A kit for detecting anti-proteasome subunit alpha 1-IgG antibody

Abstract

The present invention provides a kit for detecting anti-proteasome subunit alpha 1-IgG antibody, which is composed of antigen protein Proteasome subunit alpha type 1 (proteasome subunit alpha 1), solid phase carrier, labeled antibody, antigen and antibody diluent, sample dilution buffer, substrate chromogenic agent, washing solution, standard product, positive and negative quality control products. The kit of the present invention utilizes the reaction principle of indirect method combined with magnetic particle chemiluminescence immunoassay to detect anti-proteasome subunit alpha 1-IgG antibody in the serum to be detected. The present invention first identifies the autoantibody against the target antigen proteasome subunit alpha 1 in the serum of patients with autoimmune nephrotic syndrome. The present invention provides a basis for the research on the molecular mechanism and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to proteasome subunit alpha 1 and proteasome subunit alpha 1-IgG autoantibody at home and abroad.

Description

[0001] This application is a divisional application of the application with the application number 202110743551.7, the application date of July 1, 2021, and the invention title of a kit for detecting anti-proteasome subunit α1-IgG antibody. Technical Field

[0002] The present invention belongs to the field of biomedical technology and relates to a kit for detecting anti-proteasome subunit α1-IgG antibody. Background Art

[0003] In recent years, the types of children's kidney diseases have been increasing, among which the incidence of autoimmune nephrotic syndrome is the highest, seriously endangering the physical and mental health of children. Autoimmune nephrotic syndrome is a clinical syndrome in which the glomerular filtration membrane permeability increases, resulting in increased plasma protein filtration, causing massive proteinuria, and the main manifestations of the patient are massive proteinuria, hypoproteinemia, severe edema, etc. Scholars such as Ali observed that after transplanting the kidneys from patients with refractory minimal change nephrotic syndrome, the recipient's kidney function was normal and no proteinuria occurred. It can be seen that the cause of minimal change nephrotic syndrome is not all in the kidney itself, but may mainly be a problem with the patient's internal environment. In addition, except for some children caused by genetic defects, the conditions of most children with autoimmune nephrotic syndrome can improve after treatment with hormones and immunosuppressants, which indirectly proves that the disease is closely related to the patient's autoimmunity.

[0004] In recent years, it has been found that abnormal B cell function also plays an important role in autoimmune nephrotic syndrome. In recent years, the clinical research results of multiple multi-center studies around the world have shown that rituximab (RTX) can be successfully used in the treatment of minimal change nephrotic syndrome, especially achieving good therapeutic effects in the treatment of refractory nephrotic syndrome. However, studies have also found that during the treatment of steroid-dependent nephrotic syndrome with rituximab, its effect of clearing B cells can last for about 5 months, and the patient's condition will relapse at 6 to 7 months as the number of B cells rebounds. This suggests that there are pathological B cell clones in patients with autoimmune nephrotic syndrome. Identifying and precisely clearing these pathological B cell clones is not only beneficial to the recovery of autoimmune nephrotic syndrome, but also reduces the risk of humoral immune deficiency in patients caused by the indiscriminate clearance of B cells by means such as rituximab. However, so far, the target antigens of pathological B cells in children with autoimmune nephrotic syndrome are still not very clear. Pathologically, minimal change nephropathy or focal segmental glomerulosclerosis is considered a podocytopathy with massive proteinuria due to the loss or alteration of podocyte function. Podocytes are renal glomerular epithelial cells that attach to the outer side of the glomerular basement membrane and are the last barrier to prevent protein loss. Podocyte damage usually causes massive proteinuria.

[0005] The proteasome (Proteasome subunit) is a large molecular complex that degrades proteins within cells, and its main function is to degrade proteins that are not needed by the cells or are damaged. Proteasome subunit alpha-1 (Proteasome subunitalpha type 1, Proteasome subunit alpha type 1) is a type of proteasome family. Currently, there are relatively many studies on the Proteasome subunit alpha type 1 protein in cancer. For example, studies have shown that the Proteasome subunit alpha type 1 protein in the proteasome pathway is closely related to controlling the cell cycle progression and apoptosis. The expression of Proteasome subunit alpha type 1 is significantly downregulated in hepatocellular carcinoma tumor tissues. Proteasome subunit alpha type 1 may be a potential biomarker for hepatocellular carcinoma (Jian Qin1*, et al. Identification of proteasome subunit alpha type-1 as a novel biomarker in HBV-associated hepatocellular carcinoma tissue interstitial fluid by proteomic analysis. Int J Clin Exp Pathol 2017;10(7):7812-7820.). Yang Qian et al. reported that the Proteasome subunit alpha type 1 protein may be a potential biomarker for colon cancer (Yang Qian, Roehrl Michael H, Wang J, et al. Proteomic profiling of antibody-inducing immunogens in tumor tissue identifies Proteasome subunit alpha type 1, LAP3, ANXA3, and maspin as colon cancer markers. Journal[J] Oncotarget Volume 9, Issue 3. 2018. PP 3996-4019.).

[0006] However, the expression of Proteasome subunit alpha type 1 and the presence of autoantibodies against Proteasome subunit alpha type 1 have not been reported in nephrotic syndrome. In addition, in the prior art, there is no disclosure regarding the use of the target Proteasome subunit alpha type 1 or its autoantibodies as a serological marker in autoimmune nephrotic syndrome. The study on differentiating autoimmune nephrotic syndrome by detecting serum anti-Proteasome subunit alpha type 1-IgG antibodies is blank. Summary of the Invention

[0007] The objective of the present invention is to provide a kit for detecting anti-proteasome subunit α1-IgG antibodies, which is a detection kit based on the target Proteasome subunit alpha type 1 and its corresponding autoantibodies. The kit can detect autoantibodies in tissues (renal biopsy tissues) or body fluids (especially blood, plasma, serum) through an immune reaction with the antigen protein Proteasome subunit alpha type 1 (specifically shown according to Sequence Identification Number SEQ ID NO.1).

[0008] The kit of the present invention consists of the antigen protein Proteasome subunit alpha type 1, a solid-phase carrier, a labeled antibody such as a secondary antibody labeled with an enzyme, a chemiluminescent agent, or a biotin, an antigen diluent, a sample dilution buffer, an antibody dilution buffer, a substrate chromogenic agent, a washing solution, a standard product, a positive control product, and a negative control product.

[0009] The sequence of the antigen protein Proteasome subunit alpha type 1 is as shown in SEQ ID NO.1:

[0010] MFRNQYDNDVTVWSPQGRIHQIEYAMEAVKQGSATVGLKSKTHAVLVALKRAQSELAAHQKKILHVDNHIGISIAGLTADARLLCNFMRQECLDSRFVFDRPLPVSRLVSLIGSKTQIPTQRYGRRPYGVGLLIAGYDDMGPHIFQTCPSANYFDCRAMSIGARSQSARTYLERHMSEFMECNLNELVKHGLRALRETLPAEQDLTTKNVSIGIVGKDLEFTIYDDDDVSPFLEGLEERPQRKAQPAQPADEPAEKADEPMEH。

[0011] The Proteasome subunit alpha type 1 antigen protein of the present invention can be a fusion protein, using tags with certain biological or physical functions, especially at the N-terminus or C-terminus. The presence of these tags is beneficial for the 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. For example, the tag peptide is selected from: GST tag, c-Myc tag, His tag, Flag tag, or biotin tag.

[0012] According to the present invention, the antigen protein Proteasome subunit alpha type 1 is immobilized on a solid-phase carrier, and the Proteasome subunit alpha type 1 antigen protein is immobilized on the solid-phase carrier in a direct manner by physical adsorption, covalent bonding, or chemical bonding. Preferred solid-phase carriers include: nitrocellulose membrane, magnetic beads, and enzyme-labeled microplates.

[0013] In one embodiment of the present invention, the standard product and the positive control product are recombinant human anti-tag peptide immunoglobulin G or its fragment, or the anti-Proteasome subunit alphatype 1-IgG antibody extracted from patient serum is used as the positive control product and the standard product, and the serum of healthy subjects is used as the negative control product.

[0014] According to the present invention, the antigen protein Proteasome subunit alpha type 1 can be expressed in bacteria such as Escherichia coli, yeast, and mammalian cells.

[0015] According to the present invention, the antigen protein Proteasome subunit alpha type 1 is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, and hydrophobic column chromatography.

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

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

[0018] In a preferred embodiment, "immobilization" as described herein refers to binding to a water-insoluble solid-phase carrier of the Proteasome subunit alphatype 1 antigen protein. The solid-phase carrier or support is water-insoluble, and more preferably, it is through covalent bonding, electrostatic interaction, hydrophobic interaction, or interaction through disulfide bonds, and most preferably through one or more covalent bonds. Immobilization can be in a direct immobilization manner. For example, through filtration, centrifugation, or chromatography, the immobilized molecule is separated from the aqueous solution together with the insoluble carrier. It also includes immobilizing the Proteasome subunitalpha type 1 antigen protein in a reversible or irreversible manner. For example, the antigen protein is immobilized on the carrier through a cleavable covalent bond (such as a disulfide bond that can be cleaved by adding a thiol-containing reagent), and this immobilization is reversible. Additionally, if the antigen protein is immobilized on the carrier through a covalent bond that does not cleave in an aqueous solution (a bond formed by the reaction of an epoxide group with an amine group that couples a lysine side chain to an affinity column), the immobilization is irreversible. Immobilization can also be in an indirect manner: such as immobilizing an antibody that has specific affinity for the antigen protein, and then forming an antigen protein-antibody complex to achieve the purpose of immobilization.

[0019] The method for immobilizing the antigen protein Proteasome subunit alpha type 1 in the present invention is the direct coating method: (1) The antigen protein Proteasome subunit alpha type 1 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 combined with the amino groups of the antigen protein Proteasomesubunit alpha type 1, and the antigen protein Proteasome subunit alpha type 1 is bound to the magnetic particles by chemical coupling.

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

[0021] The present invention successfully expresses and purifies the recombinant protein Proteasomesubunit alpha type 1 by means of 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-Proteasome subunit alpha type 1-IgG antibodies in the sera of patients with autoimmune nephrotic syndrome. It is a detection kit for qualitatively or quantitatively analyzing anti-Proteasome subunit alpha type 1-IgG antibodies in human sera.

[0022] The principle of a kit for detecting anti-Proteasome subunit alpha type 1-IgG antibodies in sera utilizes the indirect reaction principle. First, the Proteasome subunit alpha type 1 antigen is adsorbed onto a solid-phase carrier as the coated antigen, then positive control products or standard products or serum samples to be tested are added for incubation, and after adding the labeled secondary antibody for reaction, if the serum to be tested contains anti-Proteasome subunit alpha type 1-IgG antibodies, a ternary complex of coated antigen Proteasome subunit alpha type 1 - serum to be tested anti-Proteasome subunit alpha type 1-IgG antibody - labeled anti-human IgG antibody is formed. Finally, light signal is detected by means of light chromogenic method, chemiluminescence method, or fluorescence luminescence method to achieve the purpose of qualitatively or quantitatively analyzing anti-Proteasome subunit alpha type 1-IgG antibodies in human sera.

[0023] Using the kit of the present invention, an anti-Proteasome subunit alpha type 1-IgG autoantibody was detected in the body of some patients with autoimmune nephrotic syndrome for the first time, and it was determined that the target antigen of this autoantibody is Proteasome subunit alpha type 1 on podocytes. Therefore, the kit of the present invention can be used for the detection of anti-Proteasome subunit alpha type 1-IgG autoantibody, providing a basis for the study of autoimmune nephrotic syndrome.

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

[0025] (1) At present, the research on Proteasome subunit alpha type 1 and anti-Proteasome subunit alpha type 1-IgG antibody in patients with kidney diseases at home and abroad is limited to the study of molecular mechanisms, and there is no quantitative detection of their levels in the sera of patients. The present invention identified the autoantibody against Proteasome subunit alpha type 1 for the first time and invented a detection kit for this anti-Proteasome subunit alpha type 1-IgG autoantibody, filling the domestic and foreign gaps.

[0026] (2) The kit of the present invention is related to the qualitative analysis of anti-Proteasome subunit alpha type 1-IgG antibody in human sera. The solid-phase membrane immunoassay for qualitative detection is simple in operation and requires less reagent, saving nearly 10 times compared with the traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely strong, approaching 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane; the NC membrane adsorbed with antigens or antibodies or existing results can be stored for a long time (it can be stored at -20°C for half a year) without affecting its activity; in addition, the kit for the solid-phase membrane immunoassay of anti-Proteasome subunit alpha type 1-IgG antibody in human sera of the present invention introduces a biotin-avidin amplification system, greatly improving the detection sensitivity.

[0027] (3) The magnetic particle chemiluminescence immunoassay quantitative detection kit for anti-Proteasome subunit alpha type 1-IgG antibody in human serum involved in the present invention uses magnetic particles as the solid-phase carrier, with a diameter of only 1.0 μm, which greatly increases the coating surface area, increases the adsorption amount of antigen, improves the reaction speed, and also makes the cleaning and separation more convenient, thereby reducing pollution and the probability of cross-infection. On the other hand, acridinium ester luminescent agent is directly labeled with anti-human IgG, and its chemical reaction is simple, rapid, and catalyst-free; the chemiluminescence of acridinium ester is of the flash type, and its emission intensity can reach the maximum after 0.4 s by starting the luminescent reagent (H2O2, NaOH), with a half-life of 0.9 s and basically ending within 2 s, which is convenient for rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The Proteasome subunit alpha type 1 protein on podocytes is the target antigen against which autoantibodies in patients with autoimmune nephrotic syndrome are directed. Figure 1 A: Two-dimensional electrophoresis protein spots with the first antibody being healthy human serum; Figure 1 B: Two-dimensional electrophoresis protein spots with the first antibody being serum from patients with autoimmune nephrotic syndrome; Figure 1 C: Mass spectrometry identification of the target antigen Proteasome subunit alpha type 1 protein.

[0029] Figure 2 : SDS-PAGE identification diagram of the expressed recombinant protein Proteasome subunit alpha type 1.

[0030] Figure 3 : Detection of anti-Proteasome subunit alpha type 1-IgG antibody in serum of patients with autoimmune nephrotic syndrome by a solid-phase membrane immunoassay kit.

[0031] Figure 4 : Schematic diagram of the principle for detecting anti-Proteasome subunit alpha type 1-IgG antibody by a magnetic particle chemiluminescence immunoassay kit.

[0032] Figure 5 : Schematic diagram of coating carboxyl magnetic particles with the antigen protein Proteasome subunit alpha type 1.

[0033] Figure 6: Detection of anti-Proteasome subunit alpha type 1-IgG antibodies in various types of nephropathy patients, where NS: Autoimmune nephrotic syndrome, HSP: Henoch-Schönlein purpura, HSPN: Henoch-Schönlein purpura nephritis, IgAN: IgA nephropathy, NC: Healthy children.

[0034] Figure 7 : Application value of anti-Proteasome subunit alpha type 1 antibody as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome evaluated by ROC curve. Specific implementation manner

[0035] 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.

[0036] Example 1 Proteasome subunit alpha type 1 protein on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome

[0037] Through a large number of previous clinical and molecular mechanism studies, the present invention first found that the serum IgG level of patients with nephrotic syndrome is relatively high, and confirmed that Proteasome subunit alpha type 1 on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Therefore, detecting the presence and quantitative level of anti-Proteasome subunit alpha type 1-IgG antibodies in serum is helpful for the early differentiation of autoimmune nephrotic syndrome, especially for screening patients with related symptoms. 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 protease inhibitor (#ab65621; Abcam, diluted 1:200). Then place the sample in a centrifuge, centrifuge at 12,000 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 the primary antibody respectively, and then add the secondary antibody for development, as shown in 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 individuals 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, incubate overnight at 37 °C, and then extract peptides from the gel with trifluoroacetic acid (0.1%). Analyze the extracted peptides using 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 the Proteasome subunit alpha type 1 protein, see Figure 1 C.

[0038] Example 2 Expression and purification of recombinant antigen protein Proteasome subunit alpha type 1

[0039] Using genetic engineering methods, with the gene encoding the Proteasome subunit alpha type 1 protein as a template, perform 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 GST 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 that the molecular weight of the recombinant protein Proteasome subunit alpha type 1 is 56 KDa, see Figure 2 .

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

[0041] According to four coating concentrations of antigen Proteasome subunit alpha type 1 (50 μg / ml, 90 μg / ml, 120 μg / ml, 150 μg / ml), each reaction time (15 min, 30 min, 45 min) and temperature (25 °C, 37 °C), and the optimal dilution of enzyme-labeled secondary antibody (four dilutions of 1:100, 1:500, 1:1000, 1:1500), an orthogonal table was selected. Each factor was repeatedly measured for standard positive serum and standard negative serum at 2 levels, and the ratio (P / N) of the highest optical signal value (P) of the positive serum and the lowest optical signal value (N) of the negative serum was selected. Through orthogonal design, the optimal coating concentration of antigen Proteasome subunit alpha type 1 in this kit was 90 μg / ml, the optimal antigen-antibody reaction temperature for the solid-phase membrane immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody was 25 °C, the optimal antigen-antibody reaction time was 30 min, and the optimal working dilution of the labeled anti-human IgG antibody was 1:1000; the optimal antigen-antibody reaction temperature for the magnetic particle chemiluminescence immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody was 37 °C, the optimal antigen-antibody reaction time was 15 min, and the optimal working dilution of the labeled anti-human IgG antibody was 1:1000.

[0042] Example 4 Preparation of a solid-phase membrane immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody

[0043] 4.1 Composition of the solid-phase membrane immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody:

[0044] 1. Antigen: Recombinant protein Proteasome subunit alpha type 1

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

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

[0047] 4. Negative control: Serum from healthy physical examination subjects

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

[0049] 6. Antigen diluent

[0050] 7. Sample dilution buffer

[0051] 8. Antibody diluent

[0052] 9. Washing liquid

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

[0054] 11. Substrate colorimetric solution: BCIP colorimetric solution.

[0055] 4.2 The detection steps of the solid phase membrane immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibodies are as follows:

[0056] 4.2.1 Coating and blocking: 8 μl of 90 μg / ml Proteasome subunit alpha type 1 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 test plate and blocked with 200 μl of 5% BSA in a 37°C incubator.

[0057] 30min, discard the blocking solution and wash twice with washing solution;

[0058] 4.2.2 Antigen incubation: Add 10 μl of antibody standard or serum to be tested diluted with diluent to the test plate, and make negative control and positive control at the same time, incubate at 25°C for 30 minutes, and set up 3 parallel wells for each sample;

[0059] 4.2.3 Secondary antibody incubation: discard the liquid in the test plate, wash 5 times with washing solution for 1 min, add 20 μl 1:1000 biotin-labeled anti-human IgG antibody, and incubate at 25°C for 30 min;

[0060] 4.2.4 Color development: discard the liquid in the test plate, wash with washing solution 5 times × 1min, add 500μl alkaline phosphatase-streptavidin, incubate at room temperature for 20min, discard the liquid in the test plate, wash with washing solution 5 times × 1min, then add BCIP color development solution, react at room temperature for 20min, 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, and use the colorimetric card to qualitatively determine the appearance of obvious brown spots as 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 level of anti-Proteasome subunit alpha type 1-IgG antibodies in serum.

[0061] Example 5. Preparation of a Magnetic Particle Chemiluminescence Immunoassay Kit for Detecting Anti-Proteasome Subunit Alpha Type 1-IgG Antibody

[0062] 5.1 Composition of the magnetic particle chemiluminescence immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody:

[0063] 1. Antigen: Recombinant protein Proteasome subunit alpha type 1

[0064] 2. Solid phase carrier: Magnetic particles with carboxyl functional groups

[0065] 3. Positive control (standard): Human anti-GST tag immunoglobulin G (purchased from Huzhou Yingchuang)

[0066] 4. Negative control: Serum from healthy physical examination subjects

[0067] 5. Labeled antibody: Acridinium ester-labeled anti-human IgG antibody

[0068] 6. Antigen diluent

[0069] 7. Sample dilution buffer

[0070] 8. Antibody diluent

[0071] 9. Washing solution

[0072] 10. Pre-excitation solution: H2O2

[0073] 11. Excitation solution: NaOH

[0074] 5.2 Detection principle of the magnetic particle chemiluminescence immunoassay kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody

[0075] The chemiluminescent immunoassay kit of the present invention is an analytical method that combines magnetic separation technology, immunoassay technology, and chemiluminescent technology. The kit of the present invention uses an indirect method for quantitative analysis and detection of anti-Proteasome subunit alpha type 1-IgG antibodies in human serum: First, the magnetic particle solution is mixed with the diluted sample, and the specific anti-Proteasome subunit alpha type 1-IgG antibody binds to the magnetic particles coated with Proteasome subunit alpha type 1 antigen. After washing, an acridinium ester-labeled anti-human IgG antibody is added to form a complex of Proteasome subunit alpha type 1 antigen-coated magnetic particles - anti-Proteasome subunit alpha type 1-IgG antibody - acridinium ester-labeled anti-human IgG antibody. Under the action of an external magnetic field, the unbound substances are separated from the complex formed by the immune reaction. After discarding the supernatant, the precipitated complex is washed, and a pre-excitation solution (H2O2) and an excitation solution (NaOH) are added for a luminescence reaction. Under alkaline conditions, the acridinium ester molecule is attacked by hydrogen peroxide to generate dioxetane, which is unstable and decomposes into CO2 and electronically excited N-methylacridone. When it returns to the ground state, it emits light with a wavelength of 430 nm, and a chemiluminescence analyzer is used to collect the luminescence intensity. The concentration of anti-Proteasome subunit alpha type 1-IgG antibody is proportional to the luminescence value, and the concentration of anti-Proteasome subunit alpha type 1-IgG antibody in the serum to be tested is calculated through a calibration curve, see Figure 4 。

[0076] 5.3 Preparation of Proteasome subunit alpha type 1 antigen-coated magnetic particles

[0077] 5.3.1 Principle of Proteasome subunit alpha type 1 antigen-coated magnetic particles: Based on the reaction of the carboxyl functional groups contained on the surface of magnetic particles with an EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution to generate an unstable amino-reactive O-acylisourea intermediate, which reacts with NHS (N-hydroxysuccinimide) to generate a semi-stable amino-reactive NHS ester, and the semi-stable amino-reactive NHS ester further reacts with the amino groups on the antigen protein Proteasome subunit alpha type 1 to form Proteasome subunit alpha type 1 antigen-coated magnetic particles, see Figure 5 。

[0078] 5.3.2 Activation of carboxyl magnetic microparticles with EDC / NHS, the specific steps are as follows:

[0079] a) Weigh 10 mg of magnetic microparticles, wash the magnetic microparticles 3 times with 20 mM MES, separate with a magnet, and discard the supernatant.

[0080] b) Resuspend the washed magnetic microparticles in 100 μl of 20 mM MES to make the final concentration of magnetic microparticles 100 mg / ml.

[0081] c) Add 50 μl of 20 mg / ml EDC and 50 μl of 24 mg / ml Sulfo-NHS prepared with phosphate buffer to the washed magnetic microparticles in sequence, mix well, and let it stand at room temperature for activation for 30 min.

[0082] d) After the action of an external magnetic field, discard the supernatant, take 400 μl of 0.05 M phosphate buffer to wash the magnetic microparticles, and add 400 μl of preservation solution to make a constant volume for storage and standby.

[0083] 5.3.3 Crosslinking of activated magnetic microparticles with antigen protein Proteasome subunit alpha type 1 Add 1 ml of pre-cooled 20 mM MES to the above-activated magnetic microparticle solution to wash the magnetic microparticles 2 more times; take 200 μl of 2 mg / ml antigen protein Proteasome subunit alpha type 1 and add it to the activated magnetic microparticles, mix well, and let it stand at room temperature for reaction for 16 hours; after the reaction, add PBS buffer with 0.2% Tween20 and pH 7.4 to wash the magnetic microparticles 2 more times; then add PBS buffer with 0.2% Tween20 and 0.2% BSA and pH 7.4 until the final concentration of magnetic microparticles is 10 mg / ml, mix well, and let it stand at room temperature for reaction for 30 min; after the reaction, discard the supernatant, and resuspend the magnetic microparticles with PBS buffer with 0.2% Tween20 and 0.2% BSA. In this way, the crosslinking of activated magnetic microparticles with antigen protein Proteasome subunit alpha type 1 is completed.

[0084] 5.4 Preparation of acridinium ester-labeled anti-human IgG antibody, the specific steps are as follows:

[0085] a) Prepare a 2 mg / mL acridinium ester solution with dimethylformamide.

[0086] b) Prepare a 1 mg / mL anti-human IgG antibody with 0.2 M (pH 8.0) carbonate buffer.

[0087] c) Take the acridinium ester and anti-human IgG antibody with a molar ratio of 4:1, mix well, and react for 40 min.

[0088] d) Add 20 μl of carbonate buffer containing 5% lysine to terminate the reaction;

[0089] e) Obtain an acridinium ester-labeled anti-human IgG antibody solution with higher purity through desalting and impurity removal.

[0090] 5.5 Steps for detecting anti-Proteasome subunit alpha type 1-IgG antibody in serum by magnetic particle chemiluminescence immunoassay kit

[0091] 5.5.1 Add 100 μl of the diluted serum to be tested or the IgG standard against GST tag to 100 μl of the magnetic particle solution coated with the antigen protein Proteasome subunit alpha type 1, and react at 37 °C for 15 min. At the same time, perform negative and positive controls.

[0092] 5.5.2 Add 400 μl of washing solution and wash 3 times for 1 min each. Then add 100 μl of acridinium ester-labeled anti-human IgG antibody diluted 1:1000 and react at 37 °C for 15 min.

[0093] 5.5.3 Discard the supernatant and wash the precipitated complex. Wash 3 times with 400 μl of washing solution for 1 min each. Then add 100 μl of pre-excitation solution (H2O2) and 100 μl of excitation solution (NaOH) for reaction. Detect the chemiluminescence signal with a chemiluminescence analyzer and record the luminescence value. The concentration of anti-Proteasome subunit alpha type 1-IgG antibody in the serum to be tested is proportional to the luminescence value. Calculate the concentration of anti-Proteasome subunit alpha type 1-IgG antibody in the serum to be tested through the standard curve.

[0094] Example 6 Clinical application of the kit for detecting anti-Proteasome subunit alpha type 1-IgG antibody in serum

[0095] 6.1 Inclusion of subjects Patients diagnosed with various kidney diseases from June 2018 to June 2020, including 466 cases of autoimmune nephrotic syndrome (NS), 168 cases of Henoch-Schönlein purpura (HSP), 137 cases of Henoch-Schönlein purpura nephritis (HSPN), 133 cases of IgA nephropathy (IgAN), and 195 healthy children (NC) during the same period. Serum samples were taken from patients with various kidney diseases and the healthy control group. All subjects underwent the first serum sample collection before immunosuppressive treatment.

[0096] 6.2 Detection of anti-Proteasome subunit alpha type 1-IgG antibodies in various types of nephropathy patients The levels of anti-Proteasome subunit alpha type 1-IgG antibodies in the sera of patients diagnosed with various types of nephropathy from June 2018 to June 2020 were detected using the kit of the present invention, including 466 cases of autoimmune nephrotic syndrome, 168 cases of allergic purpura, 137 cases of purpuric nephritis, 133 cases of IgA nephropathy, and 195 healthy children during the same period. The results showed that anti-Proteasome subunit alpha type 1-IgG antibodies were positive in patients with autoimmune nephrotic syndrome, while anti-Proteasome subunit alpha type 1-IgG antibodies were negative in patients with purpuric nephritis, allergic purpura, IgA nephropathy, and healthy children, as shown in Figure 6 .

[0097] 6.3 ROC curve evaluation of the value of anti-Proteasome subunit alpha type 1-IgG antibody as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome The detection results of anti-Proteasome subunit alpha type 1-IgG antibodies in the patients with autoimmune nephrotic syndrome in 6.2 were analyzed using the ROC curve to evaluate the application value of anti-Proteasome subunit alpha type 1-IgG antibody in the diagnosis of autoimmune nephrotic syndrome. The results showed that anti-Proteasome subunit alpha type 1-IgG antibody is a good serological marker for the diagnosis of patients with autoimmune nephrotic syndrome. The sensitivity of anti-Proteasome subunit alpha type 1-IgG antibody (using a cut-off value greater than 32.2 as the standard) as a serological marker for the diagnosis of patients with autoimmune nephrotic syndrome was 74.4%, the specificity was 80.8%, and the area under the curve was 0.832, as shown in Figure 7 . SEQUENCE LISTING <110> Zhejiang University <120> Kit for detecting anti-proteasome subunit alpha 1-IgG antibody <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 263 <212> PRT <213> Artificial Sequence (Unknow) <400> 1 Met Phe Arg Asn Gln Tyr Asp Asn Asp Val Thr Val Trp Ser Pro Gln 1 5 10 15 Gly Arg Ile His Gln Ile Glu Tyr Ala Met Glu Ala Val Lys Gln Gly 20 25 30 Ser Ala Thr Val Gly Leu Lys Ser Lys Thr His Ala Val Leu Val Ala 35 40 45 Leu Lys Arg Ala Gln Ser Glu Leu Ala Ala His Gln Lys Lys Ile Leu 50 55 60 His Val Asp Asn His Ile Gly Ile Ser Ile Ala Gly Leu Thr Ala Asp 65 70 75 80 Ala Arg Leu Leu Cys Asn Phe Met Arg Gln Glu Cys Leu Asp Ser Arg 85 90 95 Phe Val Phe Asp Arg Pro Leu Pro Val Ser Arg Leu Val Ser Leu Ile 100 105 110 Gly Ser Lys Thr Gln Ile Pro Thr Gln Arg Tyr Gly Arg Arg Pro Tyr 115 120 125 Gly Val Gly Leu Leu Ile Ala Gly Tyr Asp Asp Met Gly Pro His Ile 130 135 140 Phe Gln Thr Cys Pro Ser Ala Asn Tyr Phe Asp Cys Arg Ala Met Ser 145 150 155 160 Ile Gly Ala Arg Ser Gln Ser Ala Arg Thr Tyr Leu Glu Arg His Met 165 170 175 Ser Glu Phe Met Glu Cys Asn Leu Asn Glu Leu Val Lys His Gly Leu 180 185 190 Arg Ala Leu Arg Glu Thr Leu Pro Ala Glu Gln Asp Leu Thr Thr Lys 195 200 205 Asn Val Ser Ile Gly Ile Val Gly Lys Asp Leu Glu Phe Thr Ile Tyr 210 215 220 Asp Asp Asp Asp Val Ser Pro Phe Leu Glu Gly Leu Glu Glu Arg Pro 225 230 235 240 Gln Arg Lys Ala Gln Pro Ala Gln Pro Ala Asp Glu Pro Ala Glu Lys 245 250 255 Ala Asp Glu Pro Met Glu His 260

Claims

1. Use of a proteasome subunit alpha type 1 polypeptide capable of forming any antigen-antibody complex upon contact 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-proteasome subunit alpha type 1-IgG antibody complex; wherein the nephrotic syndrome is an autoimmune nephrotic syndrome; wherein the polypeptide sequence of the proteasome subunit alpha type 1 is as shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the nephrotic syndrome is a 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 a patient before immunotherapy.

5. Use of a proteasome subunit alpha type 1 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 with respect to Henoch-Schönlein purpura nephritis, Henoch-Schönlein purpura, and IgA nephropathy; wherein, The antigen-antibody complex includes an anti-proteasome subunit alpha type 1-IgG antibody complex; wherein the nephrotic syndrome is an autoimmune nephrotic syndrome; wherein the polypeptide sequence of the proteasome subunit alpha type 1 is as shown in SEQ ID NO.1.

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