KIT FOR DETECTING ANTI-VINCULIN-IMMUNOGLOBULIN G (IgG) ANTIBODY

Abstract

The present disclosure provides a kit for detecting an anti-vinculin-immunoglobulin G (IgG) antibody, including the antigen protein vinculin, a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color development reagent, a washing solution, a standard, a positive quality control, and negative quality control. In the present disclosure, the kit can detect the anti-Vinculin-IgG antibody in a sample to be tested by indirect reaction combined with magnetic particle-based chemiluminescence immunoassay. Autoantibodies against the target antigen vinculin are identified in the serum of a patient with autoimmune nephrotic syndrome for the first time, and a detection kit is provided for the autoantibodies. The present disclosure provides a basis for molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to vinculin and vinculin-IgG autoantibodies at home and abroad.

Claims

1. Use of an antigen protein vinculin in preparation of a kit for detecting autoimmune nephrotic syndrome, wherein the kit comprises an antigen protein vinculin, a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color development reagent, a washing solution, a standard, a positive quality control, and a negative quality control; the antigen protein vinculin has a sequence shown in SEQ ID NO: 1; the labeled antibody is selected from the group consisting of an enzyme-labeled secondary antibody, a chemiluminescent agent-labeled secondary antibody, and a biotin-labeled secondary antibody; the antigen protein vinculin has a tag peptide; the standard and the positive quality control each are selected from the group consisting of a recombinant human anti-tag peptide immunoglobulin G (IgG) and an anti- vinculin-lqG antibody extracted from serum, and the negative quality control is the serum of a healthy control; and the autoimmune nephrotic syndrome is diagnosed by detecting the anti-vinculin-lqG antibody in a serum sample of a patient.

2. (canceled)

3. (canceled)

4. The use according to claim 1, wherein the tag peptide is selected from the group consisting of a His tag, thioredoxin, a GST tag, a maltose-binding protein, an SA tag of glutathione S-transferase, a c-Myc tag, a Flag tag, and a biotin tag.

5. The use according to claim 1, wherein the antigen protein vinculin is purified by nickel column affinity chromatography, molecular sieve chromatography, gel filtration chromatography, ion exchange column chromatography, or hydrophobic interaction chromatography.

6. (canceled)

7. The use according to claim 1, wherein the antigen protein vinculin is immobilized on the solid phase carrier selected from the group consisting of a nylon membrane, an ion exchange resin, a nitrocellulose membrane, a polystyrene microplate, and magnetic particles.

8. The use according to claim 7, wherein the antigen protein vinculin is directly immobilized on the solid phase carrier by physical adsorption, covalent bonding, or chemical bonding.

9. The [[kit]] use according to claim 1, wherein the substrate color development reagent is selected from the group consisting of tetramethylbenzidine (TMB), hydrogen peroxide, 3-(2′-Spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), 4-methylumbelliferyl phosphate (4-MUP), and 5-bromo-4-chloro-3-indolyl phosphate (BCIP); the antigen diluent is a 1× PBS at a pH value of 7.4 containing 163 mM NaCl and 1% Triton X-100; the sample dilution buffer is a 0.01 M PBS at a pH value of 7.4 containing 10% bovine serum albumin (BSA); the antibody diluent is the 0.01 M PBS at a pH value of 7.4 containing 1M D-glucose, 2% glycerol, and 0.35% Tween 20; and the washing solution is the 1× PBS at a pH value of 7.4 containing the 163 mM NaCl, 10% glycerol, and the 1% Triton X-100.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows that vinculin on podocytes is the main target antigen for autoantibodies in a patient with autoimmune nephrotic syndrome; FIG. 1A: a primary antibody is a two-dimensional electrophoresis protein spot of healthy human serum; FIG. 1B: a primary antibody is a two-dimensional electrophoresis protein spot in the serum of a patient with autoimmune nephrotic syndrome; FIG. 1C: mass spectrometry identification of the target antigen Vinculin;

[0029] FIG. 2 shows SDS-PAGE identification of the expressed recombinant protein vinculin;

[0030] FIG. 3 shows the detection of an anti-Vinculin-IgG antibody in the serum of a patient with autoimmune nephrotic syndrome by a solid-phase membrane immunoassay kit.

[0031] FIG. 4 shows a schematic diagram for the detection of the anti-Vinculin-IgG antibody by a magnetic particle-based chemiluminescence immunoassay kit.

[0032] FIG. 5 shows a schematic diagram of the antigen protein vinculin coated with carboxyl magnetic particles.

[0033] FIG. 6 shows the detection of the anti-Vinculin-IgG antibody in patients with various types of renal diseases, where NS: autoimmune nephrotic syndrome, HSP: Henoch-schonlein purpura, HSPN: Henoch-schonlein purpura nephritis, IgAN: IgA nephropathy, and NC: healthy children.

[0034] FIG. 77 shows a ROC curve to evaluate the use value of the anti-Vinculin-IgG antibody as a serological marker for diagnosing patients with autoimmune nephrotic syndrome.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present disclosure will be described in further detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only intended to illustrate the disclosure rather than limiting the scope of the disclosure.

EXAMPLE 1 VINCULIN ON PODOCYTES AS THE MAIN TARGET ANTIGEN FOR AUTOANTIBODIES IN A PATIENT WITH AUTOIMMUNE NEPHROTIC SYNDROME

[0036] In the present disclosure, through many clinical and molecular mechanism studies in an early stage, it was found for the first time that patients with nephrotic syndrome have a higher serum IgG level. Furthermore, it was confirmed that vinculin on podocytes was the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative levels of the anti-Vinculin-IgG antibody in serum was helpful for the early identification of autoimmune nephrotic syndrome, especially for screening patients with related symptoms. The specific implementation included the following: (1) extraction of total protein from glomerular podocytes: a sample of a podocyte line (MPCS) was cultured, washed 2 to 3 times with PBS, and subjected to extensive lysis on ice using a focused sonicator (Covaris S220, Gene) in lysis buffer containing 30 mM Tris-HCl, 8 M urea, 4% CHAPS and a protease inhibitor (#ab65621; Abcam, 1:200 dilution), and the sample was centrifuged at 12,000 g and 4° C. for 30 min. The supernatant was collected to obtain the total protein of glomerular podocytes. The total protein concentration of the glomerular podocytes was determined using a BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: the total protein of glomerular podocytes was subjected to two-dimensional electrophoresis and transferred to a nitrocellulose membrane; sera of healthy people and patients with autoimmune nephrotic syndrome were used as primary antibodies for incubation separately, and a secondary antibody was added for development, as shown in FIG. 1A and FIG. 1B. (3) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS): after development in step (2), differential analysis was conducted on positive spots; protein spots were strongly positive in nephrotic syndrome patients, and negative or weakly positive in healthy people on the two-dimensional electrophoresis gel were selected, and the selected protein spots were cut out from the gel; a dried gel was digested with trypsin (0.1 μg/μl ), 10 μl of 25 mM ammonium bicarbonate was added to a reaction mixture, incubated overnight at 37° C., and peptides were extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptide was analyzed by a MALDI-TOF-MS mass spectrometer to obtain a mass spectrum of the peptide, which was identified as Vinculin protein, as shown in FIG. 1C.

EXAMPLE 2 EXPRESSION AND PURIFICATION OF THE RECOMBINANT ANTIGEN PROTEIN VINCULIN

[0037] Genetic engineering used a gene encoding the vinculin protein as a template for PCR amplification, and an expression carrier was constructed for protein expression. Tag peptides containing His tags were on expressed antigen proteins. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic interaction chromatography, and molecular sieve, and the molecular weight of the recombinant protein vinculin was identified by SDS-PAGE as 37 kDa, as shown in FIG. 2.

EXAMPLE 3 OPTIMIZATION OF THE REACTION CONDITIONS OF A KIT BY ORTHOGONAL EXPERIMENTAL DESIGN

[0038] According to a coating concentration of the antigen Vinculin (50 μg/ml, 80 μg/ml, 100 μg/ml, and 150 μg/ml), each reaction time (15 min, 30 min, and 45 min), temperature (25° C. and 37° C.), and an optimal dilution of enzyme-labeled secondary antibody (1:100, 1:500, 1:1000, 1:1500), an orthogonal table was selected, each factor was repeated at two levels to determine standard positive serum and standard negative serum, and a ratio (P/N) of the highest light signal value (P) of the positive serum and the lowest light signal value (N) of the negative serum was selected. By the orthogonal design, the kit had an optimal antigen Vinculin coating concentration of 80 μg/ml, the solid-phase membrane immunoassay for the anti-Vinculin-IgG antibody kit had an optimal antigen-antibody reaction temperature of 25° C., an optimal antigen-antibody reaction time of 30 min, and an optimal working dilution of the optimal biotin-labeled anti-human IgG antibody at 1:500; the magnetic particle-based chemiluminescence immunoassay for anti-Vinculin-IgG antibody kit had an optimal antigen-antibody reaction temperature of 37° C., an optimal antigen-antibody reaction time of 15 min, and an optimal working dilution of the optimal acridinium ester-labeled anti-human IgG antibody at 1:500.

EXAMPLE 4 PREPARATION OF A SOLID-PHASE MEMBRANE IMMUNOASSAY KIT FOR DETECTING AN ANTI-VINCULIN-IGG ANTIBODY

[0039] 4.1. Composition of a solid-phase membrane immunoassay kit for detection of the anti-Vinculin-IgG antibody: [0040] 1. Antigen: recombinant protein vinculin [0041] 2. Solid phase carrier: Satourius CN140 nitrocellulose membrane [0042] 3. Positive quality control (standard): human anti-His tag IgG (purchased from Huzhou Yingchuang) [0043] 4. Negative quality control: serum of healthy controls [0044] 5. Labeled antibody: biotin-labeled anti-human IgG antibody [0045] 6. Antigen diluent [0046] 7. Sample dilution buffer [0047] 8. Antibody diluent [0048] 9. Washing solution [0049] 10. Enzyme working solution: alkaline phosphatase-streptavidin [0050] 11. Substrate color development reagent: BCIP color development reagent.

[0051] 4.2. The detection steps of the solid-phase membrane immunoassay kit for detection of the anti-Vinculin-IgG antibody included the following: [0052] 4.2.1. Coating and blocking: 8 μl of Vinculin antigen with a concentration of 80 μg/ml was added dropwise directly to the nitrocellulose membrane and dried in a 37° C. incubator for 30 min, and the nitrocellulose membrane was blocked on a detection plate with 200 ul of 5% BSA in a 37° C. incubator for 30 min. After discarding the blocking solution, the nitrocellulose membrane was washed twice with a washing solution. [0053] 4.2.2. Antigen incubation: 10 μl of an antibody standard or serum to be tested diluted with diluent was added to the detection plate, while negative and positive controls were set up at the same time and then incubated at 25° C. for 30 min, where three parallel wells were set for each sample. [0054] 4.2.3. Secondary antibody incubation: The liquid in the detection plate was discarded, the plate was washed five times with a washing solution for 1 min each time, and 20 μl of a 1:500 biotin-labeled anti-human IgG antibody was added and incubated at 25° C. for 30 min. [0055] 4.2.4 Color development: The liquid in the detection plate was discarded, the plate was washed five times with the washing solution for 1 min each time, and 500 μl of alkaline phosphatase-streptavidin was added and incubated at room temperature for 20 min. The liquid in the detection plate was discarded, and the plate was washed five times with the washing solution for 1 min each time. A color development reagent BCIP was added and reacted at room temperature for 20 min, and the detection plate was rinsed with running water to stop the enzyme reaction. The nitrocellulose membrane test strip was removed and dried with a hairdryer and qualitatively determined with a colorimetric card by the naked eye. Those with prominent brown spots were positive, as shown in FIG. 3. Alternatively, the membrane strip was scanned on a developing device. Analysis software that comes with the developing device used the reference standard concentration as ordinate and the gray value read by the device as an abscissa to draw a standard curve for the semiquantitative analysis of anti-Vinculin-IgG antibody levels in serum.

EXAMPLE 5 PREPARATION OF A MAGNETIC PARTICLE-BASED CHEMILUMINESCENCE IMMUNOASSAY KIT FOR DETECTING AN ANTI-VINCULIN-IGG ANTIBODY

[0056] 5.1. Composition of a magnetic particle-based chemiluminescence immunoassay kit for detection of the anti-Vinculin-IgG antibody: [0057] 1. Antigen: recombinant protein vinculin [0058] 2. Solid phase carrier: magnetic particles with carboxyl functional groups [0059] 3. Positive quality control (standard): human anti-His tag IgG (purchased from Huzhou Yingchuang) [0060] 4. Negative quality control: serum of healthy controls [0061] 5. Labeled antibody: acridinium ester-labeled anti-human IgG antibody [0062] 6. Antigen diluent [0063] 7. Sample dilution buffer [0064] 8. Antibody diluent [0065] 9. Washing solution [0066] 10. Preexcitation solution: H.sub.2O.sub.2 [0067] 11. Excitation solution: NaOH.

[0068] 5.2. Principle of a magnetic particle-based chemiluminescence immunoassay kit for detection of the anti-Vinculin-IgG antibody

[0069] The chemiluminescence immunoassay kit is an analytical method combining magnetic separation, immunoassay, and chemiluminescence. The kit used an indirect method to quantitatively detect anti-Vinculin-IgG antibody in human serum: a Vinculin antigen-coated magnetic particle solution was mixed with a diluted sample, and specific anti-Vinculin-IgG antibodies were bound to the Vinculin antigen-coated magnetic particles; after washing, an acridinium ester-labeled anti-human IgG antibody was added to form a Vinculin antigen-coated magnetic particle-anti-Vinculin-IgG antibody-acridinium ester-labeled anti-human IgG antibody complex; under the action of an external magnetic field, the unbound substance and the complex formed by the immune reaction were separated, a supernatant was discarded, a precipitated complex was washed, and the preexcitation solution (H.sub.2O.sub.2) and the excitation solution (NaOH) were added to conduct a luminescence reaction; under alkaline conditions, the acridinium ester molecule was attacked by the hydrogen peroxide to generate dioxyethane, which was unstable and decomposed into CO.sub.2 and N-methylacridone in an electronically excited state, returning to the ground state, N-methylacridone emitted light with a wavelength of 430 nm, and a luminescence intensity was determined using a chemiluminometer. The concentration of anti-Vinculin-IgG antibody was proportional to the luminescence intensity, and the concentration of anti-Vinculin-IgG antibody in the serum to be tested was calculated by a calibration curve, as shown in FIG. 4.

[0070] 5.3. Preparation of vinculin antigen-coated magnetic particles

[0071] 5.3.1 The principle of coating magnetic particles with vinculin antigen: the carboxyl functional groups contained on the surface of the magnetic particles reacted with an EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution to generate an unstable amino-active O-acylurea intermediate; the intermediate reacted with NHS (N-hydroxysuccinimide) to form a semistable amino-reactive NHS ester; the semistable amino-reactive NHS ester reacted with amino groups on the antigen protein vinculin to form the vinculin antigen-coated magnetic particles, as shown in FIG. 5.

[0072] 5.3.2. For the EDC/NHS activation of the carboxyl magnetic particles, the specific steps were as follows: [0073] a) A total of 10 mg of magnetic particles was washed three times with 20 mM MES and separated by a magnet, and the supernatant was discarded. [0074] b) The washed magnetic particles were resuspended in 100 μl of 20 mM MES to a final 100 mg/ml concentration. [0075] c) Fifty microliters of 20 mg/ml EDC and 50 μl of 24 mg/ml Sulfo-NHS that were prepared in PBS were added to the cleaned magnetic particles in sequence, mixed well, and allowed to stand at room temperature for activation for 30 min. [0076] d) After an external magnetic field action, the supernatant was discarded, and the magnetic particles were washed with 400 μl of 0.05 M PBS, diluted with 400 μl of a preservation solution, and stored for later use.

[0077] 5.3.3 Crosslinking of activated magnetic particles with antigen protein Vinculin: precooled 1 ml of 20 mM MES was added to wash the activated magnetic particle solution twice; 200 μl of the 2 mg/ml antigen protein vinculin was added to the activated magnetic particles, mixed thoroughly, and allowed to stand for reaction at room temperature for 16 h; after the reaction, PBS containing 0.2% Tween 20 at pH 7.4 was added, and the magnetic particles were washed twice; PBS containing 0.2% Tween 20 and 0.2% BSA at pH 7.4 was added until a final concentration of the magnetic particles was 10 mg/ml, mixed well, and allowed to stand for reaction at room temperature for 30 min; after the reaction, the supernatant was discarded, and the magnetic particles were resuspended in PBS containing 0.2% Tween 20, 0.2% BSA at pH 7.4, such that the cross-linking of the activated magnetic particles and the antigen protein vinculin was completed.

[0078] 5.4. To prepare the acridinium ester-labeled anti-human IgG antibody, the specific steps were as follows: [0079] a) A 2 mg/mL acridinium ester solution was prepared by dimethylformamide. [0080] b) A 1 mg/mL anti-human IgG antibody was prepared by a 0.2 M (pH 8.0) carbonate buffer. [0081] c) The acridinium ester and the anti-human IgG antibody was thoroughly mixed at a molar ratio of 4:1 and reacted for 40 min. [0082] d) The reaction was terminated with 20 μl of carbonate buffer containing 5% lysine. [0083] e) desalting and impurity removal were conducted to obtain an acridinium ester-labeled anti-human IgG antibody solution with higher purity.

[0084] 5.5. The steps of detecting anti-Vinculin-IgG antibody in serum by a magnetic particle-based chemiluminescence immunoassay kit were as follows:

[0085] 5.5.1 Serum to be tested: 100 μl of a diluted serum or an anti-His-tag IgG standard was added to 100 μl of the magnetic particle solution coated with antigen protein Vinculin and reacted at 37° C. for 15 min. At the same time, negative and positive controls were set up.

[0086] 5.5.2 Labeled antibody: A total of 400 μl of a washing solution was added to wash three times for 1 min each time, and 100 μl of an acridinium ester-labeled anti-human IgG antibody diluted by 1:500 was added and reacted at 37° C. for 15 min.

[0087] 5.5.3 Signal detection: precipitated complexes were washed with 400 μl of the washing solution three times for 1 min each time, and 100 μl of a preexcitation solution (H.sub.2O.sub.2) and 100 μl of an excitation solution (NaOH) were added for the reaction. A chemiluminometer detected the luminescence signal, and the luminescence value was recorded. The concentration of anti-vinculin-IgG antibody was proportional to the luminescence intensity, and a standard curve calculated the concentration of anti-vinculin-IgG antibody in the serum to be tested.

EXAMPLE 6 CLINICAL USE OF A KIT FOR DETECTING AN ANTI-VINCULIN-IGG ANTIBODY IN SERUM

[0088] 6.1 Subjects included patients diagnosed with various types of nephropathies from June 2018 to June 2020, including 466 cases of NS, 168 cases of HSP, 137 cases of HSPN, 133 cases of IgAN, and 195 cases of NC during the same period. Serum samples were obtained from various nephropathy patients and healthy controls. All subjects had their first serum sample collection before immunosuppressive therapy.

[0089] 6.2 Detection of anti-Vinculin-IgG antibodies in patients with various types of nephropathies: The kit for the present disclosure was used to detect anti-Vinculin-IgG antibody levels in the serum of patients diagnosed with various types of nephropathies from June 2018 to June 2020, including 466 cases of NS, 168 cases of HSP, 137 cases of HSPN, 133 cases of IgAN, and 195 cases of NC during the same period. The results showed positive anti-Vinculin-IgG antibodies in patients with autoimmune nephrotic syndrome and negative anti-Vinculin-IgG antibodies in patients with HSPN, HSP, IgAN, and NC, as shown in FIG. 6.

[0090] 6.3 ROC curve: The value of anti-Vinculin-IgG antibody was evaluated as a serological marker in the diagnosis of patients with autoimmune nephrotic syndrome; detection results of anti-Vinculin-IgG antibody in patients with autoimmune nephrotic syndrome in 6.2 were analyzed using the ROC curve to evaluate the use value of the anti-Vinculin-IgG antibody in the diagnosis of autoimmune nephrotic syndrome. The results showed that the anti-Vinculin-IgG antibody was a desirable serological marker for the diagnosis of patients with autoimmune nephrotic syndrome; the anti-Vinculin-IgG antibody (with a cutoff value greater than 116.2 as a criterion) as a serological marker for the diagnosis of the autoimmune nephrotic syndrome had a sensitivity of 72.7%, a specificity of 83.6% and an area under the curve of 0.851, as shown in FIG. 7.