Kit for Detecting Anti-Filamentous Actin Capping Protein β-IgG Antibody

Abstract

The present invention provides a kit for detecting anti-filamentous actin capping protein β-IgG antibodies, which is composed of an antigen protein, filamentous actin capping protein β (F-actin-capping protein subunit beta), a solid-phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate chromogenic agent, a washing solution, a standard product, a positive control product, and a negative control product. The anti-filamentous actin capping protein β-IgG autoantibodies are detected by a specific kit. The present invention for the first time identifies autoantibodies against the target antigen, filamentous actin capping protein β, and provides a detection kit for such autoantibodies. The kit provided by the present invention provides a basis for the research on the molecular mechanism and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to filamentous actin capping protein β and anti-filamentous actin capping protein β-IgG autoantibodies at home and abroad.

Description

[0001] This application is a divisional application of the application with the application number 202110742935.7, the application date of July 1, 2021, and the invention title of "Kit for Detecting Anti-Filamentous Actin Capping Protein β-IgG Antibody". Technical Field

[0002] The present invention belongs to the field of biomedical technologies, and relates to a kit for detecting anti-filamentous actin capping protein β-IgG antibody, and is a kit for detecting anti-filamentous actin capping protein β-IgG antibody in a patient sample. Background Art

[0003] Autoimmune diseases are diseases caused by abnormal immune responses of the human body to substances and tissues that normally exist in the body. They may be limited to certain organs, involve specific tissues in different locations, or may be systemic. The treatment of autoimmune diseases usually involves immunosuppression, that is, drugs that reduce the immune response.

[0004] Primary nephrotic syndrome (PNS) is characterized by edema in patients, 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). The pathological types of more than 90% of children with primary nephrotic syndrome are minimal change or focal segmental glomerulosclerosis, which are mainly characterized by podocyte damage. Inflammation of podocytes increases the permeability to proteins, resulting in increased secreted proteins. When the amount of protein excreted in the urine exceeds the compensatory capacity of the liver, less protein is detected in the blood, especially albumin, which accounts for most of the circulating proteins. As the protein in the blood decreases, the osmotic pressure in the blood decreases. Since the osmotic pressure in tissues remains unchanged, edema occurs. The secretion of aldosterone by the adrenal gland exacerbates this situation, which is secreted according to the decrease in circulating blood, resulting in sodium and water retention. Hyperlipidemia is considered 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). There is evidence that the onset of the vast majority of hormone-sensitive nephrotic syndrome and 2 / 3 of hormone-resistant nephrotic syndrome in children is related to 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 F-actin-capping protein subunit beta in patients with autoimmune nephrotic syndrome. F-actin-capping protein subunit beta is a member of the F-actin capping protein family. This gene encodes the beta subunit of the barbed end actin-binding protein. This protein regulates the growth of actin filaments by covering the barbed ends of growing actin filaments.In muscle, the interaction between filamentous actin capping protein β and actin is crucial during myofibrillogenesis (Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, Solaro RJ. Actin capping protein: an essential element in protein kinase signaling to the myofilaments. Circulation Research. (2002) 90(12):1299–306).

[0005] However, there have been no reports on filamentous actin capping protein β and autoantibodies against filamentous actin capping protein β in urinary system diseases, especially in nephrotic syndrome. Additionally, in the existing technologies, there is no application involving using the target filamentous actin capping protein β or its autoantibodies as a serological marker in autoimmune nephrotic syndrome. The research on differentiating autoimmune nephrotic syndrome by detecting serum anti-filamentous actin capping protein β-IgG antibodies is blank. Summary of the Invention

[0006] The objective of the present invention is to provide a kit for detecting anti-filamentous actin capping protein β-IgG antibodies, which is a detection kit based on the target filamentous actin capping protein β and its corresponding autoantibodies. The kit can detect autoantibodies in tissues (renal biopsy tissues) or body fluids (especially blood, plasma, serum) through antigen-antibody reactions with the antigen protein filamentous actin capping protein β (specifically shown according to Sequence Identification Number SEQ ID NO.1).

[0007] The present invention relates to a detection kit for detecting anti-filamentous actin capping protein β autoantibodies, which kit contains the antigen protein filamentous actin capping protein β, a solid-phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate chromogenic agent, a washing solution, a termination solution, a standard product, a positive control product, and a negative control product.

[0008] According to the present invention, the antigen protein filamentous actin capping protein β 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 tags with certain biological or physical functions, and the presence of these tags is beneficial for the purification, immobilization, precipitation of the antigen protein or the identification of other sequence motifs or polypeptides of the polypeptides of the present invention. In a more preferred embodiment, the tag is a sequence or domain capable of specifically binding a ligand, for example, selected from: GST tag, c-Myc tag, His tag, Flag tag or biotin tag.

[0010] According to the present invention, the antigen protein filamentous actin capping protein β is immobilized on a solid-phase carrier, and the preferred solid-phase carriers include: nitrocellulose membrane, magnetic beads, enzyme-labeled microtiter plate.

[0011] In one embodiment of the present invention, the standard and positive control are recombinant human anti-tag peptide IgG or its fragment, or the anti-filamentous actin capping protein β antibody extracted from patient serum is used as the positive control and standard, and the serum of healthy subjects undergoing physical examination is used as the negative control.

[0012] According to the present invention, the antigen protein filamentous actin capping protein β is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, and hydrophobic column.

[0013] The sample herein is a sample of body fluid or tissue to be tested. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. In addition, after separation or purification procedures, such as separating whole blood into serum or plasma components, certain test samples will be easier to analyze. Therefore, in a preferred embodiment of the present invention, the sample is selected from blood samples, serum samples, plasma samples, cerebrospinal fluid samples, saliva samples, and urine samples or extracts of any of the above samples. Preferably, the sample is a blood sample, more preferably a serum sample or a plasma sample.

[0014] The detection kit further includes a substrate chromogenic agent, an antigen diluent, a sample dilution buffer, an antibody diluent, a washing solution, and a termination solution. The substrate chromogenic agent is TMB, BCIP, AMPPD, 4-MUP; the antigen diluent is pH 7.4 1xPBS, 163 mM NaCl, 1% TritonX-100; the sample dilution buffer is 0.01 M PBS buffer containing 10% BSA at pH 7.4; the antibody diluent is prepared by adding 1 M D glucose (19.82 g), 2% glycerol (2 ml) to 0.01 M PBS containing 0.2% Tween20 to 100 ml; the washing solution is: 1x PBS pH 7.4, 163 mM NaCl, 1% TritonX-100, 10% glycerol; the termination solution is: 2 M sulfuric acid.

[0015] The method for immobilizing the antigen protein filamentous actin capping protein β of the present invention is the direct coating method: 1. The antigen is physically adsorbed or non-covalently bound to a nitrocellulose membrane or a polystyrene microplate; 2. The magnetic particles with carboxyl functional groups are bound to the protein amino group, and the antigen is chemically coupled to the magnetic particles.

[0016] The labeled antibodies 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 expresses and purifies the recombinant protein filamentous actin capping protein β by gene recombination prokaryotic expression method, and uses this as the antigen protein in the kit to develop a set of kits suitable for detecting anti-filamentous actin capping protein β-IgG antibodies in patients with autoimmune nephrotic syndrome. It is a detection kit for qualitatively or quantitatively analyzing anti-filamentous actin capping protein β-IgG antibodies in human serum.

[0018] The principle of a kit for detecting anti-filamentous actin capping protein β-IgG antibodies in serum is to adsorb the antigen on a solid-phase carrier as the coated antigen, then add a positive control product or a standard product or a serum sample to be tested for incubation, and then add a labeled secondary antibody for reaction to form a ternary complex of coated antigen filamentous actin capping protein β - serum to be tested anti-filamentous actin capping protein β-IgG antibody - labeled anti-human IgG antibody. Finally, optical signal is detected by light chromogenic method, chemiluminescence method, or fluorescence luminescence method to achieve the purpose of qualitatively or quantitatively analyzing anti-filamentous actin capping protein β-IgG antibodies in human serum.

[0019] The sequence of the antigen protein filamentous actin capping protein β is shown in SEQ ID NO.1:

[0020] MSDQQLDCALDLMRRLPPQQIEKNLSDLIDLVPSLCEDLLSSVDQPLKI ARDKVVGKDYLLCDYNRDGDSYRSPWSNKYDPPLEDGAMPSARLRKLEVEANNAFDQYRDLYFEGGVSSVYLWDLDHGFAGVILIKKAGDGSKKIKGCW DSIHVVEVQEKSSGRTAHYKLTSTVMLWLQTNKSGSGTMNLGGSLTRQMEKDETVSDCSPHIANIGRLVEDENKIRSTLNEIYFGKTKDIVNGLRSIDAIPDNQ KFKQLQRELSQVLTQRQIYIQPDN.

[0021] Specific innovations can be summarized as follows:

[0022] (1) The present invention has identified for the first time autoantibodies against actin-capping protein β, and has invented a detection kit for such autoantibodies.

[0023] (2) Currently, there is no kit for detecting the level of autoantibodies against actin-capping protein β at home and abroad. This invention fills the gap in the detection kit for autoantibodies against actin-capping protein β at home and abroad.

[0024] (3) The advantage of the chemiluminescence method lies in the simplicity of the determination. 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; it has a small molecular weight, avoiding shielding of antibody binding sites, improving the overall sensitivity of the system, and having a rapid reaction; it has a low background and a high signal-to-noise ratio, and is an effective chemiluminescence label.

[0025] (4) The solid-phase membrane immunoassay greatly shortens the detection time and can achieve the effect of rapid detection. The kit for detecting actin-capping protein β-IgG antibodies introduces a biotin-avidin amplification system, greatly improving the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Actin-capping protein β protein on podocytes is one of the target antigens 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 electrophoresis protein spots of serum from patients with autoimmune nephrotic syndrome; Figure 1 C: Mass spectrometry identification of the target antigen actin-capping protein β protein.

[0027] Figure 2 : SDS-PAGE identification diagram of recombinant protein actin-capping protein β.

[0028] Figure 3 : Detection of actin-capping protein β-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome by a solid-phase membrane immunoassay kit.

[0029] Figure 4 : Schematic diagram of the detection principle of a chemiluminescence kit for detecting actin-capping protein β-IgG antibodies.

[0030] Figure 5 : Schematic diagram of carboxyl magnetic microparticles coated with the antigen protein actin-capping protein β.

[0031] Figure 6 : Detection of anti-filamentous actin capping protein β-IgG antibodies in various types of nephropathy patients, where PNS: autoimmune nephrotic syndrome, HSP: Henoch-Schönlein purpura, HSPN: Henoch-Schönlein purpura nephritis, IgAN: IgA nephropathy, NC: healthy children.

[0032] Figure 7 : Application value of ROC curve in evaluating anti-filamentous actin capping protein β antibody as a serological marker for differential diagnosis of autoimmune nephrotic syndrome. Specific implementation mode

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0034] Example 1 Filamentous actin capping protein β on podocytes is one of the target antigens of autoantibodies in patients with autoimmune nephrotic syndrome. Through a large number of previous clinical and molecular mechanism studies, the present invention first found that the serum IgG level in patients with nephrotic syndrome is relatively high, and confirmed that filamentous actin capping protein β on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative level of anti-filamentous actin capping protein β-IgG antibodies in serum is helpful for the early differential diagnosis of autoimmune nephrotic syndrome, especially for screening patients with related symptoms.

[0035] The specific implementation is as follows: (1) Extraction of total protein from glomerular podocytes: Cultivate the podocyte cell line (MPC5), wash it 2-3 times with PBS, and then use a focused ultrasound instrument (Covaris S220, Gene) to fully lyse it on ice in a lysis buffer containing 30 mM Tris-HCl, 8 M urea, 4% CHAPS, and a protease inhibitor (#ab65621; Abcam, diluted 1:200). Then place the sample in a centrifuge, centrifuge at 12000 g, 4 °C for 30 min. Collect the supernatant, which is the total protein of glomerular podocytes collected. Use a BCA protein concentration assay kit to measure the concentration of the total protein of glomerular podocytes collected. (2) Two-dimensional electrophoresis: After extracting the total protein of glomerular podocytes and performing two-dimensional electrophoresis, transfer it to a nitrocellulose membrane, incubate it with the sera of healthy people and patients with autoimmune nephrotic syndrome as primary antibodies respectively, 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 the development in step (2), perform differential analysis of positive spots. Select the protein spots that are strongly positive in patients with nephrotic syndrome and negative or weakly positive in healthy people on the two-dimensional electrophoresis gel. Cut the selected protein spots from the gel. Digest the dried gel with trypsin (0.1 μg / μl), then add 10 μl of 25 mM ammonium bicarbonate to the reaction mixture and incubate overnight at 37 °C. Then extract the peptides from the gel with trifluoroacetic acid (0.1%). Analyze the extracted peptides with a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass map, which is identified as filamentous actin capping protein β protein, as shown in Figure 1 C.

[0036] Example 2 Expression and purification of filamentous actin capping protein β antigen

[0037] Using genetic engineering methods, use the gene encoding filamentous actin capping protein β as a template for PCR amplification, and then construct an expression vector for protein expression. The antigen protein expressed in the present invention contains a tag peptide with a His tag. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc. Finally, use SDS-PAGE to identify the molecular weight of the recombinant protein filamentous actin capping protein β, and the results are shown in Figure 2 。

[0038] Example 3 Optimization of the reaction conditions of the kit by orthogonal experimental design in the present invention

[0039] According to the antigen coating concentration against filamentous actin capping protein β (four coating concentrations of 50 μg / ml, 90 μg / ml, 120 μg / ml, 150 μg / ml), each reaction time (30 min, 45 min) and temperature (25 °C, 35 °C), the optimal dilution of the enzyme-labeled secondary antibody (four dilutions of 1:100, 1:500, 1:1000, 1:1500), etc., select an orthogonal table for 4 factors. Each factor is repeated to measure the standard positive serum and standard negative serum at 2 levels. Select the ratio (P / N) of the highest luminescence value (P) of the positive serum and the lowest luminescence value (N) of the negative serum. The average P / N value of the repeated measurements is statistically processed to determine the optimal coating conditions and the optimal dilution of the secondary antibody for orthogonal optimization conditions, significantly improving the positive detection rate of the standard positive serum. Through orthogonal design, we obtained the optimal antigen coating concentration of 90 μg / ml, the optimal antigen-antibody reaction time of 30 min, and the optimal working dilution of acridinium ester-labeled anti-human IgG of 1:1000 for this kit.

[0040] Example 4 Solid-phase membrane immunoassay kit for anti-filamentous actin capping protein β-IgG antibody

[0041] 4.1 Composition of the solid phase membrane immunoassay kit for detecting anti-filamentous actin capping protein β-IgG antibodies:

[0042] Table 1

[0043]

[0044] 4.2 The detection steps of the solid phase membrane immunoassay kit for detecting anti-filamentous actin capping protein β-IgG are as follows:

[0045] (1) Coating and blocking: 8 μl of 90 ug / ml filamentous actin capping protein β antigen was directly spotted on the nitrocellulose membrane and dried in a 37°C incubator for 30 min. The nitrocellulose membrane was placed in the detection plate, and 200 μl of 5% BSA was added and blocked in a 37°C incubator for 30 min. The blocking solution was discarded and 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 3 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 NC film strip for testing and blow dry it with a hair dryer. Use a 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 and scan it. 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 to perform semi-quantitative analysis of the level of anti-filamentous actin capping protein β-IgG in serum.

[0050] Example 5 Anti-filamentous actin capping protein β-IgG antibody chemiluminescence detection kit

[0051] 5.1 Anti-filamentous actin capping protein β-IgG antibody chemiluminescent detection kit, including the following components:

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

[0053] (2) Carboxyl magnetic beads conjugated with filamentous actin capping protein β antigen;

[0054] (3) Chemiluminescence pre-excitation solution A and chemiluminescence excitation solution B;

[0055] (4) Anti-filamentous actin capping protein β-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 is Tris-HCL containing 0.5 - 5.0% BSA and 0.1 - 0.5% PC300;

[0056] (5) Cleaning solution, especially a pH 7.2, 25 mmol / L Tris-HCL solution containing 0.15 mol / L NaCl and 0.05% Tween-20.

[0057] 5.2 Preparation of magnetic beads conjugated with antigen ( Figure 4 )

[0058] (1) Take 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 and mix well, place on a magnetic rack, let stand for 5 min to separate the magnetic particles from the liquid, and discard the supernatant. Wash 3 times, then add a certain amount of MES (pH 5.0) buffer and vortex;

[0059] (2) Add 18 μL (18 μg) of filamentous actin capping protein β 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, add 200 μL of washing buffer (TBS + 0.05% Tween-20) and wash 3 times;

[0062] (5) Block with buffer 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 antibody

[0064] (1) Put a certain amount of anti-human IgG antibody into a dialysis bag, place the dialysis bag in no less than 1 L of labeling buffer for dialysis, change the buffer at least 3 times during the period, and dialyze overnight for the last time. The labeling buffer is Na2CO3-NaHCO3 buffer, pH is 10.1, and the concentration is 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) Place the dialyzed antibody solution in a 500 μL centrifuge tube, add a certain amount of 6.5 mmol / L NSP-DMAE-NHS DMF solution, with a molar ratio of acridinium ester to antibody of 7.4:1. Add 200 μL of labeling buffer and react at room temperature for 45 min. Then add 10 μL of lysine and continue to react for 15 min to terminate the labeling reaction;

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

[0068] (5) During the separation process, detect the protein peak with a chromatograph, and measure the chemiluminescence intensity and absorbance at 430 nm of the eluate respectively;

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

[0070] 5.4 Sample preparation: Dilute the sample in a certain proportion

[0071] 5.5 The detection steps of the chemiluminescence kit for detecting anti-filamentous actin capping protein β-IgG antibody are as follows:

[0072] (1) Add 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 into the reaction tube in sequence, shake well and mix, and incubate at 37 °C for 15 min;

[0073] (2) Wash and isolate 5 times;

[0074] (3) Vigorously shake the washed reaction vessel to evenly disperse the magnetic particles;

[0075] (4) Add 100 μL of chemiluminescence pre-excitation solution A, and then add 100 μL of chemiluminescence excitation solution B, and measure its relative luminescence intensity. The content of anti-filamentous actin capping protein β-IgG antibody in the sample is proportional to its luminescence intensity ( Figure 5 ).

[0076] Example 6 Application of the detection kit

[0077] 6.1 Subject inclusion: Patients diagnosed with various kidney diseases from June 2018 to June 2020, including patients with nephrotic syndrome (n = 466), patients with Henoch-Schönlein purpura (n = 168), patients with purpuric nephritis (n = 137), patients with IgA nephropathy (n = 133), and healthy individuals (n = 195). Serum samples were taken from patients with various kidney diseases and the healthy control group. The first serum sample was collected from all subjects before immunosuppressive therapy.

[0078] 6.2 Detection of anti-filamentous actin capping protein β antibody in patients with various kidney diseases. The levels of anti-filamentous actin capping protein β-IgG antibody in the sera of patients with various kidney diseases were detected using the kit of the present invention, and the detection results were analyzed by ROC curve to judge the value of this antibody in the diagnosis of nephrotic syndrome. Compared with the control group, the prevalence of Ab against filamentous actin capping protein β in the patient cohort was significantly higher ( Figure 6 ). The ROC curve evaluated the application value of anti-filamentous actin capping protein β-IgG antibody as a serological marker for the diagnosis of PNS patients. When the diagnostic cut-off value was greater than 57.3, the sensitivity of anti-filamentous actin capping protein β autoantibody in the diagnosis of nephrotic syndrome by autoantibody detection was 67.0%, and the specificity was 73.5% ( Figure 7 ). The results showed that anti-filamentous actin capping protein β-IgG antibody is a good serological marker for the differential diagnosis of autoimmune nephrotic syndrome. SEQUENCE LISTING <110> Zhejiang University <120> Kit for detecting anti-filamentous actin capping protein β-IgG antibody <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 276 <212> PRT <213> Artificial Sequence (Unknow) <400> 1 Met Ser Asp Gln Gln Leu Asp Cys Ala Leu Asp Leu Met Arg Arg Leu 1 5 10 15 Pro Pro Gln Gln Ile Glu Lys Asn Leu Ser Asp Leu Ile Asp Leu Val 20 25 30 Pro Ser Leu Cys Glu Asp Leu Leu Ser Ser Val Asp Gln Pro Leu Lys 35 40 45 Ile Ala Arg Asp Lys Val Val Gly Lys Asp Tyr Leu Leu Cys Asp Tyr 50 55 60 Asn Arg Asp Gly Asp Ser Tyr Arg Ser Pro Trp Ser Asn Lys Tyr Asp 65 70 75 80 Pro Pro Leu Glu Asp Gly Ala Met Pro Ser Ala Arg Leu Arg Lys Leu 85 90 95 Glu Val Glu Ala Asn Asn Ala Phe Asp Gln Tyr Arg Asp Leu Tyr Phe 100 105 110 Glu Gly Gly Val Ser Ser Val Tyr Leu Trp Asp Leu Asp His Gly Phe 115 120 125 Ala Gly Val Ile Leu Ile Lys Lys Ala Gly Asp Gly Ser Lys Lys Ile 130 135 140 Lys Gly Cys Trp Asp Ser Ile His Val Val Glu Val Gln Glu Lys Ser 145 150 155 160 Ser Gly Arg Thr Ala His Tyr Lys Leu Thr Ser Thr Val Met Leu Trp 165 170 175 Leu Gln Thr Asn Lys Ser Gly Ser Gly Thr Met Asn Leu Gly Gly Ser 180 185 190 Leu Thr Arg Gln Met Glu Lys Asp Glu Thr Val Ser Asp Cys Ser Pro 195 200 205 His Ile Ala Asn Ile Gly Arg Leu Val Glu Asp Glu Asn Lys Ile Arg 210 215 220 Ser Thr Leu Asn Glu Ile Tyr Phe Gly Lys Thr Lys Asp Ile Val Asn 225 230 235 240 Gly Leu Arg Ser Ile Asp Ala Ile Pro Asp Asn Gln Lys Phe Lys Gln 245 250 255 Leu Gln Arg Glu Leu Ser Gln Val Leu Thr Gln Arg Gln Ile Tyr Ile 260 265 270 Gln Pro Asp Asn 275

Claims

1. Use of filamentous actin-capping protein subunit beta polypeptide capable of forming any antigen-antibody complex by contacting with a sample obtained from a patient in the preparation of a detection reagent or kit for nephrotic syndrome; wherein, The antigen-antibody complex includes an anti-filamentous actin capping protein beta-IgG antibody (anti-F-actin-capping protein subunit beta-IgG antibody) complex; wherein the nephrotic syndrome is an autoimmune nephrotic syndrome; wherein the amino acid sequence of the filamentous actin capping protein beta is as shown in SEQ ID NO.

1.

2. The use 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.

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

5. Use of the filamentous actin-capping protein subunit beta 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 specifically detecting nephrotic syndrome relative to Henoch-Schönlein purpura nephritis, allergic purpura, and IgA nephropathy; wherein, The antigen-antibody complex includes an anti-filamentous actin capping protein beta-IgG antibody (anti-F-actin-capping protein subunit beta-IgG antibody) complex; wherein the nephrotic syndrome is an autoimmune nephrotic syndrome; wherein the amino acid sequence of the filamentous actin capping protein beta is as shown in SEQ ID NO.1.

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