester-labeled secondary antibody were added to the reaction tube in sequence, shaken to mix, and incubated at 37°C for 15 min;

[0073] (2) Isolate and wash 5 times;

[0074] (3) Shake the washed reaction container thoroughly to evenly disperse the magnetic particles;

[0075] (4) Add 100 μL of chemiluminescent pre-excitation solution A, and then add 100 μL of chemiluminescent excitation solution B, and measure the relative luminescence intensity. The content of anti-serine / arginine-rich splicing factor 9-IgG antibody in the sample is proportional to its luminescence intensity ( Figure 5 ).

[0076] Example 6 Detection Kit Application

[0077] 6.1 Subject Enrollment: Patients diagnosed with various renal diseases between June 2018 and June 2020, including patients with nephrotic syndrome (n = 466), Henoch-Schonlein purpura (n = 168), purpuric nephritis (n = 137), and IgA nephropathy (n = 133), as well as healthy controls (n = 195), were enrolled. Serum samples were obtained from patients with various renal diseases and healthy controls. All subjects underwent the first serum sample collection before immunosuppressive therapy was initiated.

[0078] 6.2 Detection of Anti-Ser / Arg-Rich Splicing Factor 9 Antibodies in Patients with Various Kidney Diseases. The kit of the present invention was used to detect the level of anti-Ser / Arg-Rich Splicing Factor 9-IgG antibodies in the serum of patients with various kidney diseases. The test results were analyzed using ROC curves to determine the value of the antibodies in diagnosing nephrotic syndrome. The results showed that some patients with autoimmune nephrotic syndrome had autoantibodies to Ser / Arg-Rich Splicing Factor 9 ( Figure 6 ). The ROC curve evaluated the application value of anti-serine / arginine-rich splicing factor 9-IgG antibodies as a serological marker for the differential diagnosis of autoimmune nephrotic syndrome. When anti-serine / arginine-rich splicing factor 9 autoantibodies greater than 21.9 were used as the diagnostic cutoff for diagnosing autoimmune nephrotic syndrome, its sensitivity was 69.3% and specificity was 60.9% ( Figure 7 The results showed that anti-serine / arginine-rich splicing factor 9-IgG antibody is a good serological marker for the differential diagnosis of patients with autoimmune nephrotic syndrome. SEQUENCE LISTING <110> Zhejiang University <120> Kit for detecting anti-serine / arginine-rich splicing factor 9-IgG antibodies <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 221 <212> PRT <213> Artificial sequence (Unknow) <400> 1 Met Ser Gly Trp Ala Asp Glu Arg Gly Gly Glu Gly Asp Gly Arg Ile 1 5 10 15 Tyr Val Gly Asn Leu Pro Thr Asp Val Arg Glu Lys Asp Leu Glu Asp 20 25 30 Leu Phe Tyr Lys Tyr Gly Arg Ile Arg Glu Ile Glu Leu Lys Asn Arg 35 40 45 His Gly Leu Val Pro Phe Ala Phe Val Arg Phe Glu Asp Pro Arg Asp 50 55 60 Ala Glu Asp Ala Ile Tyr Gly Arg Asn Gly Tyr Asp Tyr Gly Gln Cys 65 70 75 80 Arg Leu Arg Val Glu Phe Pro Arg Thr Tyr Gly Gly Arg Gly Gly Trp 85 90 95 Pro Arg Gly Gly Arg Asn Gly Pro Pro Thr Arg Arg Ser Asp Phe Arg 100 105 110 Val Leu Val Ser Gly Leu Pro Pro Ser Gly Ser Trp Gln Asp Leu Lys 115 120 125 Asp His Met Arg Glu Ala Gly Asp Val Cys Tyr Ala Asp Val Gln Lys 130 135 140 Asp Gly Val Gly Met Val Glu Tyr Leu Arg Lys Glu Asp Met Glu Tyr 145 150 155 160 Ala Leu Arg Lys Leu Asp Asp Thr Lys Phe Arg Ser His Glu Gly Glu 165 170 175 Thr Ser Tyr Ile Arg Val Tyr Pro Glu Arg Ser Thr Ser Tyr Gly Tyr 180 185 190 Ser Arg Ser Arg Ser Gly Ser Arg Gly Arg Asp Ser Pro Tyr Gln Ser 195 200 205 Arg Gly Ser Pro His Tyr Phe Ser Pro Phe Arg Pro Tyr 210 215 220

Claims

1. Use of a serine / arginine-rich splicing factor 9 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-serine / arginine-rich splicing factor 9-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The polypeptide sequence of the serine / arginine-rich splicing factor 9 is shown as SEQ ID NO.

1.

2. The method according to claim 1, wherein the nephrotic syndrome is childhood autoimmune nephrotic syndrome.

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

5. Use of a serine / arginine-rich splicing factor 9 polypeptide capable of forming any antigen-antibody complex upon contact with a sample obtained from a patient in the preparation of a reagent or kit for the specific detection of nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy; wherein: The antigen-antibody complex comprises an anti-serine / arginine-rich splicing factor 9-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The polypeptide sequence of the serine / arginine-rich splicing factor 9 is shown as SEQ ID NO.

1.

6. A reagent for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy, comprising: A Serine / arginine-rich splicing factor 9 polypeptide, which is capable of forming any antigen-antibody complex with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-Serine / arginine-rich splicing factor 9-IgG antibody complex; and Carboxyl magnetic beads, wherein the carboxyl magnetic beads are coupled to a serine / arginine-rich splicing factor 9 antigen; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The polypeptide sequence of the serine / arginine-rich splicing factor 9 is shown as SEQ ID NO.

1.

7. A kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy, comprising: A Serine / arginine-rich splicing factor 9 polypeptide, which is capable of forming any antigen-antibody complex with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-Serine / arginine-rich splicing factor 9-IgG antibody complex; and Carboxyl magnetic beads, wherein the carboxyl magnetic beads are coupled to a serine / arginine-rich splicing factor 9 antigen; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The polypeptide sequence of the serine / arginine-rich splicing factor 9 is shown as SEQ ID NO.1.

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