Anti-Nephrin autoantibody assay kit, detection method, and preparation method thereof

Abstract

The present invention relates to magnetic microparticles that specifically bind to anti-nephrin antibodies in a sample, comprising: magnetic beads coated with a polypeptide or fragment thereof that specifically binds to mammalian anti-nephrin autoantibodies; wherein the magnetic beads are carboxyl magnetic beads; the particle size of the carboxyl magnetic beads is 1-5 μm; preferably, the particle size of the carboxyl magnetic beads is 1-3 μm; more preferably, the particle size of the magnetic beads is 3 μm. The anti-nephrin antibody detection method of the present application has a short detection time, high sensitivity, strong specificity, stable and reliable results, high uniformity, and is easy to promote clinically.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, in particular to an anti-Nephrin autoantibody detection kit and a detection method and a preparation method thereof. Background Art

[0002] Nephrin protein is mainly found in the slit diaphragm of podocytes in the glomerulus. It is a membrane protein that is essential for maintaining the normal function of the glomerular filtration barrier. As early as 2021, when we first discovered and screened for podocyte autoantibodies specific to idiopathic nephrotic syndrome, we discovered the presence of anti-Nephrin autoantibodies. 1 . However, the concentration of anti-Nephrin autoantibodies in serum is relatively low compared to other podocyte autoantibodies we have found. Therefore, the detection rate is low due to the limited sensitivity of conventional enzyme-linked immunosorbent assay and immunoblotting. Subsequently, further studies found that post-transplant relapse in patients with focal segmental glomerulosclerosis nephrotic syndrome is associated with nephrin autoantibodies, indicating that anti-Nephrin autoantibodies may be an important pathogenic factor. 2,3 Recent clinical cohort studies have further found that anti-Nephrin autoantibodies are widely present in patients with minimal change disease and idiopathic nephrotic syndrome, and are closely related to disease activity, making them an effective biomarker for monitoring disease changes and treatment response. 4,5 In summary, accumulating evidence indicates that anti-nephrin autoantibodies have important clinical value. There is an urgent need to establish and develop anti-nephrin antibody detection methods and kits that are quick to detect, highly sensitive, specific, stable and reliable, highly uniform, and easily applicable in clinical practice.

[0003] Existing methods for detecting anti-nephrin antibodies include enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). ELISA suffers from poor sensitivity, a lengthy experimental process, and significant inter-batch variability in reagents. While RIA offers high sensitivity and specificity, the use of radioactive tracers can lead to radioactive contamination and hazards. Furthermore, the short half-life of commonly used radionuclides limits the shelf life of test kits, making automated analysis impossible and challenging to implement in clinical practice. There is an urgent need for routine, reliable, efficient, and clinically accessible methods and kits for the quantitative detection of anti-nephrin autoantibodies.

[0004] [1]Ye Q,Zhang Y,Zhuang J,Bi Y,Xu H,Shen Q,Liu J,Fu H,Wang J,Feng C,Tang X,Liu F,Gu W,Zhao F,Zhang J,Qin Y,Shang S,Shen H,Chen X,Shen H,Liu A,XiaY,Lu Z,Shu Q,Mao J.The important roles and molecular mechanisms of annexin A2autoantibody in children with nephrotic syndrome.Ann Transl Med.2021Sep;9(18):1452.

[0005] [2]Hattori M.Anti-nephrin autoantibodies:novel predictors of post-transplant recurrence of focal segmental glomerular sclerosis.KidneyInt.2024Oct;106(4):570-572;

[0006] [3]Hattori M,Shirai Y,Kanda S,et al.Circulating nephrinautoantibodies and posttransplant recurrence of primary focal segmentalglomerulosclerosis.Am JTransplant.2022Oct;22(10):2478-2480;

[0007] [4]Watts AJB,Keller KH,Lerner G,et al.Discovery of AutoantibodiesTargeting Nephrin in Minimal Change Disease Supports a Novel AutoimmuneEtiology.J Am Soc Nephrol.2022Jan;33(1):238-252.doi:10.1681 / ASN.2021060794;

[0008] [5] Tomas NM, Hengel FE, Huber TB. Autoantibodies Targeting Nephrin in Podocytopathies. Reply. N Engl J Med. 2024 Oct 10; 391(14): 1367-1368. doi: 10.1056 / NEJMc2410840. Summary of the Invention

[0009] In view of the technical problems existing in the prior art, the first object of the present invention is to provide an anti-Nephrin autoantibody determination kit, which can solve the problems of low accuracy, low sensitivity and low signal-to-noise ratio of the existing kits.

[0010] The second object of the present invention is to provide a detection method for an anti-Nephrin autoantibody determination kit, which can solve the problems of complex detection methods and long detection time of existing kits.

[0011] The third object of the present invention is to provide a method for preparing an anti-Nephrin autoantibody assay kit, which can solve the problems of low stability, difficult operation, high production cost and difficulty in clinical promotion of existing kit preparation processes.

[0012] The first and third objectives of the present invention are achieved by the following technical solutions:

[0013] A magnetic particle that specifically binds to an anti-nephrin antibody in a sample, the magnetic particle comprising a nephrin antigen and magnetic beads, wherein the nephrin antigen is coated on the surface of the magnetic beads; wherein the magnetic beads are carboxyl magnetic beads; the particle size of the carboxyl magnetic beads is 1-5 μm; preferably, the particle size of the carboxyl magnetic beads is 1-3 μm; more preferably, the particle size of the magnetic beads is 3 μm.

[0014] The magnetic particles as described above, wherein the nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies, and its sequence is shown in SEQ ID NO.1.

[0015] The magnetic particles as described above are blocked by a blocking agent; preferably, the blocking agent comprises: 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin.

[0016] A method for preparing magnetic microparticles that specifically bind to anti-nephrin antibodies in a sample comprises: obtaining a mixed solution comprising activated magnetic beads; adding nephrin antigen to the mixed solution and mixing uniformly; wherein the nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies; allowing the mixture to suspend and react for 1-2 hours in an environment at a temperature of 20-25°C; adding the reaction product to a blocking agent for washing; wherein the blocking agent comprises: 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin; and obtaining nephrin antigen-coated magnetic beads.

[0017] The method as described above further comprises: diluting the nephrin antigen-coated magnetic beads in a diluent to obtain a magnetic particle diluent; wherein the concentration of the magnetic particles in the magnetic particle diluent is 0.1-0.2 mg / mL.

[0018] According to the method described above, the diluent comprises: (0.80-1.30) g / L tris(hydroxymethyl)aminomethane; (5.00-8.00) g / L tris(hydroxymethyl)aminomethane hydrochloride; (7.00-11.00) g / L sodium chloride; (1.00-5.00) g / L Triton-X100; (0.20-0.70) g / L Tween-20; and (0.20-0.70) g / L ProClin300; preferably, the diluent comprises: 1.12 g / L tris(hydroxymethyl)aminomethane; 6.42 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; 3.00 g / L Triton-X 100; 0.50 g / L Tween-20; and 0.50 g / L ProClin 300.

[0019] The method as described above, wherein the blocking agent comprises fish gelatin.

[0020] In the above method, the ratio of adding the magnetic beads and the nephrin antigen is: adding 5-20 μg of nephrin antigen to the magnetic bead diluent containing about 1 mg of magnetic beads; preferably, adding 10-20 μg of nephrin antigen.

[0021] In the method as described above, the magnetic beads are carboxyl magnetic beads.

[0022] In the method described above, the particle size of the magnetic beads is 1-5 μm; preferably, the particle size of the magnetic beads is 1-3 μm; more preferably, the particle size of the magnetic beads is 3 μm.

[0023] A kit for measuring anti-Nephrin autoantibodies comprises: the magnetic particles as described above or the magnetic particles prepared by the methods described above.

[0024] The kit as described above further comprises: an IgG antibody labeled with a chemiluminescent marker; preferably, the concentration of the IgG antibody labeled with a chemiluminescent marker is 0.02-0.04 μg / mL.

[0025] In the kit as described above, the chemiluminescent marker is selected from acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide or acridinium trifluoromethanesulfonamide; preferably, the chemiluminescent marker is acridinium ester.

[0026] In the kit as described above, the IgG antibody labeled with the chemiluminescent marker is diluted in a labeling diluent, wherein: the labeling diluent includes: (0.30-0.70) g / L sodium dihydrogen phosphate; (15.00-18.00) g / L sodium dihydrogen phosphate; (35.00-45.00) g / L sodium chloride; (7.00-13.00) g / L bovine serum albumin; (3.00-7.00) g / L Triton-X405; and (0.30-0.70) g / L ProClin 300; preferably, the labeling diluent includes: 0.50 g / L sodium dihydrogen phosphate; 16.75 g / L sodium dihydrogen phosphate; 40.00 g / L sodium chloride; 10.00 g / L bovine serum albumin; 5.00 g / L Triton-X405; and 0.50 g / L ProClin300.

[0027] The kit as described above further comprises: a sample diluent; the sample diluent comprises: (0.50-1.50) g / L tris(hydroxymethyl)aminomethane; (5.00-8.00) g / L tris(hydroxymethyl)aminomethane hydrochloride); (7.00-11.00) g / L sodium chloride; and (17.00-23.00) g / L bovine serum albumin; preferably, the sample diluent comprises: 1.00 g / L tris(hydroxymethyl)aminomethane; 6.58 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; and 20.00 g / L bovine serum albumin;

[0028] Preferably, the sample diluent further comprises (0.30-0.70) g / L Tween 20 and (0.30-0.70) g / L ProClin300; preferably, the sample diluent further comprises 0.50 g / L Tween 20 and 0.50 g / L ProClin300.

[0029] The kit as described above further comprises: a calibrator, which comprises an anti-Nephrin autoantibody; and a calibrator buffer; preferably, the calibrator buffer comprises: (0.10-0.30) g / L tris(hydroxymethyl)aminomethane; (1.00-1.60) g / L tris(hydroxymethyl)aminomethane hydrochloride; (7.00-11.00) g / L sodium chloride; (3.00-7.00) g / L bovine serum albumin; (0.30-0.70) g / L Tween; and (0.30-0.70) g / L ProClin300; further preferably, the calibrator buffer comprises: 0.20 g / L tris(hydroxymethyl)aminomethane; 1.32 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; 5.00 g / L bovine serum albumin; 0.50 g / L Tween; and 0.50 g / L ProClin300.

[0030] The second object of the present invention is achieved by the following technical solutions:

[0031] A method for detecting anti-nephrin autoantibodies in a sample using nephrin antigen-coated magnetic beads prepared according to any of the above methods, the nephrin antigen-coated magnetic beads as described above, or the kit as described above, comprising: adding a sample to be tested to a sample diluent and mixing uniformly; adding nephrin antigen-coated magnetic beads or magnetic particle diluent, reacting for a predetermined time; adding acridinium ester-labeled mouse anti-human IgG antibodies or acridinium ester-labeled mouse anti-human IgG antibodies diluted in labeling diluent, reacting for a predetermined time; adding a pre-excitation solution and an excitation solution; and detecting relative luminescence intensity.

[0032] In the method described above, the pre-excitation solution includes (0.80-1.70)% (W / V) hydrogen peroxide; the excitation solution includes (0.2-0.5) mol / L sodium hydroxide; preferably, the pre-excitation solution includes 1.32% (W / V) hydrogen peroxide; and the excitation solution includes 0.35 mol / L sodium hydroxide.

[0033] The method as described above further comprises: obtaining the concentration and luminescence intensity values ​​of the calibration sample and determining a calibration curve, see attached Figure 21 , wherein the concentrations of the calibrators are CAL1: 0.42 AU / mL, CAL2: 39.3 AU / mL; the luminescence intensity value of the sample is matched with the standard curve of the luminescence intensity value of the calibrator to obtain the content of the anti-Nephrin antibody in the sample to be tested.

[0034] In the above method, the volume ratio of the test sample, magnetic particle diluent, acridinium ester-labeled mouse anti-human IgG antibody diluted in the labeling diluent, and sample diluent is (15-25): (40-60): (80-120): (80-120); preferably 20:50:100:100.

[0035] In the above method, the reaction time of adding the nephrin antigen-coated magnetic beads or magnetic microparticle dilution is 10-20 minutes.

[0036] In the method described above, the sample is serum.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The ratio of the nephrin antigen-coated magnetic microparticle working solution (R1 component) to the mouse anti-human IgG labeled acridinium ester working solution (R2 component) in the kit of the present invention enables the magnetic beads to bind more antigens at the same ratio, thereby achieving a better capture effect on the antibodies in the sample to be tested, thereby significantly improving the signal-to-noise ratio and sensitivity of the detection, and being able to quantitatively detect the concentration of anti-nephrin antibodies in the sample.

[0039] The detection method of the kit of the present invention selectively captures target molecules through magnetic beads, has a simple and rapid operation process, is easy to automate and promote clinical application, and can obtain uniform and highly accurate results within 30 minutes, greatly improving the processing efficiency of samples. At the same time, multiple dilution is performed, reducing the cost of use and expanding the reportable range.

[0040] The preparation method of the kit of the present invention has a simple process flow, and the prepared product has good sensitivity, specificity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Below, the preferred embodiments of the present invention will be further described in detail with reference to the accompanying drawings, in which:

[0042] Figures 1-10 1 is an indirect immunofluorescence image of positive samples 1 to 10 according to one embodiment of the present invention;

[0043] Figures 11-20 1 is an indirect immunofluorescence image of negative sample 11 to negative sample 20 according to one embodiment of the present invention;

[0044] Figure 21 is a calibration curve of an anti-Nephrin autoantibody kit according to one embodiment of the present invention; and

[0045] Figure 221 is a ROC curve of the anti-Nephrin autoantibody test results of the sample in this example. DETAILED DESCRIPTION

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] In this application, "anti-nephrin autoantibodies" are key proteins in the complex podocyte slit diaphragm structure, with extensive signaling functions; severe podocyte damage occurs when the gene is mutated or experimentally knocked out. Anti-nephrin autoantibodies are widely present in patients with nephrotic syndrome of all ages, inducing nephrotic syndrome, nephrin phosphorylation, and cellular rearrangements at the proteomic level, leading to the typical ultrastructural changes of minimal change disease.

[0048] In this application, "Nephrin antigen" refers to any polypeptide or fragment thereof that can specifically bind to anti-Nephrin autoantibodies in mammals, including humans. In some embodiments, the Nephrin antigen refers to the extracellular segment of a human recombinant Nephrin protein. Furthermore, in some embodiments, the sequence of the Nephrin antigen is as shown in SEQ ID NO.1 or SEQ ID NO.2:

[0049] SEQ ID NO.1:

[0050]

[0051] SEQ ID NO.2:

[0052]

[0053] In this application, "bead coating amount" refers to the ratio of activated magnetic beads to the extracellular segment of human recombinant nephrin protein (nephrin antigen) when preparing magnetic microparticles. For example, if 10 μg of nephrin antigen is added per milliliter of activated magnetic beads, the bead coating amount is considered to be 10 μg / ml.

[0054] In this application, "sample" refers to the serum of a subject.

[0055] The present application relates to magnetic particles that specifically bind to anti-Nephrin antibodies in a sample, wherein the magnetic particles include Nephrin antigens and magnetic beads, wherein the Nephrin antigens are coated on the surface of the magnetic beads; wherein the magnetic beads are carboxyl magnetic beads; the particle size of the carboxyl magnetic beads is 1-5 μm; preferably, the particle size of the carboxyl magnetic beads is 1-3 μm; more preferably, the particle size of the magnetic beads is 3 μm.

[0056] In some embodiments, the nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies, and its sequence is shown in SEQ ID NO.1.

[0057] In some embodiments, the magnetic particles are blocked by a blocking agent; preferably, the blocking agent comprises: 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin.

[0058] The present application relates to a method for preparing magnetic microparticles that specifically bind to anti-nephrin antibodies in a sample, comprising: obtaining a mixture comprising 1 mg of activated magnetic beads; adding 5-20 μg of nephrin antigen, preferably 10-20 μg of nephrin antigen, to the mixture and mixing uniformly; wherein the nephrin antigen is a polypeptide or fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies; suspending and reacting the mixture at a temperature of 20-25°C for 1-2 hours; adding the reaction product to a blocking agent for washing; wherein the blocking agent comprises: 2% (w / v) bovine serum albumin, 2% (w / v) casein, or 2% (w / v) fish gelatin; preferably, the blocking agent comprises fish gelatin; and obtaining nephrin antigen-coated magnetic beads.

[0059] In some embodiments, the method of coating magnetic beads with nephrin antigen further comprises: diluting the magnetic beads coated with nephrin antigen in a diluent to obtain a magnetic particle diluent; wherein the concentration of the magnetic particles in the magnetic particle diluent is 0.1-0.2 mg / mL.

[0060] In some embodiments, the diluent comprises: (0.80-1.30) g / L tris(hydroxymethyl)aminomethane; (5.00-8.00) g / L tris(hydroxymethyl)aminomethane hydrochloride; (7.00-11.00) g / L sodium chloride; (1.00-5.00) g / L Triton-X100; (0.20-0.70) g / L Tween-20; and (0.20-0.70) g / L ProClin300; preferably, the diluent comprises: 1.12 g / L tris(hydroxymethyl)aminomethane; 6.42 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; 3.00 g / L Triton-X 100; 0.50 g / L Tween-20; and 0.50 g / L ProClin 300.

[0061] In some embodiments, the magnetic beads are carboxyl magnetic beads. In some embodiments, the magnetic beads have a particle size of 1-5 μm; preferably, the magnetic beads have a particle size of 1-3 μm; more preferably, the magnetic beads have a particle size of 3 μm.

[0062] The present application relates to an anti-Nephrin autoantibody assay kit, comprising: magnetic particles as described above or magnetic particles prepared by any of the preparation methods described above.

[0063] In some embodiments, the kit further comprises: an IgG antibody labeled with a chemiluminescent marker; preferably, the concentration of the IgG antibody labeled with a chemiluminescent marker is 0.02-0.04 μg / mL.

[0064] In some embodiments, the chemiluminescent marker is selected from acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide or acridinium trifluoromethanesulfonamide; preferably, the chemiluminescent marker is an acridinium ester.

[0065] In some embodiments, the IgG antibody labeled with the chemiluminescent marker is diluted in a labeling diluent, wherein: the labeling diluent includes: (0.30-0.70) g / L sodium dihydrogen phosphate; (15.00-18.00) g / L sodium dihydrogen phosphate; (35.00-45.00) g / L sodium chloride; (7.00-13.00) g / L bovine serum albumin; (3.00-7.00) g / L Triton-X405; and (0.30-0.70) g / L ProClin 300; preferably, the labeling diluent includes: 0.50 g / L sodium dihydrogen phosphate; 16.75 g / L sodium dihydrogen phosphate; 40.00 g / L sodium chloride; 10.00 g / L bovine serum albumin; 5.00 g / L Triton-X405; and 0.50 g / L ProClin 300.

[0066] In some embodiments, the kit further comprises: a sample diluent; the sample diluent comprises: (0.50-1.50) g / L tris(hydroxymethylaminomethane); (5.00-8.00) g / L tris(hydroxymethylaminomethane) hydrochloride; (7.00-11.00) g / L sodium chloride; and (17.00-23.00) g / L bovine serum albumin; preferably, the sample diluent comprises: 1.00 g / L tris(hydroxymethylaminomethane); 6.58 g / L tris(hydroxymethylaminomethane) hydrochloride; 9.00 g / L sodium chloride; and 20.00 g / L bovine serum albumin;

[0067] Preferably, the sample diluent further comprises (0.30-0.70) g / L Tween 20 and (0.30-0.70) g / L ProClin300; preferably, the sample diluent further comprises 0.50 g / L Tween 20 and 0.50 g / L ProClin300.

[0068] In some embodiments, the kit further comprises: a calibrator comprising an anti-Nephrin autoantibody; and a calibrator buffer; preferably, the calibrator buffer comprises: (0.10-0.30) g / L tris(hydroxymethyl)aminomethane; (1.00-1.60) g / L tris(hydroxymethyl)aminomethane hydrochloride; (7.00-11.00) g / L sodium chloride; (3.00-7.00) g / L bovine serum albumin; (0.30-0.70) g / L Tween; and (0.30-0.70) g / L ProClin300; further preferably, the calibrator buffer comprises: 0.20 g / L tris(hydroxymethyl)aminomethane; 1.32 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; 5.00 g / L bovine serum albumin; 0.50 g / L Tween; and 0.50 g / L ProClin300.

[0069] In some embodiments, the calibrator comprises anti-Nephrin autoantibodies. In some embodiments, the calibrator comprises human anti-Nephrin autoantibodies. In some embodiments, the anti-Nephrin autoantibodies in the calibrator are obtained from a clinically positive patient for anti-Nephrin autoantibodies. In some embodiments, the anti-Nephrin autoantibodies in the subject sample are detected, and the anti-Nephrin autoantibody in the patient sample is obtained.

[0070] The present application relates to a method for detecting anti-nephrin autoantibodies in a sample using nephrin antigen-coated magnetic beads prepared according to any of the above methods, the nephrin antigen-coated magnetic beads as described above, or the kit as described above, comprising: adding a sample to be tested to a sample diluent and mixing uniformly; adding nephrin antigen-coated magnetic beads or magnetic microparticle diluent, and reacting for a certain time; adding acridinium ester-labeled mouse anti-human IgG antibodies or acridinium ester-labeled mouse anti-human IgG antibodies diluted in labeling diluent, and reacting for a certain time; adding a pre-excitation solution and an excitation solution; and detecting relative luminescence intensity.

[0071] In some embodiments, the pre-excitation solution includes (0.80-1.70)% (W / V) hydrogen peroxide; the excitation solution includes (0.2-0.5) mol / L sodium hydroxide; preferably, the pre-excitation solution includes 1.32% (W / V) hydrogen peroxide; the excitation solution includes 0.35 mol / L sodium hydroxide.

[0072] In some embodiments, the method for detecting anti-Nephrin autoantibodies in a sample further comprises: obtaining the concentration and luminescence intensity values ​​of a calibrator, and determining a calibration curve, see attached. Figure 22 , wherein the concentrations of the calibrators are CAL1: 0.42 AU / mL, CAL2: 39.3 AU / mL; the luminescence intensity value of the sample is matched with the standard curve of the luminescence intensity value of the calibrator to obtain the content of the anti-Nephrin antibody in the sample to be tested.

[0073] In some embodiments, the added volume ratio of the test sample, magnetic particle diluent, acridinium ester-labeled mouse anti-human IgG antibody diluted in label diluent, and sample diluent is (15-25):(40-60):(80-120):(80-120); preferably 20:50:100:100.

[0074] In some embodiments, the reaction time of adding the nephrin antigen-coated magnetic beads or magnetic microparticle dilution is 10-20 minutes.

[0075] Hereinafter, preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings. The detection standard reference of the embodiments of the present invention is YY / T 1789.4-202 2 Performance Evaluation Method for In Vitro Diagnostic Test Systems.

[0076] In the following examples, samples from subjects clinically confirmed to be positive for anti-Nephrin autoantibodies were selected as the experimental group, and samples from subjects clinically confirmed to be negative for anti-Nephrin autoantibodies were selected as the control group. Both the experimental and control groups were children aged 3-18 years old, and all had varying degrees of nephrotic syndrome.

[0077] Furthermore, the present application relates to a method for measuring anti-nephrin autoantibodies in an ex vivo sample from a subject. This method utilizes a complex of magnetic beads coated with a polypeptide or fragment thereof that specifically binds to anti-nephrin autoantibodies (hereinafter referred to as magnetic particles) and an IgG antibody labeled with a chemiluminescent marker (hereinafter referred to as the labeled antigen). When the magnetic particles react with the labeled antigen to form a magnetic particle-labeled antigen complex, the acridinium ester decomposes and emits light in an alkaline environment formed by the oxidizing agents hydrogen peroxide and sodium hydroxide, thereby determining the detection result of anti-nephrin autoantibodies.

[0078] Example 1: Preparation of an Anti-Nephrin Autoantibody Assay Kit

[0079] 1. Preparation of R1 Reagent-Magnetic Particles

[0080] In this example, the magnetic beads can be Toysl magnetic beads or carboxyl magnetic beads of different particle sizes. The adjustment of the magnetic bead particle size and the adjustment of the magnetic bead coating amount are described in Example 3 and will not be repeated in this example. In this example, the Toysl magnetic beads used were purchased from JSR Corporation with the product batch number MS300 / Tosyl; the carboxyl magnetic beads used were purchased from JSR Corporation with the product batch number MS300 / Carboxyl. The polypeptide or fragment thereof (antigen) that specifically binds to the anti-nephrin autoantibody involved in this example is the extracellular segment of human recombinant nephrin protein.

[0081] Furthermore, during the screening and optimization of magnetic beads in Example 3, only the corresponding parameters were replaced based on this example, while the other parameters remained unchanged.

[0082] In this embodiment, the method for preparing magnetic particles includes:

[0083] The magnetic beads were added to the chemical crosslinking agents EDC and Sulfo-NHS to activate the functional groups on their surface.

[0084] 1 mg of activated magnetic beads was coated with 5 μg, 10 μg, 15 μg, or 20 μg of the extracellular segment of human recombinant nephrin protein (nephrin antigen) to achieve a coating concentration of 5 μg / mg, 10 μg / mg, 15 μg / mg, or 20 μg / mg, respectively. The coating reaction conditions were as follows: suspension reaction at 20-25°C for 1-2 hours;

[0085] After blocking and washing, magnetic beads coated with Nephrin antigen are obtained; wherein, the components of the blocking agent can refer to Example 3;

[0086] Then, diluent (1 L containing 950.00 g of ultrapure water, 1.12 g of Tris(hydroxymethyl)aminomethane, 6.42 g of Tris(hydroxymethyl)aminomethane hydrochloride, 9.00 g of sodium chloride, 3.00 g of Triton-X 100, 0.50 g of Tween-200, and 0.50 g of ProClin 3000) was added to obtain a working solution of nephrin antigen-coated magnetic beads. According to one embodiment of the present application, the working solution concentration of the magnetic particles is 0.1 mg / mL.

[0087] 2. Preparation of R2 Reagent-Labeled Antibody

[0088] In this embodiment, a chemiluminescent marker is used to label an IgG antibody. Among them, the IgG antibody used in this embodiment is a mouse anti-human IgG antibody, purchased from GenScript, with a product batch number of A01855. In this application, the selection of chemiluminescent markers is not limited, such as acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide, acridinium trifluoromethanesulfonamide, etc. can be used to label IgG antibodies. Further, in this embodiment, the chemiluminescent marker for labeling IgG antibodies is acridinium ester, which is purchased from Seebio, with a product batch number of EKY0980A.

[0089] In this embodiment, the method of labeling mouse anti-human IgG antibody with acridinium ester includes:

[0090] Mouse anti-human IgG antibody and acridinium ester were incubated and combined, and the coupling ratio of mouse anti-human IgG antibody and acridinium ester was 8 μg acridinium ester per 100 μg antibody;

[0091] The coupling conditions were as follows: the acridinium ester and antibody were reacted in the above ratio at room temperature in the dark for 2 hours, followed by the addition of a stop solution containing 0.03 M lysine. The reaction was continued at room temperature in the dark for 1 hour. After completion of the reaction, the acridinium ester marker was purified using a desalting column.

[0092] After purification, an acridinium ester diluent (1 L containing 965.00 g of ultrapure water, 0.50 g of sodium dihydrogen phosphate, 16.75 g of sodium hydrogen phosphate, 40.00 g of sodium chloride, 10.00 g of bovine serum albumin, 5.00 g of Triton-X405, and 0.50 g of ProClin300) was added to obtain an acridinium ester-labeled mouse anti-human IgG working solution. In some embodiments, the concentration of the labeled antibody working solution is 0.02 μg / mL.

[0093] 3. Preparation of R3 Reagent-Sample Diluent

[0094] In this embodiment, the sample diluent includes: per liter of sample diluent, 976.00 g of ultrapure water, 1.00 g of Tris(hydroxymethyl)aminomethane, 6.58 g of Tris(hydroxymethyl)aminomethane hydrochloride, 9.00 g of sodium chloride, 20.00 g of bovine serum albumin, 0.50 g of Tween, and 0.50 g of ProClin300. The sample diluent formula concentration is selected according to conventional concentrations.

[0095] 4. Preparation of R4 Reagent-Calibrator Buffer

[0096] In this example, high-value serum samples were diluted fivefold with calibration buffer. The calibration buffer was prepared as follows: per liter, it contained 990.00 g of ultrapure water, 0.20 g of Tris(OH), 1.32 g of Tris(OH) hydrochloride, 9.00 g of sodium chloride, 5.00 g of bovine serum albumin, 0.50 g of Tween, and 0.50 g of ProClin300. Store at 2-8°C until needed.

[0097] Example 2 Method for Determining Anti-Nephrin Autoantibodies in Samples

[0098] 1. Chemiluminescence

[0099] The sample in this embodiment is the serum of the subject.

[0100] The method for detecting a subject's sample using the anti-Nephrin autoantibody assay kit of Example 1 comprises:

[0101] Dilute 20 μL of the sample to be tested into 100 μL of the sample diluent of R3 reagent;

[0102] The diluted sample to be tested was incubated with 50 μL of R1 reagent magnetic particle working solution at room temperature for 5-20 minutes, and the first washing number was 4 times;

[0103] Then, 100 μL of R2 reagent-labeled antibody working solution was added and incubated at room temperature for 10 min, and the second washing number was 4 times;

[0104] Finally, the pre-excitation solution and the excitation solution were added, and the maximum luminescence intensity was reached in 0.4 s; wherein the pre-excitation solution contained 1.32% (W / V) hydrogen peroxide and the excitation solution contained 0.35 mol / L sodium hydroxide;

[0105] The luminescence value (RLU) of the final reaction product was detected at a wavelength of 430 nm.

[0106] 3. Indirect Immunofluorescence

[0107] The indirect immunofluorescence method for detecting antibodies in samples is the gold standard for detection, and its detection results can be used as a control for the chemiluminescence detection results of the present application.

[0108] The specific experimental steps of indirect immunofluorescence include:

[0109] 1. Culture and Preparation of Normal Human Podocytes: Normal human podocytes were seeded on coverslips and cultured in appropriate culture medium until they reached a monolayer. The cells were washed three times with PBS for 5 minutes each time. Normal human podocytes were obtained from an immortalized human podocyte cell line.

[0110] 2. Fix cells: Fix cells with 4% paraformaldehyde at room temperature for 10-15 minutes. After fixation, wash cells three times with PBS for 5 minutes each time.

[0111] 3. Blocking: Block nonspecific binding sites with PBS containing 5-10% normal goat serum or BSA and incubate at room temperature for 30 minutes;

[0112] 4. Incubate with primary antibody: Place coverslip in sample serum and incubate overnight at 4°C.

[0113] 5. Washing: After incubation, wash with PBS three times, 5 minutes each time, to remove unbound antibodies;

[0114] 6. Incubate with secondary antibody: Use fluorescently labeled anti-human IgG secondary antibody and incubate in the dark at room temperature for 1 hour;

[0115] 7. Washing: After incubation, wash with PBS three times, 5 minutes each time, to remove unbound antibodies;

[0116] 8. Sealing and observation: Mount the coverslip with anti-fluorescence quenching mounting medium, and observe and capture cell fluorescence images under a laser confocal microscope.

[0117] Example 3: Screening and optimization of magnetic beads

[0118] In this example, various parameters for preparing magnetic microparticles were optimized to obtain optimal nephrin antigen-coated magnetic beads.

[0119] 1. Effects of different types of magnetic beads on sample reactivity

[0120] In this example, 10 samples with clinically positive and clinically negative anti-Nephrin autoantibodies were selected, and the Nephrin antigen was bound to Toysl magnetic beads and carboxyl magnetic beads, respectively. The other reagents in the kit of Example 1 of the present application and the chemiluminescence method of Example 2 were used to detect the anti-Nephrin autoantibodies in the samples, and the luminescence value RLU was obtained.

[0121] In this embodiment, the magnetic beads coating amount is 5 μg / mg when preparing magnetic microparticles, the particle size of the carboxyl magnetic beads is 1.5 μm, and the blocking agent is bovine serum albumin;

[0122] Furthermore, when detecting anti-Nephrin autoantibodies in samples, the working concentration of magnetic particles was 0.2 mg / ml, the concentration of labeled antibody working solution was 0.04 μg / mL, the sample addition volume was 5 μL, and the first step reaction time in the reaction conditions was about 10 minutes.

[0123] Table 1 shows the effects of different types of magnetic beads on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0124] Table 1 Optimization of different types of magnetic beads

[0125]

[0126] Wherein, S represents the mean value of the luminescence value detection result of experimental group sample, and N represents the mean value of the luminescence value detection result of control sample, and S / N refers to signal to noise ratio, wherein, the value of S / N is larger, when representing using the test kit to detect sample, the discrimination degree of positive sample detection result and negative sample detection result is larger, the more difficult false (such as false positive or false negative) result is, the sensitivity and accuracy of the test kit are better. In the examples below, the same letters have similar meanings to the present embodiment, and will not be repeated hereafter. As can be seen from Table 1, the S / N value of carboxyl magnetic beads reaches 1.34, much larger than Toysl magnetic beads, and its signal to noise ratio is better, which can be used as preferred magnetic bead types.

[0127] 2. Effect of different antigen coating amounts on sample reactivity

[0128] In this example, 4 samples were selected for each of clinically positive and clinically negative anti-Nephrin autoantibodies, and magnetic microparticles with different carboxyl magnetic bead coating amounts of Nephrin antigens were obtained. The other reagents in the kit of Example 1 of the present application and the chemiluminescence method of Example 2 were used to detect anti-Nephrin autoantibodies in the samples, and the luminescence value RLU was obtained.

[0129] In this embodiment, the particle size of the carboxyl magnetic beads is 1.5 μm, and the blocking agent is bovine serum albumin;

[0130] Furthermore, when detecting anti-Nephrin autoantibodies in samples, the working concentration of magnetic particles was 0.2 mg / ml, the concentration of labeled antibody working solution was 0.04 μg / mL, the sample addition volume was 5 μL, and the first step reaction time in the reaction conditions was about 10 minutes.

[0131] Table 2 shows the effects of different magnetic bead coating amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0132] Table 2 Optimization of antigen coating amount of different magnetic beads

[0133]

[0134]

[0135] As can be seen from Table 2, the signal-to-noise ratio is best when the magnetic bead coating amount is 10 μg / mg. Therefore, it is determined that the preferred magnetic bead coating amount in this embodiment is 10 μg / mg.

[0136] 3. Effect of different magnetic bead sizes on sample reactivity

[0137] In this example, three samples were selected for each of clinically positive and clinically negative anti-Nephrin autoantibodies. Magnetic microparticles of carboxyl magnetic beads of different sizes coated with Nephrin antigens were obtained. The other reagents in the kit of Example 1 of the present application and the chemiluminescence method of Example 2 were used to detect anti-Nephrin autoantibodies in the samples, and the luminescence value RLU was obtained.

[0138] In this embodiment, the magnetic bead coating amount is 10 μg / mg when preparing magnetic microparticles, and the blocking agent is bovine serum albumin;

[0139] Furthermore, when detecting anti-Nephrin autoantibodies in samples, the working concentration of magnetic particles was 0.2 mg / ml, the concentration of labeled antibody working solution was 0.04 μg / mL, the sample addition volume was 5 μL, and the first step reaction time in the reaction conditions was about 10 minutes.

[0140] Table 3 shows the effects of magnetic particles of different particle sizes on the luminescence value of anti-Nephrin autoantibodies in a sample according to one embodiment of the present application.

[0141] Table 3 Optimization of different magnetic bead particle sizes

[0142]

[0143]

[0144] As can be seen from Table 3, the signal-to-noise ratio is best when the magnetic bead particle size is 3.0 μm. Therefore, it is determined that the preferred magnetic bead particle size in this embodiment is 3.0 μm.

[0145] 4. Effects of different blocking processes on sample reactivity

[0146] In this example, two samples were selected, one clinically positive and one clinically negative for anti-Nephrin autoantibodies, and carboxyl magnetic particles were obtained under different blocking processes. The other reagents in the kit of Example 1 of the present application and the chemiluminescence method of Example 2 were used to detect anti-Nephrin autoantibodies in the samples to obtain the luminescence value RLU.

[0147] In this example, the effects of magnetic particles prepared under three blocking agents on the detection results of anti-Nephrin antibodies in samples were tested.

[0148] Blocking agent 1 included: 1% (w / v) bovine serum albumin;

[0149] Blocking agent 2 included: 0.5% (w / v) casein;

[0150] Blocking agent 3 included: 0.5% (w / v) fish gelatin.

[0151] Furthermore, in this embodiment, when preparing magnetic microparticles, the magnetic bead coating amount is 10 μg / mg, the magnetic bead particle size is 3 μm, and the blocking agent is bovine serum albumin;

[0152] Furthermore, when detecting anti-Nephrin autoantibodies in samples, the working concentration of magnetic particles was 0.2 mg / ml, the concentration of labeled antibody working solution was 0.04 μg / mL, the sample addition volume was 5 μL, and the first step reaction time in the reaction conditions was about 10 minutes.

[0153] Table 4 shows the effects of magnetic particles under different blocking processes on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0154] Table 4 Optimization of different sealing processes

[0155]

[0156] As can be seen from Table 4, the signal-to-noise ratio of blocking agent 3 is the best, so it is determined that the preferred magnetic bead blocking agent in this embodiment is fish gelatin.

[0157] Example 4: Optimization of reagent working concentrations

[0158] In this example, the magnetic particles used were the optimal magnetic particles obtained under the optimized conditions in Example 3. The remaining reagents were the same as those obtained in Example 1.

[0159] When using the kit as in Example 1 to detect anti-Nephrin autoantibodies in a sample, the mass ratio of magnetic particles to labeled antibodies is set to 250:1, the working solution concentrations of the magnetic particles of the R1 reagent are set to 0.1 and 0.2 mg / mL, and the working solution concentrations of the labeled antibodies of the R2 reagent are set to 0.02 and 0.04 μg / mL. The optimal dilution concentration is selected based on the signal-to-noise ratio of the luminescence value.

[0160] Furthermore, when detecting anti-Nephrin autoantibodies in a sample, the sample addition volume is 5 μL, and the first step reaction time in the reaction conditions is about 10 minutes.

[0161] Table 5 shows the effects of different amounts of magnetic particles and labeled antibodies added on the luminescence value of anti-Nephrin autoantibodies in a sample according to one embodiment of the present application.

[0162] Table 5 Optimization of different working concentrations of reagents in the kit

[0163]

[0164] As shown in Table 5, the kit of the present invention has the highest signal-to-noise ratio when the working solution concentration of the R1 reagent magnetic particles is preferably 0.1 mg / mL and the working solution concentration of the R2 reagent labeled antibody is preferably 0.02 μg / mL.

[0165] Example 5: Effect of sample loading volume on sample reactivity

[0166] In this example, the magnetic particles used were the optimal magnetic particles obtained under the optimized conditions in Example 3. The remaining reagents were the same as those obtained in Example 1.

[0167] When the kit in Example 1 is used to detect anti-Nephrin autoantibodies in a sample, different sample addition amounts are set, and the optimal dilution concentration is selected based on a comprehensive consideration of the signal-to-noise ratio of the luminescence value.

[0168] Furthermore, when detecting anti-Nephrin autoantibodies in a sample, the working concentration of the magnetic particles is 0.1 mg / ml, the concentration of the labeled antibody working solution is 0.02 μg / mL, and the first step reaction time in the reaction conditions is about 10 minutes.

[0169] Table 6 shows the effects of different sample addition amounts on the luminescence value of anti-Nephrin autoantibody detection in the sample according to one embodiment of the present application.

[0170] Table 6 Optimization of sample addition amount

[0171]

[0172] As can be seen from Table 6, the optimal sample addition volume after dilution is 20 μl. In this case, the signal-to-noise ratio of the kit is the highest.

[0173] Example 6: Optimization of reaction conditions

[0174] In this example, the magnetic particles used were the optimal magnetic particles obtained under the optimized conditions in Example 3. The remaining reagents were the same as those obtained in Example 1.

[0175] When the kit in Example 1 is used to detect anti-Nephrin autoantibodies in a sample, different first step reaction times are set, while the second step reaction time is kept constant at 10 min. The optimal reaction time is selected based on the signal-to-noise ratio of the luminescence value.

[0176] Furthermore, when detecting anti-Nephrin autoantibodies in a sample, the working concentration of the magnetic particles was 0.1 mg / ml, the concentration of the labeled antibody working solution was 0.02 μg / mL, and the sample addition volume was 20 μl.

[0177] Table 7 shows the effects of different reaction times on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0178] Table 7 Optimization of different reaction times

[0179]

[0180] As can be seen from Table 7, when the second-step reaction time remains unchanged at 10 min, there is no significant difference in the signal-to-noise ratio between the first-step reaction time of 10 min and 20 min. Taking all factors into consideration, choosing a first-step reaction time of 10 min can shorten the reaction time and improve the working efficiency of the kit.

[0181] Example 7: Anti-Nephrin Autoantibody Kit Performance Test

[0182] The anti-Nephrin antibody assay kit was used to test the sera of 100 healthy children undergoing physical examinations, where the maximum signal value was less than 15 AU / mL. In addition, the sera of 100 children with kidney disease were tested, where the minimum signal value was greater than 20 AU / mL. Therefore, the cut-off value of this detection kit was selected as 20 AU / mL. If the sample test result is greater than or equal to 20 AU / mL, it is judged to be reactive, i.e., positive; if the sample test result is less than 20 AU / mL, it is judged to be non-reactive, i.e., negative.

[0183] In this example, 20 samples were tested using the indirect immunofluorescence method (gold standard) in Example 2, of which 10 samples were positive (e.g. Figure 1-10 As shown), 10 samples were negative (as shown Figure 11-20The anti-Nephrin antibody content was detected using the kit prepared in Example 1 and the chemiluminescence method of Example 2. The results are shown in Table 8.

[0184] Table 8 Anti-Nephrin autoantibody detection results

[0185]

[0186] As shown in Table 8, the positive accuracy rate and negative accuracy rate of the kit of the present application detected by chemiluminescence method were both 80%.

[0187] Further, Figure 21 This is a calibration curve for an anti-Nephrin autoantibody kit according to one embodiment of the present invention. A serum sample with a high luminescence intensity of anti-Nephrin antibodies was selected as a calibrator and diluted in multiples (luminescence values ​​RLU were 2000, 4000, 6000, 8000, and 10000, respectively) to obtain the signal value corresponding to the calibrator. A scatter plot of standard RLU (x-axis) and signal (y-axis) was drawn. Based on the scatter plot, a linear trend line (least squares method) was added, and the linear equation and determination coefficient (R) were drawn. 2 ), R 2 =1, indicating a good linear relationship. The concentrations of the calibrators are CAL1: 0.42 AU / mL and CAL2: 39.3 AU / mL. The luminescence intensity values ​​of the samples are matched to the standard curve of the luminescence intensity values ​​of the calibrators to obtain the content (AU value) of the anti-nephrin antibody in the sample to be tested.

[0188] Figure 22 : is the ROC curve of the anti-Nephrin autoantibody test results of the sample in this embodiment. According to the ROC curve, its AUC is 0.997.

[0189] Example 8: Sensitivity and specificity of anti-Nephrin autoantibody kit detection

[0190] In this example, the inventors tested over 1,000 clinically confirmed positive and negative samples and obtained their test results. A cut-off value of 20 AU / mL or greater was set as positive, and less than 20 AU / mL was set as negative. In this example, the detection accuracy of over 1,000 clinically confirmed positive and negative samples reached over 99%. The test results for 50 positive and 50 negative samples are further listed in Table 9.

[0191] Table 9 Anti-Nephrin autoantibody detection results

[0192]

[0193]

[0194] In this example, the detection rates of both negative and positive samples were close to 100%. The applicant further calculated the number of positive samples that remained positive after testing with the kit of this application as TP, and the number of negative samples after testing as FN; the number of negative samples that remained positive after testing with the kit of this application as FP, and the number of negative samples after testing as TN.

[0195] The test kit detection sensitivity formula is based on: Sensitivity = TP / (TP+FN)*100%.

[0196] The test kit detection specificity formula is based on: specificity = TN / (TN+FP)*100%.

[0197] According to statistics and calculations, the sensitivity and specificity of the kit are close to 100%.

[0198] The above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. A magnetic particle that specifically binds to an anti-Nephrin antibody in a sample, characterized in that: The magnetic particles include Nephrin antigen and magnetic beads, wherein the Nephrin antigen is coated on the surface of the magnetic beads; wherein the magnetic beads are carboxyl magnetic beads; and the particle size of the carboxyl magnetic beads is 1-5 μm; The nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies, and the sequence of the nephrin antigen is shown in SEQ ID NO.

1.

2. The magnetic particle according to claim 1, wherein The particle size of the carboxyl magnetic beads is 1-3 μm.

3. The magnetic particle according to claim 1, wherein The particle size of the magnetic beads is 3 μm.

4. A method for preparing magnetic particles according to any one of claims 1 to 3, characterized in that: include: obtaining a mixed solution including activated magnetic beads; Add Nephrin antigen to the mixture and mix well; Wherein the nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-nephrin autoantibodies; Suspend and react in an environment with a temperature of 20-25°C for 1-2 hours; The reaction product is added to a blocking agent for washing; wherein the blocking agent includes: 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin; and magnetic beads coated with nephrin antigen are obtained.

5. The preparation method according to claim 4, wherein Further including: The magnetic beads coated with the nephrin antigen are diluted in a diluent to obtain a magnetic particle diluent; wherein the concentration of the magnetic particles in the magnetic particle diluent is 0.1-0.2 mg / mL.

6. The preparation method according to claim 5, characterized in that The diluent comprises: 0.80-1.30 g / L Tris; 5.00-8.00 g / L Tris hydrochloride; 7.00-11.00 g / L sodium chloride; 1.00-5.00 g / L Triton-X 100; 0.20-0.70 g / L Tween-20; and 0.20-0.70 g / L ProClin 300.

7. The preparation method according to claim 6, characterized in that The diluent comprises: 1.12 g / L Tris; 6.42 g / L Tris hydrochloride; 9.00 g / L sodium chloride; 3.00 g / L Triton-X 100; 0.50 g / L Tween-20; and 0.50 g / L ProClin 300.

8. The preparation method according to claim 4, wherein The addition ratio of the magnetic beads and the nephrin antigen is: 5-20 μg of nephrin antigen is added to the magnetic bead diluent containing about 1 mg of magnetic beads.

9. The preparation method according to claim 4, wherein The addition ratio of the magnetic beads and the nephrin antigen is: 10-20 μg of nephrin antigen is added to the magnetic bead diluent containing about 1 mg of magnetic beads.

10. A kit for measuring anti-Nephrin autoantibodies, characterized in that: include: The magnetic particles according to any one of claims 1 to 3 or the magnetic particles prepared by the preparation method according to any one of claims 4 to 9.

11. The kit according to claim 10, characterized in that Further including: Chemiluminescent labeled IgG antibody and its dilution.

12. The kit according to claim 11, characterized in that The concentration of the IgG antibody labeled with the chemiluminescent marker in its diluent is 0.02-0.04 μg / mL.

13. The kit according to claim 11, characterized in that The chemiluminescent label is selected from acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide or acridinium trifluoromethanesulfonamide.

14. The kit according to claim 11, characterized in that The chemiluminescent marker is acridinium ester.

15. The kit according to claim 11, characterized in that The IgG antibody labeled with the chemiluminescent marker is diluted in a labeling diluent, wherein: The labeled diluent includes: 0.30-0.70 g / L sodium dihydrogen phosphate; 15.00-18.00 g / L sodium dihydrogen phosphate; 35.00-45.00 g / L sodium chloride; 7.00-13.00 g / L bovine serum albumin; 3.00-7.00 g / L Triton-X 405; and 0.30-0.70 g / L ProClin 300.

16. The kit according to claim 15, wherein The labeled diluent includes: 0.50 g / L sodium dihydrogen phosphate; 16.75 g / L sodium dihydrogen phosphate; 40.00 g / L sodium chloride; 10.00 g / L bovine serum albumin; 5.00 g / L Triton-X 405; and 0.50 g / L ProClin 300.

17. The kit according to claim 10, characterized in that Further including: Sample diluent; The sample diluent includes: 0.50-1.50 g / L tris(hydroxymethyl)aminomethane; 5.00-8.00 g / L tris(hydroxymethyl)aminomethane hydrochloride; 7.00-11.00 g / L sodium chloride; and 17.00-23.00 g / L bovine serum albumin.

18. The kit according to claim 17, wherein The sample diluent includes: 1.00 g / L tris(hydroxymethyl)aminomethane; 6.58 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; and 20.00 g / L bovine serum albumin.

19. The kit according to claim 17 or 18, wherein The sample diluent further includes 0.30-0.70 g / L Tween 20 and 0.30-0.70 g / L ProClin300.

20. The kit according to claim 19, characterized in that The sample diluent further includes 0.50 g / L Tween 20 and 0.50 g / L ProClin 300.

21. The kit according to claim 10, characterized in that Further including: A calibrator comprising an anti-Nephrin autoantibody; and a calibrator buffer.

22. The kit according to claim 21, wherein The calibrator buffer includes: 0.10-0.30 g / L tris(hydroxymethyl)aminomethane; 1.00-1.60 g / L tris(hydroxymethyl)aminomethane hydrochloride; 7.00-11.00 g / L sodium chloride; 3.00-7.00 g / L bovine serum albumin; 0.30-0.70 g / L Tween; and 0.30-0.70 g / L ProClin300.

23. The kit according to claim 22, characterized in that The calibration buffer includes: 0.20 g / L tris(hydroxymethyl)aminomethane; 1.32 g / L tris(hydroxymethyl)aminomethane hydrochloride; 9.00 g / L sodium chloride; 5.00 g / L bovine serum albumin; 0.50 g / L Tween; and 0.50 g / L ProClin300.

24. Use of the magnetic particles according to any one of claims 1 to 3, or the magnetic particles prepared by the preparation method according to any one of claims 4 to 9, or the kit according to any one of claims 10 to 23 in preparing a reagent for detecting anti-Nephrin autoantibodies in a sample, characterized in that: The method of the kit for detecting anti-Nephrin autoantibodies in a sample comprises: Add 20-50 μL of the sample to be tested to the sample diluent and mix well; Add nephrin antigen-coated magnetic beads or magnetic microparticle dilution and react for 10-20 minutes; Add acridinium ester-labeled mouse anti-human IgG antibody or acridinium ester-labeled mouse anti-human IgG antibody diluted in labeling diluent and react for about 10 minutes; Adding pre-excitation solution and excitation solution; and detecting relative luminescence intensity; wherein The added volume ratio of the sample to be tested, magnetic particle diluent, acridinium ester-labeled mouse anti-human IgG antibody diluted in the labeling diluent, and sample diluent is (15-25): (40-60): (80-120): (80-120). 25 . The use according to claim 24 , wherein the volume ratio of the test sample, magnetic particle diluent, acridinium ester-labeled mouse anti-human IgG antibody diluted in labeling diluent, and sample diluent is 20:50:100:

100.

26. The use according to claim 24, characterized in that The pre-stimulation solution includes 0.80-1.70% (W / V) hydrogen peroxide; the stimulation solution includes 0.2-0.5 mol / L sodium hydroxide.

27. The use according to claim 26, characterized in that The pre-stimulation solution includes 1.32% (W / V) hydrogen peroxide; the stimulation solution includes 0.35 mol / L sodium hydroxide.

28. The use according to claim 24, characterized in that Further including: Determine the calibration curve based on the concentration and luminescence intensity values ​​of the calibrator; Matching the sample luminescence intensity value with the luminescence intensity value standard curve of the calibrator; as well as Obtain the content of anti-Nephrin antibodies in the test sample.

29. The use according to claim 24, characterized in that The sample is serum.

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