A kit for detecting anti-focal adhesion protein-IgG antibodies

Abstract

The present invention provides a kit for detecting anti-vinculin-IgG antibodies, which consists of an antigen protein Vinculin (vinculin), 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 substance, and a negative quality control substance. The kit of the present invention utilizes the indirect reaction principle combined with magnetic particle chemiluminescence immunoassay to detect anti-Vinculin-IgG antibodies in a sample to be tested. The present invention identifies for the first time autoantibodies against the target antigen Vinculin in the serum of patients with autoimmune nephrotic syndrome, 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 Vinculin and Vinculin-IgG autoantibodies at home and abroad.

Description

[0001] This application is a divisional application of application number 202110743524.X, application date July 1, 2021, and invention name A kit for detecting anti-adhesion protein-IgG antibodies. Technical Field

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

[0003] In recent years, the incidence of pediatric kidney diseases has increased, with autoimmune nephrotic syndrome (ANS) being the most prevalent, and seriously endangering children's physical and mental health. ANS is a clinical syndrome characterized by increased proteinuria, hypoproteinemia, and severe edema, caused by increased permeability of the glomerular filtration membrane. Ali et al. observed that transplanted kidneys from patients with refractory minimal change disease (MCH) resulted in normal renal function and no proteinuria in the recipients. This suggests that MCH is not primarily due to the kidneys themselves, but rather to problems in the patient's internal environment. Furthermore, with the exception of some children with genetic defects, the majority of children with ACH improve with steroids and immunosuppressive therapy, indirectly demonstrating a close link between the disease and the patient's autoimmune system.

[0004] In recent years, B cell dysfunction has been shown to play a significant role in autoimmune nephrotic syndrome. In recent years, multicenter clinical studies worldwide have demonstrated the successful use of rituximab (RTX) in the treatment of minimal change disease, particularly in refractory nephrotic syndrome. However, studies have also shown that the B cell depletion effect of rituximab in steroid-dependent nephrotic syndrome persists for approximately five months, with relapse occurring after six to seven months as B cell counts recover. This suggests that pathological B cell clones exist in patients with autoimmune nephrotic syndrome. Identifying and precisely eliminating these pathological B cell clones not only facilitates recovery from autoimmune nephrotic syndrome but also reduces the risk of humoral immune deficiency associated with indiscriminate B cell depletion with rituximab and other treatments. However, the target antigens of these pathological B cells in children with autoimmune nephrotic syndrome remain unclear. Pathologically, minimal change disease or focal segmental glomerulosclerosis is believed to be a podocyte disease caused by loss or alteration of podocyte function, leading to high levels of proteinuria. Podocytes are renal glomerular epithelial cells that are attached to the outer side of the glomerular basement membrane and serve as the final barrier to prevent protein loss. Podocyte damage often leads to high levels of proteinuria.

[0005] Vinculin (focal adhesion protein) is a cytoplasmic protein that binds to actin and participates in cell adhesion. There are numerous studies on vinculin, including one study that found that anti-vinculin autoantibody titers were inversely correlated with the density of interstitial cells of Cajal (ICC) in the gastric myogenic plexus of gastric cancer patients (Kim JH, Nam SJ, Park SC, et al. Association between interstitial cells of Cajal and anti-vinculin antibodies in human stomach. Korean J Physiol Pharmacol 24:185–191). Vinculin protein has also been studied in autoimmune diseases. For example, Brittany L. Adler et al. found that systemic sclerosis patients had higher levels of anti-vinculin autoantibodies compared to healthy controls, and vinculin autoantibodies may also play an important role in the pathogenesis of gastrointestinal dysfunction in scleroderma (Brittany L, Adler1, Zsuzsanna McMahan. Anti-vinculin autoantibodies in systemic sclerosis: a step toward a novel biomarker? Clinical Rheumatology (2021) 40:809–811). Vinculin antibodies have also been studied in irritable bowel syndrome. For example, Luo Chaoling et al. developed a vinculin antibody detection kit (Luo Chaoling, Chen Xiao, Dong Min. A vinculin antibody detection kit for an irritable bowel syndrome marker and its preparation method. Application number: 201610325264.3).

[0006] However, there are currently no reports on the expression of Vinculin and the presence of Vinculin autoantibodies in nephrotic syndrome. In addition, the prior art does not involve the use of target Vinculin or its autoantibodies as a serological marker in autoimmune nephrotic syndrome. Research on identifying autoimmune nephrotic syndrome by detecting serum anti-Vinculin-IgG antibodies is blank. Compared with the irritable bowel syndrome marker Vinculin antibody detection kit developed by Luo Chaoling et al., the kit of the present invention can both qualitatively and quantitatively detect anti-Vinculin-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome. The kit of the present invention uses human anti-tag peptide IgG antibodies as a standard and combines a biotin-avidin amplification system and magnetic microparticle chemiluminescence immunoassay to greatly improve the accuracy, sensitivity, specificity and detection speed of the test. Summary of the Invention

[0007] The present invention provides a kit for detecting anti-vinculin IgG antibodies, specifically targeting vinculin and its corresponding autoantibodies. The kit can detect autoantibodies in tissues (e.g., renal biopsy tissue) or body fluids (e.g., blood, plasma, serum) through an immune reaction with the antigenic protein vinculin (particularly as represented by SEQ ID NO. 1).

[0008] The kit consists of an antigen protein Vinculin, a solid phase carrier, a labeled antibody such as an enzyme-labeled or chemiluminescent-labeled or biotin-labeled secondary antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color developer, a washing solution, a standard, a positive quality control product, and a negative quality control product.

[0009] The sequence of the antigen protein Vinculin is shown in SEQ ID NO.1:

[0010] PKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLDSGYRILGAVAKVREAFQPQEPDFPPPPQLRLTDELAPPKPPLPEGEVPPPRPPPPEEKDEEFPEQKAGEVINQPMMMAARQLHDEARKWSSKGNDIIAAAKRMALLMAEMSRLVRGGSGTKRALIQCAKDIAKASDEVTRLAK EVAKQCTDKRLLQVCERIPTISTQLKILSTVKATMLGRTNISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRTDAGFTLRWVRKTPWYQ.

[0011] The Vinculin antigen protein of the present invention may be a fusion protein, using a tag with certain biological or physical functions, particularly at the N-terminus or C-terminus. The presence of such a tag facilitates purification, immobilization, and precipitation of the antigen protein. In a preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, and the tag peptide is selected from the group consisting of: a His tag, thioredoxin, a GST tag, maltose binding protein, the SA tag of glutathione transferase, a c-Myc tag, a Flag tag, or a biotin tag.

[0012] According to the present invention, the antigen protein Vinculin is fixed on a solid phase carrier, and preferred solid phase carriers include: nitrocellulose membrane, magnetic particles, and enzyme-labeled microplates.

[0013] In one embodiment of the present invention, the standard and positive control are recombinant human anti-tag peptide immunoglobulin G or its fragments, or anti-Vinculin-IgG antibodies extracted from patient serum as positive control and standard, and the serum of healthy subjects is used as negative control.

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

[0015] According to the present invention, the antigen protein Vinculin is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, and hydrophobic column.

[0016] According to the present invention, the biological sample is a sample containing autoantibodies, selected from whole blood, serum, plasma, urine, lymph, pleural effusion and ascites, preferably mammalian (human) serum.

[0017] The detection kit also includes a substrate colorimeter, an antigen diluent, a sample dilution buffer, an antibody diluent, and a washing solution. The substrate colorimeter includes TMB, hydrogen peroxide, AMPPD, 4-MUP, and BCIP; the antigen dilution includes 1x PBS pH 7.4 containing 163mM NaCL and 1% Triton X-100; the sample dilution buffer includes 0.01M PBS pH 7.4 containing 10% BSA; the antibody dilution includes 0.01M PBS pH 7.4 containing 1M D-glucose, 2% glycerol, and 0.35% Tween 20; and the washing solution includes 1x PBS pH 7.4 containing 163mM NaCL, 10% glycerol, and 1% Triton X-100.

[0018] In a preferred embodiment, as described herein, "immobilization" refers to binding to a water-insoluble solid support of the Vinculin antigen protein, wherein the solid support or support is water-insoluble, more preferably by covalent bonding, electrostatic interaction, hydrophobic interaction, or disulfide interaction, and most preferably by one or more covalent bonds. Immobilization can be a direct immobilization method, such as separating the immobilized molecule from the aqueous solution together with the insoluble support by filtration, centrifugation or chromatography. It also includes reversible or irreversible immobilization of the Vinculin antigen protein. For example, the antigen protein is immobilized to the support by a cleavable covalent bond (such as a disulfide bond that can be cleaved by adding a thiol-containing reagent), and this immobilization is reversible. In addition, if the antigen protein is immobilized to the support by a covalent bond that will not be cleaved in aqueous solution (a bond formed by the reaction of an epoxide group with an amine group that couples the lysine side chain to an affinity column), then the immobilization is irreversible. Immobilization can also be an indirect method: such as immobilizing an antibody with a specific affinity for the antigen protein, and then forming an antigen protein-antibody complex to achieve the effective purpose of immobilization.

[0019] The method for fixing the antigen protein Vinculin described in the present invention is a direct coating method: (1) the antigen protein Vinculin is bound to a nitrocellulose membrane or a polystyrene microplate by physical adsorption or non-covalent bonding; (2) magnetic particles with carboxyl functional groups are bound to the amino groups of the antigen protein Vinculin, and the antigen protein Vinculin is bound to the magnetic particles by chemical coupling.

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

[0021] The present invention successfully expressed and purified the recombinant protein Vinculin using a prokaryotic expression method. This protein serves as the antigen in a test kit, developing a kit suitable for detecting anti-Vinculin-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome. The kit includes a qualitative or quantitative assay for detecting anti-Vinculin-IgG antibodies in human serum.

[0022] The principle of a kit for detecting anti-Vinculin-IgG antibodies in serum utilizes the indirect reaction principle. First, Vinculin antigen is adsorbed on a solid phase carrier as a coating antigen. Then, a positive quality control product or a standard product or a serum sample to be tested is added for incubation. After the labeled secondary antibody is added for reaction, if the serum to be tested contains anti-Vinculin-IgG antibodies, a ternary complex of coating antigen Vinculin-anti-Vinculin-IgG antibodies in the serum to be tested-labeled anti-human IgG antibodies is formed. Finally, light signals are detected using a photochromic method, a chemiluminescence method, or a fluorescence method to achieve the purpose of qualitative or quantitative analysis of anti-Vinculin-IgG antibodies in human serum.

[0023] The kit of the present invention was used to detect, for the first time, an anti-Vinculin-IgG autoantibody in some patients with autoimmune nephrotic syndrome. The target antigen of the autoantibody was determined to be vinculin, a focal adhesion protein on podocytes. Therefore, the kit of the present invention can be used to detect anti-Vinculin-IgG autoantibodies, providing a basis for studying the molecular mechanisms and clinical diagnosis and treatment of autoimmune nephrotic syndrome.

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

[0025] (1) Currently, research on vinculin and anti-vinculin-IgG antibodies in patients with kidney disease is limited to molecular mechanism studies, and there is no quantitative detection of their levels in patient serum. The present invention identifies autoantibodies against vinculin for the first time and invents a detection kit for the vinculin-IgG autoantibodies, filling this gap both domestically and internationally. The kit of the present invention was used to detect anti-vinculin-IgG antibodies in the serum of 466 patients with nephrotic syndrome. The results showed that 262 patients were positive for anti-vinculin-IgG antibodies, i.e., the positive detection rate for anti-vinculin-IgG antibodies was 56.2%.

[0026] (2) The kit of the present invention relates to a solid-phase membrane immunoassay for qualitative analysis of anti-Vinculin-IgG antibodies in human serum, using human anti-tag peptide IgG antibodies as a standard, which greatly improves the accuracy of the test. The solid-phase membrane immunoassay is simple to operate and uses less reagents, saving nearly 10 times the traditional ELISA. In addition, the NC membrane has an extremely strong adsorption capacity close to 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane. The NC membrane that has adsorbed antigens or antibodies or has a result can be stored for a long time (can be stored at -20°C for half a year) without affecting its activity. In addition, the kit of the present invention for qualitative detection of anti-Vinculin-IgG antibodies in human serum by solid-phase membrane immunoassay introduces a biotin-avidin amplification system, which greatly improves the detection sensitivity.

[0027] (3) The present invention relates to a magnetic microparticle chemiluminescence immunoassay kit for quantitatively detecting anti-Vinculin-IgG antibodies in human serum. The kit utilizes magnetic microparticles as solid-phase carriers with a diameter of only 1.0 μm. This significantly increases the coating surface area, increases the amount of antigen adsorbed, speeds up the reaction, and simplifies cleaning and separation, thereby reducing contamination and the probability of cross-infection. Furthermore, the anti-human IgG is directly labeled with an acridinium ester luminescent agent, resulting in a simple, rapid, and catalyst-free chemical reaction. Acridinium ester chemiluminescence is a flash-type agent, reaching maximum emission intensity 0.4 seconds after initiation of the luminescent reagent (H2O2, NaOH), with a half-life of 0.9 seconds and essentially complete emission within 2 seconds, facilitating rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Vinculin protein on podocytes is the main 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 Vinculin protein.

[0029] Figure 2 : SDS-PAGE identification of the expressed recombinant protein Vinculin.

[0030] Figure 3 : Solid phase membrane immunoassay kit for detecting anti-Vinculin-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome.

[0031] Figure 4 : Schematic diagram of the principle of detecting anti-Vinculin-IgG antibodies using magnetic particle chemiluminescence immunoassay kit.

[0032] Figure 5 : Schematic diagram of antigen protein Vinculin coated carboxyl magnetic particles.

[0033] Figure 6 :Detection of anti-Vinculin-IgG antibodies in patients with various kidney diseases, including NS: autoimmune nephrotic syndrome, HSP: Henoch-Schonlein purpura, HSPN: purpuric nephritis, IgAN: IgA nephropathy, NC: healthy children.

[0034] Figure 7 : Receiver operating characteristic (ROC) curve was used to evaluate the application value of anti-Vinculin-IgG antibody as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome. DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1 Vinculin protein on podocytes is the main target antigen of autoantibodies in patients with autoimmune nephrotic syndrome

[0037] Through a large number of clinical and molecular mechanism studies in the early stage, the present invention has found for the first time that the serum IgG level of patients with nephrotic syndrome is high, and confirmed that Vinculin on podocytes is the main target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence of anti-Vinculin-IgG antibodies in serum and their quantitative levels are helpful for the early identification of autoimmune nephrotic syndrome, especially for screening patients with related symptoms. The specific implementation is as follows (1) Extraction of total protein of glomerular podocytes: culture podocyte cell line (MPC5), wash with PBS 2-3 times, and then use focused ultrasound instrument (Covaris S220, Gene) to fully lyse on ice in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitors (#ab65621; Abcam, 1:200 dilution), and then place the sample in a centrifuge, 12000g, 4℃, centrifuge for 30min. Collect the supernatant, which is the collected total protein of glomerular podocytes. The total protein concentration of the collected glomerular podocytes was determined using a BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: The total protein of the glomerular podocytes was extracted and subjected to two-dimensional electrophoresis and then transferred to a nitrocellulose membrane. The membrane was incubated with the serum of healthy individuals and patients with autoimmune nephrotic syndrome as the primary antibody, and then developed with a secondary antibody. Figure 1 A, 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 Vinculin protein. Figure 1 C.

[0038] Example 2 Expression and purification of recombinant antigen protein Vinculin

[0039] The gene encoding Vinculin 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 Vinculin, which was 37KDa. Figure 2 .

[0040] Example 3 The present invention uses orthogonal experimental design to optimize the reaction conditions of the kit

[0041] An orthogonal table was selected based on four factors: the coating concentration of the antigen Vinculin (four coating concentrations of 50μg, 80μg, 100μg, and 150μg), the reaction time (15min, 30min, and 45min), the temperature (25℃ and 37℃), and the optimal dilution of the enzyme-labeled secondary antibody (four dilutions of 1:100, 1:500, 1:1000, and 1:1500). For each factor, the standard positive serum and the standard negative serum were repeatedly measured at two levels, and the ratio (P / N) of the highest light signal value (P) of the positive serum to the lowest light signal value (N) of the negative serum was selected. Through orthogonal design, we obtained that the optimal antigen Vinculin coating concentration of this kit is 80μg / ml, the optimal antigen-antibody reaction temperature of the solid phase membrane immunoassay anti-Vinculin-IgG antibody kit is 25℃, the optimal antigen-antibody reaction time is 30min, and the optimal working dilution of the optimal biotin-labeled anti-human IgG antibody is 1:500; the optimal antigen-antibody reaction temperature of the magnetic particle chemiluminescence immunoassay anti-Vinculin-IgG antibody kit is 37℃, the optimal antigen-antibody reaction time is 15min, and the optimal working dilution of the optimal acridinium ester-labeled anti-human IgG antibody is 1:500.

[0042] Example 4 Preparation of a Solid-Phase Membrane Immunoassay Kit for Detecting Anti-Vinculin-IgG Antibodies

[0043] 4.1 Composition of the solid phase membrane immunoassay kit for detecting anti-Vinculin-IgG antibodies:

[0044] 1. Antigen: Recombinant protein Vinculin

[0045] 2. Solid phase support: Satourius CN140 nitrocellulose membrane

[0046] 3. Positive quality control (standard): human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang)

[0047] 4. Negative quality control: serum of healthy people

[0048] 5. Labeled antibody: biotin-labeled anti-human IgG antibody

[0049] 6. Antigen dilution

[0050] 7. Sample dilution buffer

[0051] 8. Antibody diluent

[0052] 9. Washing liquid

[0053] 10. Enzyme working solution: alkaline phosphatase-streptavidin

[0054] 11. Substrate color development solution: BCIP color development solution.

[0055] 4.2 The detection steps of the solid phase membrane immunoassay kit for detecting anti-Vinculin-IgG antibodies are as follows:

[0056] 4.2.1 Coating and Blocking: Spot 8 μl of 80 μg / ml Vinculin antigen directly onto the nitrocellulose membrane and incubate at 37°C to dry for 30 min. Place the nitrocellulose membrane in the assay plate and block with 200 μl of 5% BSA in a 37°C incubator for 30 min. Discard the blocking solution and wash twice with washing buffer.

[0057] 4.2.2 Antigen Incubation: Add 10 μl of antibody standard or serum to be tested diluted in diluent to the test plate, and perform negative and positive controls simultaneously. Incubate at 25°C for 30 min. Set up three parallel wells for each sample.

[0058] 4.2.3 Secondary Antibody Incubation: Discard the liquid in the test plate, wash with washing buffer 5 times for 1 min, add 20 μl of 1:500 biotin-labeled anti-human IgG antibody, and incubate at 25°C for 30 min;

[0059] 4.2.4 Color development: Discard the liquid in the test plate, wash with washing solution 5 times for 1 minute, add 500μl alkaline phosphatase-streptavidin, incubate at room temperature for 20 minutes, discard the liquid in the test plate, wash with washing solution 5 times for 1 minute, then add BCIP color development solution, react at room temperature for 20 minutes, rinse the test plate with running water to terminate the enzyme reaction. Take out the test nitrocellulose membrane strip and blow dry the membrane strip with a hair dryer. Use the colorimetric card to determine the quality of the test by naked eye. The one with obvious brown spots is positive. 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 for semi-quantitative analysis of the anti-Vinculin-IgG antibody level in the serum.

[0060] Example 5 Preparation of a Magnetic Particle Chemiluminescent Immunoassay Kit for Detecting Anti-Vinculin-IgG Antibodies

[0061] 5.1 Composition of the magnetic microparticle chemiluminescence immunoassay kit for detecting anti-Vinculin-IgG antibodies:

[0062] 1. Antigen: Recombinant protein Vinculin

[0063] 2. Solid phase carrier: magnetic particles with carboxyl functional groups

[0064] 3. Positive quality control (standard): human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang)

[0065] 4. Negative quality control: serum of healthy people

[0066] 5. Labeled antibody: Acridinium ester labeled anti-human IgG antibody

[0067] 6. Antigen dilution

[0068] 7. Sample dilution buffer

[0069] 8. Antibody diluent

[0070] 9. Washing liquid

[0071] 10. Pre-excitation solution: H2O2

[0072] 11. Excitation solution: NaOH.

[0073] 5.2 Detection Principle of the Magnetic Particle Chemiluminescent Immunoassay Kit for Detecting Anti-Vinculin-IgG Antibodies

[0074] The chemiluminescence immunoassay kit of the present invention is an analysis method that combines magnetic separation technology, immunoassay technology and chemiluminescence technology. The kit of the present invention uses an indirect method to quantitatively analyze and detect anti-Vinculin-IgG antibodies in human serum: first, a Vinculin antigen-coated magnetic microparticle solution is mixed with a diluted sample, and specific anti-Vinculin-IgG antibodies bind to the Vinculin antigen-coated magnetic microparticles. After washing, acridinium ester-labeled anti-human IgG antibodies are added to form a Vinculin antigen-coated magnetic microparticle-anti-Vinculin-IgG antibody-acridinium ester-labeled anti-human IgG antibody complex. Under the action of an external magnetic field, unbound substances are separated from the complex formed by the immune reaction. After discarding the supernatant and washing the precipitated complex, a pre-excitation solution (H2O2) and an excitation solution (NaOH) are added to carry out a luminescent reaction. Under alkaline conditions, the acridinium ester molecules are attacked by hydrogen peroxide to generate ethylene dioxide. The ethylene dioxide is unstable and decomposes into CO2 and electronically excited N-methylacridone. When the ethylene dioxide returns to the ground state, it emits light with a wavelength of 430 nm, and the luminescence intensity is collected using a chemiluminescence instrument. The concentration of anti-Vinculin-IgG antibodies in the serum to be tested is proportional to the luminescence value. The concentration of anti-Vinculin-IgG antibodies in the serum to be tested is calculated using the calibration curve. Figure 4 .

[0075] 5.3 Preparation of Vinculin Antigen-Coated Magnetic Microparticles

[0076] 5.3.1 Principle of Vinculin Antigen Coated Magnetic Microparticles: Based on the reaction of the carboxyl functional groups on the surface of the magnetic microparticles with EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution to generate an unstable amino-reactive O-acylurea intermediate, which then reacts with NHS (N-hydroxysuccinimide) to generate a semi-stable amino-reactive NHS ester. The semi-stable amino-reactive NHS ester then reacts with the amino groups on the antigen protein Vinculin to form Vinculin antigen-coated magnetic microparticles, see Figure 5 .

[0077] 5.3.2 EDC / NHS activation of carboxyl magnetic particles. The specific steps are as follows:

[0078] a) Weigh 10 mg of magnetic particles, wash them three times with 20 mM MES, separate them with a magnet, and discard the supernatant;

[0079] b) resuspending the washed magnetic particles in 100 μl of 20 mM MES to a final concentration of 100 mg / ml;

[0080] c) adding 50 μl of 20 mg / ml EDC and 50 μl of 24 mg / ml Sμlfo-NHS prepared in phosphate buffer to the washed magnetic particles, mixing thoroughly, and incubating at room temperature for 30 minutes for activation;

[0081] d) After applying the magnetic field, discard the supernatant, take 400 μl of 0.05 M phosphate buffer to wash the magnetic particles, and add 400 μl of preservation solution to make up the volume for future use.

[0082] 5.3.3 Activation of magnetic particles and cross-linking with antigen protein Vinculin. Add pre-cooled 1 ml of 20 mM MES to the above-mentioned activated magnetic particle solution and continue to wash the magnetic particles twice; take 200 μl of 2 mg / ml antigen protein Vinculin and add it to the activated magnetic particles, mix thoroughly, and let it stand at room temperature for 16 hours; after the reaction is completed, add pH 7.4 PBS buffer containing 0.2% Tween20 and repeat washing the magnetic particles twice; then add pH 7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA to a final concentration of 10 mg / ml of magnetic particles, mix thoroughly, and let it stand at room temperature for 30 minutes; after the reaction is completed, discard the supernatant and resuspend the magnetic particles with pH 7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA. The cross-linking of the activated magnetic particles and the antigen protein Vinculin is completed.

[0083] 5.4 Preparation of acridinium ester-labeled anti-human IgG antibody. The specific steps are as follows:

[0084] a) preparing a 2 mg / mL acridinium ester solution in dimethylformamide;

[0085] b) preparing 1 mg / mL anti-human IgG antibody using 0.2 M (pH 8.0) carbonate buffer;

[0086] c) thoroughly mixing acridinium ester and anti-human IgG antibody at a molar ratio of 4:1 and reacting for 40 minutes;

[0087] d) Add 20 μl of carbonate buffer containing 5% lysine to terminate the reaction;

[0088] e) obtaining a high-purity acridinium ester-labeled anti-human IgG antibody solution by desalting and removing impurities.

[0089] 5.5 Steps for detecting anti-Vinculin-IgG antibodies in serum using a magnetic microparticle chemiluminescence immunoassay kit

[0090] 5.5.1 Add 100 μl of the diluted serum to be tested or anti-His-tagged IgG standard to 100 μl of the Vinculin antigen-coated magnetic microparticle solution and react at 37°C for 15 min. Perform a positive / negative control at the same time.

[0091] 5.5.2 Labeled Antibody: Wash three times for 1 minute with 400 μl of washing buffer, then add 100 μl of 1:500 diluted acridinium ester-labeled anti-human IgG antibody and react at 37°C for 15 minutes.

[0092] 5.5.3 Signal Detection: Wash three times for 1 minute with 400 μl of washing buffer. Add 100 μl of pre-stimulation buffer (H₂O₂) and 100 μl of stimulation buffer (NaOH) to initiate the reaction. Detect the luminescence signal using a chemiluminescence analyzer and record the luminescence value. The concentration of anti-vinculin IgG antibodies in the serum to be tested is proportional to the luminescence value. Calculate the concentration of anti-vinculin IgG antibodies in the serum to be tested using a standard curve.

[0093] Example 6 Clinical Application of a Kit for Detecting Serum Anti-Vinculin-IgG Antibodies

[0094] 6.1 Subjects: Patients diagnosed with various renal diseases between June 2018 and June 2020 were enrolled, including 466 cases of nephrotic syndrome (NS), 168 cases of Henoch-Schönlein purpura (HSP), 137 cases of purpuric nephritis (HSPN), and 133 cases of IgA nephropathy (IgAN), as well as 195 healthy children (NC) from the same period. Serum samples were obtained from patients with various renal diseases and healthy controls. All subjects had their first serum sample collected before starting immunosuppressive therapy.

[0095] 6.2 Detection of anti-Vinculin-IgG antibodies in patients with various kidney diseases The kit of the present invention was used to detect the levels of anti-Vinculin-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 anti-Vinculin-IgG was positive in patients with autoimmune nephrotic syndrome, while anti-Vinculin-IgG was negative in patients with purpuric nephritis, Henoch-Schönlein purpura, IgA nephropathy, and healthy children. Figure 6 .

[0096] 6.3 ROC curve evaluation of the value of anti-Vinculin-IgG antibody as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome. The ROC curve analysis of the detection results of anti-Vinculin-IgG antibody in patients with autoimmune nephrotic syndrome in 6.2 was used to evaluate the application value of anti-Vinculin-IgG antibody in the diagnosis of autoimmune nephrotic syndrome. The results showed that anti-Vinculin antibody is a good serological marker for the diagnosis of patients with autoimmune nephrotic syndrome. The sensitivity of anti-Vinculin-IgG antibody (using a cutoff value greater than 116.2 as the standard) as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome was 72.7%, the specificity was 83.6%, and the area under the curve was 0.851. Figure 7 . SEQUENCE LISTING <110> Zhejiang University <120> A kit for detecting anti-focal adhesion protein-IgG antibodies <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 282 <212> PRT <213> Artificial sequence (Unknow) <400> 1 Pro Lys Phe Arg Glu Ala Val Lys Ala Ala Ser Asp Glu Leu Ser Lys 1 5 10 15 Thr Ile Ser Pro Met Val Met Asp Ala Lys Ala Val Ala Gly Asn Ile 20 25 30 Ser Asp Pro Gly Leu Gln Lys Ser Phe Leu Asp Ser Gly Tyr Arg Ile 35 40 45 Leu Gly Ala Val Ala Lys Val Arg Glu Ala Phe Gln Pro Gln Glu Pro 50 55 60 Asp Phe Pro Pro Pro Pro Gln Leu Arg Leu Thr Asp Glu Leu Ala Pro 65 70 75 80 Pro Lys Pro Pro Leu Pro Glu Gly Glu Val Pro Pro Pro Arg Pro Pro 85 90 95 Pro Pro Glu Glu Lys Asp Glu Glu Phe Pro Glu Gln Lys Ala Gly Glu 100 105 110 Val Ile Asn Gln Pro Met Met Met Ala Ala Arg Gln Leu His Asp Glu 115 120 125 Ala Arg Lys Trp Ser Ser Lys Gly Asn Asp Ile Ile Ala Ala Ala Lys 130 135 140 Arg Met Ala Leu Leu Met Ala Glu Met Ser Arg Leu Val Arg Gly Gly 145 150 155 160 Ser Gly Thr Lys Arg Ala Leu Ile Gln Cys Ala Lys Asp Ile Ala Lys 165 170 175 Ala Ser Asp Glu Val Thr Arg Leu Ala Lys Glu Val Ala Lys Gln Cys 180 185 190 Thr Asp Lys Arg Leu Leu Gln Val Cys Glu Arg Ile Pro Thr Ile Ser 195 200 205 Thr Gln Leu Lys Ile Leu Ser Thr Val Lys Ala Thr Met Leu Gly Arg 210 215 220 Thr Asn Ile Ser Asp Glu Glu Ser Glu Gln Ala Thr Glu Met Leu Val 225 230 235 240 His Asn Ala Gln Asn Leu Met Gln Ser Val Lys Glu Thr Val Arg Glu 245 250 255 Ala Glu Ala Ala Ser Ile Lys Ile Arg Thr Asp Ala Gly Phe Thr Leu 260 265 270 Arg Trp Val Arg Lys Thr Pro Trp Tyr Gln 275 280

Claims

1. Use of a focal adhesion protein 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-vinculin-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The adhesion focal protein polypeptide sequence is shown in 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 focal adhesion protein 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-vinculin-IgG antibody complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The adhesion focal protein polypeptide sequence is shown in SEQ ID NO.1.

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