Anti-Nephrin autoantibody determination kit and detection method and preparation method thereof

Abstract

The invention relates to a magnetic particle capable of being specifically combined with an anti-Nephrin antibody in a sample. The magnetic particle comprises a magnetic bead which can be specifically combined with an anti-Nephrin autoantibody of a mammal and is coated with a polypeptide or a fragment of the polypeptide; wherein the magnetic beads are carboxyl magnetic beads; the particle size of the carboxyl magnetic beads is 1-5 [mu] m; preferably, the particle size of the carboxyl magnetic beads is 1-3 [mu] m; more preferably, the particle size of the magnetic beads is 3 microns. The anti-Nephrin antibody detection method disclosed by the invention is short in detection time, high in sensitivity, strong in specificity, stable and reliable in result, high in uniformity and easy to clinically popularize.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro detection, and 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 the podocytes of the glomerulus and 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 existence of anti-Nephrin autoantibodies. 1 . However, the concentration of anti-Nephrin autoantibodies in serum is relatively low compared to other podocyte autoantibodies we have discovered. 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 was 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 effective biomarkers for monitoring disease changes and treatment responses. 4,5 In summary, the accumulating evidence shows that anti-Nephrin autoantibodies have important clinical value. It is urgent to establish and develop anti-Nephrin antibody detection methods and detection kits with short detection time, high sensitivity, strong specificity, stable and reliable results, high uniformity, and easy clinical application.

[0003] Existing methods for detecting anti-Nephrin antibodies include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), etc. Among them, ELISA has poor sensitivity, a long experimental process, and large batch-to-batch differences in reagents. Although RIA has high sensitivity and specificity, the use of radioactive tracers can lead to radioactive contamination and hazards, and the half-life of commonly used radionuclides is short, the validity period of the kit is limited, and automated analysis cannot be performed, making it difficult to promote clinical applications. There is an urgent need to find a conventional, reliable, efficient, and clinically accessible quantitative detection method and kit for 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 of 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 the existing kit preparation process.

[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 a magnetic bead, wherein the Nephrin antigen is coated on the surface of the magnetic bead; wherein the magnetic bead is a carboxyl magnetic bead; the particle size of the carboxyl magnetic bead is 1-5 μm; preferably, the particle size of the carboxyl magnetic bead is 1-3 μm; more preferably, the particle size of the magnetic bead 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 including activated magnetic beads; adding Nephrin antigen to the mixed solution and mixing evenly; wherein the Nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-Nephrin autoantibodies; suspending and reacting in an environment 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: 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin; and obtaining magnetic beads coated with Nephrin antigen.

[0017] The method as described above further comprises: diluting the magnetic beads coated with the 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.

[0018] In the method as 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 sealing agent comprises fish gelatin.

[0020] In the method described above, 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: any of the magnetic particles described above or magnetic particles prepared by any of the preparation 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 toluene sulfonamide, acridinium p-methyl sulfonamide or acridinium trifluoromethane sulfonamide; 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 includes (0.30-0.70) g / L Tween 20; and (0.30-0.70) g / L ProClin300; preferably, the sample diluent further includes 0.50 g / L Tween 20; and 0.50 g / L ProClin300.

[0029] The kit as described above further comprises: a calibrator, the 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.

[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, Nephrin antigen-coated magnetic beads as described above, or any of the above kits, comprising: adding a sample to be tested into a sample diluent and mixing evenly; adding Nephrin antigen-coated magnetic beads or magnetic particle diluent, 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 a labeling diluent, reacting for a certain 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 exciting solution includes (0.2-0.5) mol / L sodium hydroxide; preferably, the pre-excitation solution includes 1.32% (W / V) hydrogen peroxide; the exciting 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 substance, and determining a calibration curve, see attached Fig.21 , wherein the concentrations of the calibrators are CAL1: 0.42AU / mL, CAL2: 39.3AU / mL; the luminescence intensity value of the sample is matched with the luminescence intensity value standard curve of the calibrator to obtain the content of the anti-Nephrin antibody in the sample to be tested.

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

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

[0036] In the method as 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 of the R1 component to the mouse anti-human IgG labeled acridinium ester working solution of the R2 component in the kit of the present invention enables the magnetic beads to bind more antigens at the same ratio, so that the antibody capture effect in the sample to be tested is better, thereby significantly improving the signal-to-noise ratio and sensitivity of the detection, and being able to quantitatively detect the concentration of the anti-nephrin antibody in the sample.

[0039] The detection method of the kit of the present invention selectively captures the target molecules through magnetic beads, and the operation process is simple and rapid, and it is easy to automate and promote clinical application. Results with high uniformity and accuracy can be obtained within 30 minutes, which greatly improves the processing efficiency of samples. At the same time, multiple dilution is performed, which reduces the cost of use and expands 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] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

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

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

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

[0045] Fig. 22It is a ROC curve of the test results of the anti-Nephrin autoantibody kit for the samples in this example. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 a wide range of 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 cell rearrangement at the proteomic level, leading to ultrastructural changes typical of minimal change nephropathy.

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

[0049] SEQ ID NO.1:

[0050]

[0051] SEQ ID NO.2:

[0052]

[0053] In this application, "magnetic 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 to each milliliter of activated magnetic beads, the magnetic bead coating amount can be considered to be 10 μg / ml.

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

[0055] The present application relates to magnetic microparticles that specifically bind to anti-Nephrin antibodies in a sample, wherein the magnetic microparticles 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 mixed solution comprising 1 mg of activated magnetic beads; adding 5-20 μg of Nephrin antigen, preferably 10-20 μg of Nephrin antigen, to the mixed solution and mixing them evenly; wherein the Nephrin antigen is a polypeptide or a fragment thereof that can specifically bind to mammalian anti-Nephrin autoantibodies; suspending and reacting in an environment 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 magnetic beads coated with Nephrin antigen.

[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 toluene sulfonamide, 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(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;

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

[0068] In some embodiments, the kit further includes: a calibrator, the calibrator including anti-Nephrin autoantibodies; and a calibrator buffer; preferably, 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; further preferably, the calibrator 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.

[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 autoantibodies in the clinically positive patient sample for anti-Nephrin autoantibodies are 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, Nephrin antigen-coated magnetic beads as described above, or a kit as described above, comprising: adding a sample to be tested into a sample diluent and mixing evenly; adding Nephrin antigen-coated magnetic beads or magnetic particle diluent, 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 a labeling diluent, 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 exciting solution includes (0.2-0.5) mol / L sodium hydroxide; preferably, the pre-excitation solution includes 1.32% (W / V) hydrogen peroxide; the exciting 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 the calibrator, and determining a calibration curve, see Attachment Fig. 22 , wherein the concentrations of the calibrators are CAL1: 0.42AU / mL, CAL2: 39.3AU / mL; the luminescence intensity value of the sample is matched with the luminescence intensity value standard curve 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 sample to be tested, the magnetic particle diluent, the acridinium ester-labeled mouse anti-human IgG antibody diluted in the label diluent, and the 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] Below, the preferred embodiments of the present invention will be further described in 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 System.

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

[0077] Furthermore, the present application relates to a method for determining anti-Nephrin autoantibodies in a sample isolated from a subject, the method using a complex of magnetic beads coated with a polypeptide or a fragment thereof that can specifically bind to anti-Nephrin autoantibodies (hereinafter referred to as magnetic particles) and IgG antibodies labeled with a chemiluminescent marker (hereinafter referred to as 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 agent hydrogen peroxide and sodium hydroxide, thereby obtaining the detection result of the anti-Nephrin autoantibodies.

[0078] Example 1: Preparation method of anti-Nephrin autoantibody assay kit

[0079] 1. Preparation method of R1 reagent-magnetic particles

[0080] In this embodiment, the magnetic beads can be Toysl magnetic beads or carboxyl magnetic beads of different particle sizes. The adjustment of the particle size of the magnetic beads and the adjustment of the coating amount of the magnetic beads refer to Example 3 and will not be repeated in this embodiment. In this embodiment, the Toysl magnetic beads used were purchased from JSR Company, and the product batch number is MS300 / Tosyl; the carboxyl magnetic beads used were purchased from JSR Company, and the product batch number is MS300 / Carboxyl. The polypeptide or fragment thereof (antigen) involved in this embodiment that can specifically bind to anti-Nephrin autoantibodies 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 on the basis of this example, while the other parameters remained unchanged.

[0082] In this embodiment, the preparation method of 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 and 5 μg, 10 μg, 15 μg or 20 μg of human recombinant Nephrin protein extracellular segment (Nephrin antigen) were coated and bound, so that the coating amount of magnetic beads was 5 μg / mg, 10 μg / mg, 15 μg / mg or 20 μg / mg respectively. The reaction conditions for coating and binding were: 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 add diluent (1L dosage contains 950.00g ultrapure water, 1.12g tris(hydroxymethyl)aminomethane, 6.42g tris(hydroxymethyl)aminomethane hydrochloride, 9.00g sodium chloride, 3.00g Triton-X 100, 0.50g Tween-200, and 0.50g ProClin 300) to dilute and obtain Nephrin antigen coated magnetic bead working solution. According to one embodiment of the present application, the working solution concentration of magnetic particles is 0.1mg / mL.

[0087] 2. Preparation of R2 reagent-labeled antibody

[0088] In this embodiment, a chemiluminescent marker is used to label IgG antibodies. Among them, the IgG antibody used in this embodiment is a mouse anti-human IgG antibody, purchased from GenScript, and the product batch number is A01855. In this application, the selection of chemiluminescent markers is not limited, such as acridinium esters, acridinium sulfonamides, acridinium toluene sulfonamides, acridinium p-methyl sulfonamides, acridinium trifluoromethanesulfonamides, etc. can all be used to label IgG antibodies. Further, in this embodiment, the chemiluminescent marker for labeling IgG antibodies is acridinium esters, which are purchased from Seebio, and the product batch number is EKY0980A.

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

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

[0091] The coupling conditions are as follows: the acridinium ester and antibody are reacted at room temperature in the dark for 2 hours according to the above ratio, and then a reaction stop solution is added, which includes 0.03Mol lysine. The reaction is carried out at room temperature in the dark for 1 hour. After the reaction is completed, the acridinium ester marker is purified using a desalting column;

[0092] After purification, add acridinium ester diluent (1L dosage contains 965.00g ultrapure water, 0.50g sodium dihydrogen phosphate, 16.75g disodium hydrogen phosphate, 40.00g sodium chloride, 10.00g bovine serum albumin, 5.00g Triton-X405, 0.50g ProClin300) to dilute to obtain 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: 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 per 1 L of the sample diluent. The concentration of the sample diluent formula is selected according to the conventional concentration.

[0095] 4. Preparation of R4 reagent-calibrator buffer

[0096] In this embodiment, the high-value serum sample is diluted 5 times with the calibration buffer. The preparation method of the calibration buffer comprises: 990.00 g of ultrapure water, 0.20 g of tris(hydroxymethyl)aminomethane, 1.32 g of tris(hydroxymethyl)aminomethane 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 are contained in each 1 L amount. The sample is stored at 2-8°C for future use.

[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 test 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 and 50 μL of R1 reagent magnetic particle working solution were incubated 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 for incubation 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 at 0.4 s; wherein the pre-excitation solution included 1.32% (W / V) hydrogen peroxide, and the excitation solution included 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 method include:

[0109] 1. Culture and preparation of normal human podocytes: Normal human podocytes were seeded on coverslips, cultured in a suitable culture medium to a monolayer, and washed three times with PBS, each time for 5 minutes; wherein, 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 3 times with PBS, 5 minutes each time.

[0111] 3. Blocking: Use PBS containing 5-10% normal goat serum or BSA to block non-specific binding sites and incubate at room temperature for 30 minutes;

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

[0113] 5. Washing: After incubation, wash with PBS 3 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 dark at room temperature for 1 hour;

[0115] 7. Washing: After incubation, wash with PBS 3 times, 5 minutes each time to remove unbound antibodies;

[0116] 8. Sealing and observation: Use anti-fluorescence quenching sealing medium to install the cover fragment, observe and take 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 combined with 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 to obtain the luminescence value RLU.

[0121] In this embodiment, when preparing magnetic microparticles, the magnetic bead coating amount is 5 μg / mg, 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 min.

[0123] Table 1 shows the effects of different types of magnetic beads on the luminescence value of anti-Nephrin autoantibodies in a sample according to an 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 the experimental group sample, N represents the mean value of the luminescence value detection result of the control sample, and S / N refers to signal-to-noise ratio, wherein, the greater the value of S / N, the greater the discrimination between the positive sample detection result and the negative sample detection result when the kit is used to detect the sample, the less likely it is to have false (such as false positive or false negative) results, and the better the sensitivity and accuracy of the kit. The same letters in the following examples have similar meanings to the present embodiment, and will not be repeated hereinafter. As can be seen from Table 1, the S / N value of carboxyl magnetic beads reaches 1.34, which is much larger than Toysl magnetic beads, and its signal-to-noise ratio is better, which can be used as a preferred magnetic bead type.

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

[0128] In this example, 4 samples with clinically positive and clinically negative anti-Nephrin autoantibodies were selected, and magnetic microparticles with different carboxyl magnetic beads coating amounts of Nephrin antigen 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 to obtain the luminescence value RLU.

[0129] Among them, 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 min.

[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, so 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, 3 samples with clinically positive and clinically negative anti-Nephrin autoantibodies were selected, and magnetic particles 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 to obtain the luminescence value RLU.

[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 min.

[0140] Table 3 shows the effect of magnetic particles of different magnetic bead diameters on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0141] Table 3 Optimization of different magnetic bead sizes

[0142]

[0143]

[0144] It can be seen from Table 3 that 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. Effect of different blocking processes on sample reactivity

[0146] In this example, 2 samples with clinically positive and clinically negative anti-Nephrin autoantibodies were selected, and carboxyl magnetic particles obtained under different blocking processes 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 the 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] Further, 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 min.

[0153] Table 4 shows the effects of magnetic particles under different sealing 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 sealant 3 is the best, so it is determined that the preferred magnetic bead sealant in this embodiment is fish gelatin.

[0157] Example 4: Optimization of reagent working concentrations

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

[0159] When the kit as in Example 1 is used 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 concentration of R1 reagent magnetic particles is set to 0.1 and 0.2 mg / mL, and the working solution concentration of R2 reagent labeled antibodies is set to 0.02 and 0.04 μg / mL. The optimal dilution concentration is selected based on the comprehensive consideration of 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 min.

[0161] Table 5 shows the effects of different amounts of magnetic particles and labeled antibodies added on the luminescence value of anti-Nephrin autoantibodies detected 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 can be seen from Table 5, the signal-to-noise ratio of the kit of the present invention is the highest 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 are the optimal magnetic particles obtained under the optimized conditions in Example 3. The other reagents are 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 a better 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 samples, the working concentration of magnetic particles is 0.1 mg / ml, the concentration of 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 which 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 are the optimal magnetic particles obtained under the optimized conditions in Example 3. The other reagents are 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, and the second step reaction time is kept constant at 10 min for testing. The optimal reaction time is selected based on the comprehensive consideration of the signal-to-noise ratio of the luminescence value.

[0176] Furthermore, when detecting anti-Nephrin autoantibodies in samples, the working concentration of magnetic particles was 0.1 mg / ml, the concentration of 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] It can be seen from Table 7 that when the reaction time of the second step 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. After comprehensive consideration, choosing the 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 detect the sera of 100 healthy children undergoing physical examinations, where the maximum signal value was less than 15AU / mL. In addition, the sera of 100 children with kidney disease were tested, where the minimum signal value was greater than 20AU / mL. Therefore, the cut-off value of this detection kit was selected as 20AU / mL. If the sample test result is greater than or equal to 20AU / mL, it is judged to be reactive, i.e. positive; if the sample test result is less than 20AU / 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 (eg Figure 1-10 ), 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, Fig.21 This is a calibration curve of an anti-Nephrin autoantibody kit according to an embodiment of the present invention. A serum sample with a high luminescence intensity of anti-Nephrin antibodies was selected as a calibrator, and multiple dilutions were performed (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. According to the scatter plot, a linear trend line (least squares method) was added, and a linear equation and determination coefficient (R 2 ), R 2 =1, the linear relationship is good. The concentration of the calibrator is CAL1: 0.42AU / mL, CAL2: 39.3AU / mL; and the luminescence intensity value of the sample is matched with the luminescence intensity value standard curve of the calibrator to obtain the content (AU value) of the anti-Nephrin antibody in the sample to be tested.

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

[0189] Example 8: Anti-Nephrin Autoantibody Kit Detection Sensitivity and Specificity

[0190] In this embodiment, the inventors detected more than 1000 clinically confirmed positive samples and negative samples, and obtained their test results. Set the cut-off value to be greater than or equal to 20AU / mL as positive, and less than 20AU / mL as negative. In this embodiment, the detection accuracy of more than 1000 clinical positive samples and negative samples can reach more than 99%. In this embodiment, the test results of 50 positive samples and 50 negative samples are further listed, as shown in Table 9.

[0191] Table 9 Anti-Nephrin autoantibody detection results

[0192]

[0193]

[0194] In this embodiment, the detection rates of negative samples and positive samples are close to 100%. The applicant further counted the number of positive samples that were still positive after detection by the kit of this application as TP, and the number of negative samples after detection as FN; the number of negative samples that were positive after detection by the kit of this application as FP, and the number of negative samples after detection 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, but not 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, the magnetic particle comprising a Nephrin antigen and a magnetic bead, wherein the Nephrin antigen is coated on the surface of the magnetic bead; 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. 2 . The magnetic particle according to claim 1 , 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.

3. A method for preparing magnetic particles according to claim 1 or 2, comprising: 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 an anti-nephrin autoantibody of a mammal; 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.

4. The preparation method according to claim 3, further comprising: 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.

5. The preparation method according to claim 3, wherein 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-X 100; (0.20-0.70) g / L Tween-20; and (0.20-0.70) g / L ProClin 300; 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.

6. The preparation method according to claim 3, wherein the magnetic beads and the nephrin antigen are added in a ratio of: 5-20 μg of nephrin antigen is added to the magnetic bead diluent containing about 1 mg of magnetic beads; preferably, 10-20 μg of nephrin antigen is added.

7. A kit for determining anti-Nephrin autoantibodies, comprising: The magnetic particles as described in claim 1 or 2 or the magnetic particles prepared by the preparation method as described in any one of claims 3-6.

8. The kit according to claim 7, further comprising: IgG antibody labeled with chemiluminescent marker and its dilution solution; Preferably, the concentration of the IgG antibody labeled with the chemiluminescent marker in its diluent is 0.02-0.04 μg / mL.

9. The kit according to claim 8, wherein the chemiluminescent marker is selected from acridinium ester, acridinium sulfonamide, acridinium toluene sulfonamide, acridinium p-methylsulfonamide or acridinium trifluoromethanesulfonamide; preferably, the chemiluminescent marker is acridinium ester.

10. The kit according to claim 8, wherein the IgG antibody labeled with a 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 labeled diluent comprises: 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.

11. The kit according to claim 7, further comprising: 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; Preferably, the sample diluent further includes (0.30-0.70) g / L Tween 20; and (0.30-0.70) g / L ProClin300; preferably, the sample diluent further includes 0.50 g / L Tween 20; and 0.50 g / L ProClin300.

12. The kit according to claim 7, further comprising: A calibrator comprising an anti-Nephrin autoantibody; and a calibrator buffer.

13. The kit according to claim 12, wherein 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; Preferably, the calibration 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.

14. A method for detecting anti-Nephrin autoantibodies in a sample using the magnetic particles according to claim 1 or 2, or the magnetic particles prepared by the preparation method according to any one of claims 3 to 6, or the kit according to any one of claims 7 to 13, comprising: 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 dilutions 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); preferably 20:50:100:

100.

15. The method according to claim 14, wherein the pre-excitation solution comprises (0.80-1.70)% (W / V) hydrogen peroxide; the exciting solution comprises (0.2-0.5) mol / L sodium hydroxide; preferably, the pre-excitation solution comprises 1.32% (W / V) hydrogen peroxide; and the exciting solution comprises 0.35 mol / L sodium hydroxide.

16. The method according to claim 14, further comprising: Determine a 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 The content of anti-Nephrin antibodies in the sample to be tested is obtained. The method according to claim 14 , wherein the sample is serum.

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