A kit for detecting anti-peroxiredoxin-1-igG antibody

Abstract

This invention provides a kit for detecting anti-peroxidase-1-IgG antibodies, comprising the antigen protein peroxiredoxin-1 (peroxidase-1), a solid-phase carrier, labeled antibody, antigen dilution buffer, sample dilution buffer, antibody dilution buffer, substrate chromogenic agent, washing buffer, standards, positive control, and negative control. This kit utilizes an indirect reaction principle combined with magnetic microparticle chemiluminescence immunoassay to detect anti-peroxidase-1-IgG antibodies in the serum of the test sample. This invention is the first to identify autoantibodies against the target antigen peroxidase-1 in the serum of patients with autoimmune nephrotic syndrome. The provided kit provides a basis for domestic and international research on the molecular mechanisms and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to peroxidase-1 and peroxidase-1-IgG autoantibodies.

Description

[0001] This application is a divisional application of application number 202110743523.5, filed on July 1, 2021, entitled "A Kit for Detecting Anti-peroxidase-1-IgG Antibody". Technical Field

[0002] This invention belongs to the field of biomedical technology and relates to a kit for detecting anti-peroxidase-1-IgG antibodies. Background Technology

[0003] In recent years, the types of kidney diseases in children have been increasing, with autoimmune nephrotic syndrome (ANS) having the highest incidence, seriously endangering children's physical and mental health. ANS is a syndrome characterized by increased glomerular filtration membrane permeability, leading to increased plasma protein filtration and massive proteinuria. Patients typically present with massive proteinuria, hypoalbuminemia, and severe edema. Researchers such as Ali observed that after kidney transplantation from patients with refractory minimal change disease (MND), the recipients' kidney function remained normal without any proteinuria. This suggests that the cause of MND is not solely within the kidneys themselves, but may primarily stem from problems in the patient's internal environment. Furthermore, aside from some children with ANS caused by genetic defects, most children with ANS show improvement after treatment with hormones and immunosuppressants, indirectly demonstrating a close link between the disease and the patient's autoimmune system.

[0004] In recent years, B-cell dysfunction has been found to play an important role in autoimmune nephrotic syndrome. Multiple multicenter clinical studies worldwide have demonstrated the successful use of rituximab (RTX) in the treatment of minimal change disease (MDR), particularly in refractory MDR. However, studies have also found that rituximab's B-cell-clearing effect in treating hormone-dependent nephrotic syndrome lasts approximately five months, with relapse occurring around six to seven months as B-cell counts recover. This suggests the presence of pathological B-cell clones in MDR patients. Identifying and precisely eliminating these pathological B-cell clones is beneficial for MDR recovery and reduces the risk of humoral immune deficiencies caused by indiscriminate B-cell clearance using rituximab and other methods. However, the target antigens of these pathological B-cells in children with MDR remain unclear to date. Pathologically, minimal change disease or focal segmental glomerulosclerosis is considered a podocyte disease caused by loss or alteration of podocyte function, leading to massive proteinuria. Podocytes are glomerular epithelial cells of the kidney, attached to the outer side of the glomerular basement membrane, and are the last barrier preventing protein loss. Damage to podocytes usually causes massive proteinuria.

[0005] Peroxiredoxin (peroxide reductase) participates in enzymatic reactions that constitute the body's antioxidant system, playing an important role in the organism's innate immune response. Peroxiredoxin-1 is a subunit of the peroxiredoxin family. It has been used in cancer research, including studies on oral squamous cell carcinoma (Yanagawa T, Iwasa S, Ishii T, et al. PeroxiredoxinⅠ expression in oral cancer: A potential new tumor marker. [J] Cancer Lett, 2000, 156(1): 27-35.), thyroid cancer (Kim YJ, Ahn JY, Liang P, et al. Human prdx1 gene is a target of Nrf2 and is up-regulated by hypoxia / reoxygenation: Implication to tumor biology. Cancer Res, 2007, 67(2): 546-554.), and lung cancer (Kim JH, Bogner PN, Ramnath N, et al. Elevated peroxiredoxin 1, but not NF-E2-related factor 2, is an independent prognostic factor for disease recurrence and reduced survival in...). Overexpression of peroxiredoxin-1 can be detected in stage I nonsmall cell lung cancer. Clin Cancer Res, 2007, 13(13): 3875-3882. Rho et al. first found that peroxiredoxin-1 is upregulated in human colorectal cancer tissues, and its expression is closely related to the proliferation, differentiation, invasion and metastasis of colorectal cancer, and plays a bidirectional regulatory role in rectal cancer (Rho J, Qin S, Wang J, et al. Proteomicexpression analysis of surgical human colorectal cancer tissues: Up-regulation of PSB7, peroxiredoxin-1, and SRP9 and hypoxic adaptation in cancer. J Proteome Res, 2008, 7(7): 2959-2972.).

[0006] However, there are currently no reports on the expression of peroxiredoxin-1 or the presence of peroxiredoxin-1 autoantibodies in nephrotic syndrome. Furthermore, existing techniques do not address the application of peroxiredoxin-1 or its autoantibodies as serological markers in autoimmune nephrotic syndrome. Research on identifying autoimmune nephrotic syndrome by detecting serum anti-peroxiredoxin-1-IgG antibodies is lacking. Summary of the Invention

[0007] The purpose of this invention is to provide a kit for detecting anti-peroxidase-1-IgG antibodies, which is a detection kit based on the target peroxiredoxin-1 and its corresponding autoantibodies. The kit can detect autoantibodies from tissues (kidney biopsy tissue) or body fluids (especially blood, plasma, and serum) through an immune reaction with the antigen protein peroxiredoxin-1 (particularly according to the sequence identification number SEQ ID NO.1).

[0008] The kit consists of the antigen protein peroxiredoxin-1, a solid-phase carrier, labeled antibodies such as enzyme-labeled, chemiluminescent-labeled, or biotin-labeled secondary antibodies, antigen dilution buffer, sample dilution buffer, antibody dilution buffer, substrate chromogenic agent, washing buffer, standards, positive control, and negative control.

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

[0010] MSSGNAKIGHPAPNFKATAVMPDGQFKDISLSDYKGKYVVFFFYPLDFTFVCPTEIIAFSDRAEEFKKLNCQVIGASVDSHFCHLAWVNTPKKQGGLGPMNIPLVSDPKRTIAQDYGVLKADEGISFRGLFIIDDKGILRQITVNLPVGRSVDETLRLVQAFQFTDKHGEVCPAGWKPGSDTIKPDVQKSKEYFSKQK.

[0011] The peroxiredoxin-1 antigen protein of the present invention can be a fusion protein, using a tag with certain biological or physical functions, particularly an N-terminal or C-terminal tag. The presence of these tags facilitates 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, and the tag peptide is selected from: His tag, thioredoxin, GST tag, maltose-binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag, or biotin tag.

[0012] According to the present invention, the antigen protein peroxiredoxin-1 is immobilized on a solid support, preferably including: nitrocellulose membrane, magnetic microparticles, and enzyme-labeled microplate.

[0013] In one embodiment of the present invention, the standard and positive control are recombinant human anti-tag peptide immunoglobulin G or a fragment thereof, or anti-peroxiredoxin-1-IgG antibody extracted from patient serum as positive control and standard, and serum from healthy examinees as negative control.

[0014] According to the present invention, the antigen protein peroxiredoxin-1 can be expressed in bacteria such as Escherichia coli, fungi such as yeast, and mammalian cells.

[0015] According to the present invention, the antigen protein peroxiredoxin-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, selected from whole blood, serum, plasma, urine, lymph, and pleural or peritoneal fluid. Preferably, it is mammalian (human) serum.

[0017] The detection kit also includes a substrate chromogenic agent, an antigen diluent, a sample dilution buffer, an antibody diluent, and a washing buffer. The substrate chromogenic agent is TMB, hydrogen peroxide, 4-MUP, AMPPD, 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; and the washing buffer is 1x PBS (pH 7.4) containing 163 mM NaCl, 10% glycerol, and 1% Triton X-100.

[0018] In a preferred embodiment, as used herein, “immobilization” refers to binding to a water-insoluble solid support for the peroxiredoxin-1 antigen protein. This solid support or substrate is insoluble in water, more preferably through covalent bonding, electrostatic interactions, hydrophobic interactions, or disulfide interactions, and most preferably through one or more covalent bonds. Immobilization can be a direct immobilization method, such as by filtration, centrifugation, or chromatography, separating the immobilized molecules from the aqueous solution along with the insoluble support. Reversible or irreversible immobilization of the peroxiredoxin-1 antigen protein is also included. For example, the antigen protein is immobilized to the support via cleavable covalent bonds (such as disulfide bonds that can be cleaved by the addition of a thiol-containing reagent), which is reversible. Conversely, if the antigen protein is immobilized to the support via covalent bonds that do not cleave in aqueous solution (bonds 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. Fixation can also be done indirectly: for example, fixing an antibody with a specific affinity for the antigen protein and then forming an antigen protein-antibody complex to achieve the purpose of fixation.

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

[0020] The labeled antibody described in this invention can be horseradish peroxidase (HRP) labeled anti-human IgG antibody, biotin labeled anti-human IgG antibody, or acridine ester labeled anti-human IgG antibody.

[0021] This invention utilizes a recombinant prokaryotic expression method to successfully express and purify the recombinant protein peroxiredoxin-1. Using this as the antigen protein in a kit, a suitable kit for detecting serum anti-peroxiredoxin-1-IgG antibodies in patients with autoimmune nephrotic syndrome has been developed. This includes a detection kit for qualitative or quantitative analysis of anti-peroxiredoxin-1-IgG antibodies in human serum.

[0022] A kit for detecting anti-peroxiredoxin-1-IgG antibodies in serum utilizes an indirect reaction principle. First, the peroxiredoxin-1 antigen is adsorbed onto a solid-phase carrier as the coating antigen. Then, positive control samples, standards, or the serum sample to be tested are added and incubated. Next, a labeled secondary antibody is added, and if the serum sample contains anti-peroxiredoxin-1-IgG antibodies, a ternary complex is formed: coating antigen peroxiredoxin-1 - serum anti-peroxiredoxin-1-IgG antibody - labeled anti-human IgG antibody. Finally, the light signal is detected using photoluminescence, chemiluminescence, or fluorescence luminescence methods to achieve qualitative or quantitative analysis of anti-peroxiredoxin-1-IgG antibodies in human serum.

[0023] Using the kit of this invention, an anti-peroxiredoxin-1-IgG autoantibody was detected for the first time in some patients with autoimmune nephrotic syndrome, and the target antigen of this autoantibody was identified as peroxiredoxin-1 on podocytes. Therefore, the kit of this invention can be used for the detection of anti-peroxiredoxin-1-IgG autoantibodies, providing a basis for the study of autoimmune nephrotic syndrome.

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

[0025] (1) Currently, research on peroxiredoxin-1 and anti-peroxiredoxin-1-IgG antibodies in patients with kidney disease, both domestically and internationally, is limited to molecular mechanism studies, and quantitative detection of their levels in patient serum is not available. This invention is the first to identify an autoantibody against peroxiredoxin-1 and has developed a detection kit for this peroxiredoxin-1-IgG autoantibody, filling a gap both domestically and internationally.

[0026] (2) The kit of the present invention relates to the qualitative analysis of anti-peroxiredoxin-1-IgG antibodies in human serum. The solid-phase membrane immunoassay is simple to operate and requires less reagent, saving nearly 10 times compared with the traditional ELISA. In addition, the NC membrane has extremely strong adsorption capacity, close to 100%, and trace amounts of antigen can be completely adsorbed and fixed on the NC membrane. The NC membrane with adsorbed antigen or antibody or with existing results can be stored for a long time (six months at -20℃) without affecting its activity. Furthermore, the kit of the present invention for the solid-phase membrane immunoassay of anti-peroxiredoxin-1-IgG antibodies in human serum introduces a biotin-avidin amplification system, which greatly improves the detection sensitivity.

[0027] (3) The magnetic microparticle chemiluminescence immunoassay kit for quantitative detection of anti-peroxiredoxin-1-IgG antibodies in human serum, which relates to this invention, utilizes magnetic microparticles as a solid-phase carrier with a diameter of only 1.0 μm. This greatly increases the coating surface area, the amount of antigen adsorbed, and the reaction rate, and also makes washing and separation simpler, thereby reducing contamination and lowering the probability of cross-infection. On the other hand, the acridinium ester luminescent agent is used to directly label anti-human IgG, and its chemical reaction is simple, rapid, and requires no catalyst. The acridinium ester chemiluminescence is flash-type, and its emission intensity reaches its maximum 0.4s after being activated by the luminescent reagent (H2O2, NaOH), with a half-life of 0.9s, and basically ends within 2s, which facilitates rapid detection. Attached Figure Description

[0028] Figure 1 The peroxiredoxin-1 protein on podocytes is a target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Figure 1 A: Two-dimensional electrophoretic protein spot pattern of serum from healthy individuals with primary antibody; 1B: Two-dimensional electrophoretic protein spot pattern of serum from patients with autoimmune nephrotic syndrome with primary antibody; Figure 1 C: Mass spectrometry identification of the target antigen peroxiredoxin-1 protein.

[0029] Figure 2 SDS-PAGE image of the expressed recombinant protein peroxiredoxin-1.

[0030] Figure 3 Solid-phase membrane immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome.

[0031] Figure 4 Schematic diagram of the principle of the magnetic microparticle chemiluminescent immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibody.

[0032] Figure 5 Schematic diagram of antigen protein peroxiredoxin-1 coated with carboxyl magnetic microparticles.

[0033] Figure 6 The detection status of anti-peroxiredoxin-1-IgG antibodies in patients with various types of kidney diseases, including NS: autoimmune nephrotic syndrome, HSP: Henoch-Schönlein purpura, HSPN: Henoch-Schönlein purpura nephritis, IgAN: IgA nephropathy, and NC: healthy children.

[0034] Figure 7 ROC curve analysis was used to evaluate the application value of anti-peroxiredoxin-1-IgG antibody as a serological marker for the diagnosis of PNS patients. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0036] Example 1: The peroxiredoxin-1 protein on podocytes is a target antigen of autoantibodies in patients with autoimmune nephrotic syndrome.

[0037] This invention, through extensive preliminary clinical and molecular mechanism studies, first discovered that patients with nephrotic syndrome have higher serum IgG levels and confirmed that peroxiredoxin-1 on podocytes is the target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. The specific implementation is as follows: (1) Extraction of total protein from glomerular podocytes: Culture podocytes (MPC5), wash 2-3 times with PBS, and then perform thorough lysis on ice using a focused ultrasound instrument (Covaris S220, Gene) in lysis buffer containing 30 mM Tris-HCl, 8 mM urea, 4% CHAPS and protease inhibitor (#ab65621; Abcam, 1:200 dilution). Then place the sample in a centrifuge at 12000g, 4℃, for 30 min. Collect the supernatant, which is the collected total protein from glomerular podocytes. The concentration of the collected total protein from glomerular podocytes is determined using a BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: Total protein extracted from glomerular podocytes was subjected to two-dimensional electrophoresis and then transferred to a nitrocellulose membrane. The membrane was incubated with primary antibodies from serum of healthy individuals and patients with autoimmune nephrotic syndrome, respectively, followed by secondary antibody addition and imaging. (See figure) Figure 1 A, 1B. (3) Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis: After development in 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 individuals were selected from the two-dimensional electrophoresis gel. The selected protein spots were cut off 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 overnight at 37°C. The peptide was then extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptide was analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain a peptide mass spectrum. It was identified as peroxiredoxin-1 protein. See Figure 1 C.

[0038] Example 2: Expression and purification of recombinant antigen protein peroxiredoxin-1

[0039] Using genetic engineering methods, the gene encoding peroxiredoxin-1 protein was used as a template for PCR amplification, followed by the construction of an expression vector for protein expression. The expressed antigen protein contains a His-tagged peptide. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column chromatography, and molecular sieve analysis. Finally, SDS-PAGE analysis confirmed the molecular weight of the recombinant protein peroxiredoxin-1 to be 27 kDa. Figure 2 .

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

[0041] An orthogonal array was selected based on four factors: antigen peroxiredoxin-1 coating concentration (50 μg, 80 μg, 100 μg, and 150 μg), reaction time (15 min, 30 min, and 45 min), temperature (25℃ and 37℃), and optimal enzyme-labeled secondary antibody dilution (1:100, 1:500, 1:1000, and 1:1500). Each factor was tested in duplicate at two levels with standard positive and negative sera. The ratio of the highest light signal value (P) of positive serum to the lowest light signal value (N) of negative serum (P / N) was selected. Through orthogonal design, we determined the optimal antigen-coating concentration of peroxiredoxin-1 for this kit to be 80 μg / ml, the optimal antigen-antibody reaction temperature for the solid-phase membrane immunoassay kit for anti-peroxiredoxin-1-IgG antibody to be 25℃, the optimal antigen-antibody reaction time to be 30 min, and the optimal working dilution of biotin-labeled anti-human IgG antibody to be 1:500. For the magnetic microparticle chemiluminescent immunoassay kit for anti-peroxiredoxin-1-IgG antibody to be detected, we determined the optimal antigen-antibody reaction temperature to be 37℃, the optimal antigen-antibody reaction time to be 15 min, and the optimal working dilution of acridine ester-labeled anti-human IgG antibody to be 1:500.

[0042] Example 4: Preparation of a solid-phase membrane immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibodies

[0043] 4.1 Composition of the solid-phase membrane immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibodies:

[0044] 1. Antigen: Recombinant protein peroxiredoxin-1

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

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

[0047] 4. Negative control: Serum from healthy individuals undergoing physical examinations

[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. Detergent

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

[0054] 11. Substrate and developing solution: BCIP developing solution.

[0055] 4.2 The detection steps for the solid-phase membrane immunoassay kit used to detect anti-peroxiredoxin-1-IgG antibodies are as follows:

[0056] 4.2.1 Coating and blocking: 8 μl of peroxiredoxin-1 antigen at a concentration of 80 μg / ml was directly spotted onto the nitrocellulose membrane and dried in an incubator at 37°C for 30 min. The nitrocellulose membrane was then placed in the detection plate, and 200 μl of 5% BSA was added and blocked in a box at 37°C for 30 min. The blocking solution was discarded and the membrane was washed twice with washing solution.

[0057] 4.2.2 Antigen Incubation: Add 10 μl of antibody standard or serum to be tested diluted with diluent to the detection plate, and...

[0058] Negative and positive controls were prepared at the same time, and the samples were incubated at room temperature for 30 minutes. Three parallel wells were set up for each sample.

[0059] 4.2.3 Secondary antibody incubation: Discard the liquid in the test plate, wash 5 times with washing buffer for 1 min each time, add 20 μl of 1:500 biotin-labeled anti-human IgG antibody, and incubate at room temperature for 30 min;

[0060] 4.2.4 Color Development: Discard the liquid in the test plate, wash 5 times with washing buffer for 1 min each time, add 500 μl of alkaline phosphatase-streptavidin, incubate at room temperature for 20 min, discard the liquid in the test plate, wash 5 times with washing buffer for 1 min each time, then add BCIP colorimetric solution, react at room temperature for 20 min, rinse the test plate with running water to terminate the enzyme reaction. Remove the nitrocellulose membrane strip and dry it with a hair dryer. Visually determine the color using a colorimetric card; a positive result is indicated by the appearance of obvious brown spots. Figure 3Alternatively, the membrane strip can be placed on a developing instrument for scanning. The analytical software built into the developing instrument uses the concentration of the reference standard as the ordinate and the gray value read by the instrument as the abscissa to plot a standard curve for semi-quantitative analysis of the level of anti-peroxiredoxin-1-IgG antibody in serum.

[0061] Example 5: Preparation of a magnetic microparticle chemiluminescent immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibodies

[0062] 5.1 Composition of the magnetic microparticle chemiluminescent immunoassay kit for detecting anti-peroxiredoxin-1-IgG antibodies:

[0063] 1. Antigen: Recombinant protein peroxiredoxin-1

[0064] 2. Solid support: Magnetic microparticles with carboxyl functional groups

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

[0066] 4. Negative control: Serum from healthy individuals undergoing physical examinations

[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. Detergent

[0072] 10. Pre-activation solution: H2O2

[0073] 11. Activation solution: NaOH.

[0074] 5.2 Detection Principle of the Magnetic Microparticle Chemiluminescent Immunoassay Kit for Detecting Anti-peroxiredoxin-1-IgG Antibody

[0075] The chemiluminescence immunoassay kit of this invention is an analytical method that combines magnetic separation technology, immunoassay technology, and chemiluminescence technology. The kit of this invention uses an indirect method to quantitatively analyze and detect anti-peroxiredoxin-1-IgG antibodies in human serum: First, magnetic microparticle solution is mixed with diluted sample. Specific anti-peroxiredoxin-1-IgG antibody binds to magnetic microparticles coated with peroxiredoxin-1 antigen. After washing, acridinium ester-labeled anti-human IgG antibody is added to form a peroxiredoxin-1 antigen-coated magnetic microparticle-anti-peroxiredoxin-1-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, the precipitated complex is washed, and pre-excitation solution (H2O2) and excitation solution (NaOH) are added to initiate a luminescence reaction. Under alkaline conditions, acridinium ester molecules are attacked by hydrogen peroxide to generate ethylenedioxane. Ethylenedioxane 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. The luminescence intensity is collected by a chemiluminescence analyzer. The concentration of anti-peroxiredoxin-1-IgG antibody in the serum being tested is directly proportional to the luminescence value. The concentration of anti-peroxiredoxin-1-IgG antibody in the serum being tested was calculated using a calibration curve. (See figure) Figure 4 .

[0076] 5.3 Preparation of peroxiredoxin-1 antigen-coated magnetic microparticles

[0077] 5.3.1 The principle of coating peroxiredoxin-1 antigen with 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-active O-acylurea intermediate. This intermediate 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 peroxiredoxin-1 to form peroxiredoxin-1 antigen-coated magnetic microparticles. See [link to documentation]. Figure 5 .

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

[0079] a) Weigh 10mg of magnetic microparticles, wash the magnetic microparticles three times with 20mM MES, separate the magnets, and discard the supernatant;

[0080] b) The cleaned magnetic microparticles were resuspended 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 Sμlfo-NHS prepared in phosphate buffer to the cleaned magnetic microparticles in sequence, mix thoroughly, and let stand at room temperature for 30 min to activate.

[0082] d) After applying an external magnetic field, discard the supernatant, wash the magnetic microparticles with 400 μl of 0.05 M phosphate buffer, add 400 μl of preservation solution to make up the volume and store for later use.

[0083] 5.3.3 Crosslinking of Activated Magnetic Microparticles with Peroxiredoxin-1 Antigen Protein: Add 1 ml of pre-cooled 20 mM MES to the activated magnetic microparticle solution and wash the magnetic microparticles twice. Add 200 μl of 2 mg / ml peroxiredoxin-1 antigen protein to the activated magnetic microparticles, mix thoroughly, and incubate at room temperature for 16 hours. After the reaction, add pH 7.4 PBS buffer containing 0.2% Tween 20 and wash the magnetic microparticles twice. Then add pH 7.4 PBS buffer containing 0.2% Tween 20 and 0.2% BSA until the final concentration of magnetic microparticles is 10 mg / ml, mix thoroughly, and incubate at room temperature for 30 minutes. After the reaction, discard the supernatant and resuspend the magnetic microparticles in pH 7.4 PBS buffer containing 0.2% Tween 20 and 0.2% BSA. This completes the crosslinking of the activated magnetic microparticles with the peroxiredoxin-1 antigen protein.

[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 acridine ester solution using dimethylformamide;

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

[0087] c) Mix acridine ester with anti-human IgG antibody at a molar ratio of 4:1 thoroughly and react for 40 min;

[0088] d) The reaction was terminated by adding 20 μl of carbonate buffer containing 5% lysine;

[0089] e) By desalting and removing impurities, a high-purity acridine ester-labeled anti-human IgG antibody solution is obtained.

[0090] 5.5 Steps for detecting anti-peroxiredoxin-1-IgG antibodies in serum using a magnetic microparticle chemiluminescent immunoassay kit

[0091] 5.5.1 Add 100 μl of diluted serum or anti-His-tagged IgG standard to 100 μl of peroxiredoxin-1 antigen-coated magnetic microparticle solution and react at 37°C for 15 min. Simultaneously, perform positive and negative controls.

[0092] 5.5.2 Addition of labeled antibody: Wash 3 times x 1 min with 400 μl washing buffer, add 100 μl of acridine ester-labeled anti-human IgG antibody diluted 1:500, and react at 37℃ for 15 min;

[0093] 5.5.3 Signal Detection: Wash three times with 400 μl of washing buffer for 1 min each time. Add pre-activation solution (H2O2) and activation solution (NaOH) to initiate the reaction. Detect the luminescence signal using a chemiluminescence analyzer and record the luminescence value. The concentration of anti-peroxiredoxin-1-IgG antibody in the test serum is directly proportional to the luminescence value. Calculate the concentration of anti-peroxiredoxin-1-IgG antibody in the test serum using a standard curve.

[0094] Example 6: Clinical application of the reagent kit for detecting serum anti-peroxiredoxin-1-IgG antibodies

[0095] 6.1 Participants included patients diagnosed with various kidney diseases between June 2018 and June 2020, including 466 patients with nephrotic syndrome (NS), 168 patients with Henoch-Schönlein purpura (HSP), 137 patients with Henoch-Schönlein purpura nephritis (HSPN), 133 patients with IgA nephropathy (IgAN), and 195 healthy children (NC) during the same period. Serum samples were collected from patients with various kidney diseases and healthy controls. All participants underwent their first serum sample collection before receiving immunosuppressive therapy.

[0096] 6.2 Detection of Anti-peroxiredoxin-1-IgG Antibodies in Patients with Various Kidney Diseases The levels of anti-peroxiredoxin-1-IgG antibodies in the serum of patients diagnosed with various kidney diseases from June 2018 to June 2020 were detected using the kit of this invention. This included 466 cases of autoimmune nephrotic syndrome, 168 cases of Henoch-Schönlein purpura, 137 cases of Henoch-Schönlein purpura nephritis, 133 cases of IgA nephropathy, and 195 healthy children during the same period. The results showed that patients with autoimmune nephrotic syndrome were positive for anti-peroxiredoxin-1-IgG antibodies, while patients with Henoch-Schönlein purpura nephritis, Henoch-Schönlein purpura, IgA nephropathy, and healthy children were negative for anti-peroxiredoxin-1-IgG antibodies. (See [link to original text]). Figure 6 .

[0097] 6.3 ROC Curve Evaluation of the Value of Anti-peroxiredoxin-1-IgG Antibody as a Serological Marker in the Diagnosis of Autoimmune Nephrotic Syndrome The detection results of anti-peroxiredoxin-1-IgG antibody in patients with autoimmune nephrotic syndrome in Example 6.2 were analyzed using ROC curves to evaluate the application value of anti-peroxiredoxin-1-IgG antibody in the diagnosis of autoimmune nephrotic syndrome. The results showed that anti-peroxiredoxin-1-IgG antibody is a good serological marker for the diagnosis of autoimmune nephrotic syndrome. The sensitivity of anti-peroxiredoxin-1-IgG antibody (using a cutoff value greater than 172.5 as the standard) as a serological marker for the diagnosis of autoimmune nephrotic syndrome was 77.3%, the specificity was 65.3%, and the area under the curve was 0.777. (See [link to ROC curve analysis]). Figure 7 . SEQUENCE LISTING <110> Zhejiang University <120> A kit for detecting anti-peroxidase-1-IgG antibodies <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 199 <212> PRT <213> Artificial sequence (Unknown) <400> 1 Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn Phe Lys 1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser 20 25 30 Asp Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe 35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Glu 50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser 65 70 75 80 His Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys Gln Gly Gly 85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys Arg Thr Ile 100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg 115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Val 130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr Leu Arg Leu Val 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Gln Lys Ser Lys 180 185 190 Glu Tyr Phe Ser Lys Gln Lys 195

Claims

1. Use of a peroxiredoxin-1 polypeptide or an antibody binding fragment thereof capable of forming any antigen-antibody complex with a sample obtained from a patient in the manufacture of a test reagent or kit for nephrotic syndrome; wherein, The antigen-antibody complex comprises an anti-peroxiredoxin-1-IgG antibody complex; wherein the nephrotic syndrome is a pediatric autoimmune nephrotic syndrome; wherein the peroxiredoxin-1 antibody binding fragment sequence is set forth in SEQ ID NO.

1.

2. The use according to claim 1, wherein the sample is serum.

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

4. Use of a peroxiredoxin-1 polypeptide or antibody binding fragment capable of forming any antigen-antibody complex with a sample obtained from a patient in the manufacture of a reagent or kit for the specific detection of nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, IgA nephropathy; wherein, The antigen-antibody complex comprises an anti-peroxiredoxin-1-IgG antibody complex.

5. A reagent for diagnosing a pediatric autoimmune renal syndrome or specifically detecting a pediatric autoimmune renal syndrome relative to purpura nephritis, anaphylactic purpura, IgA nephropathy, comprising: A peroxiredoxin-1 polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex upon contact with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-peroxiredoxin-1-IgG antibody complex; wherein the peroxiredoxin-1 antibody binding fragment sequence is set forth in SEQ ID NO.

1.

6. A kit for diagnosing a pediatric autoimmune renal syndrome or detecting a pediatric autoimmune renal syndrome specifically with respect to purpura nephritis, anaphylactoid purpura, IgA nephropathy, comprising: A peroxiredoxin-1 polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex upon contact with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-peroxiredoxin-1-IgG antibody complex; wherein the peroxiredoxin-1 antibody binding fragment sequence is set forth in SEQ ID NO.

1. A peroxiredoxin-1 polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex upon contact with a sample obtained from a patient; wherein the antigen-antibody complex comprises an anti-peroxiredoxin-1-IgG antibody complex; wherein the peroxiredoxin-1 antibody binding fragment sequence is set forth in SEQ ID NO. 1.

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