Anti-nephrin autoantibody assay kit, detection method therefor and preparation method therefor

Abstract

The present application relates to a magnetic particle that specifically binds to an anti-Nephrin antibody in a sample, the magnetic particle comprising a magnetic bead coated with a polypeptide or a fragment thereof that can specifically bind to an anti-Nephrin autoantibody of a mammal, wherein the magnetic bead is a carboxyl magnetic bead, and a particle size of the carboxyl magnetic bead is 1-5 μm.

Description

An anti-nephrin autoantibody assay kit and its detection method and preparation method

[0001] Related applications

[0002] The present application claims priority to the Chinese patent application No. 202411462363.7, filed on October 18, 2024, entitled "An anti-nephrin autoantibody assay kit and its detection method and preparation method", the contents of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of in vitro detection technology, in particular to an anti-nephrin autoantibody assay kit and its detection method and preparation method. BACKGROUND

[0004] Nephrin protein mainly exists in the slit diaphragm of podocyte in glomerulus, and is a membrane protein that is essential for maintaining the normal function of glomerular filtration barrier. As early as 2021, when we first discovered and screened podocyte autoantibodies specific to idiopathic nephrotic syndrome, we found the existence of anti-nephrin autoantibodies1. However, compared with other podocyte autoantibodies we discovered, the concentration of anti-nephrin autoantibodies in serum is relatively low. Therefore, due to the limited sensitivity, the detection based on conventional enzyme-linked immunosorbent assay and immunoblotting results in a low detection rate. Subsequently, further studies found that the recurrence of patients with focal segmental glomerulosclerosis after transplantation was related to nephrin autoantibodies, indicating that anti-nephrin autoantibodies may be an important pathogenic factor2,3. 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, and are effective biomarkers for monitoring disease changes and treatment response4,5. In summary, the accumulating evidence suggests that anti-nephrin autoantibodies have important clinical value. There is an urgent need in the clinic 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 to be applied in clinical practice.

[0005] The existing anti-Nephrin antibody detection methods include enzyme-linked immunosorbent assay, radioimmunoassay, etc. Among them, the enzyme-linked immunosorbent assay has poor sensitivity, and the experimental process is long, and the reagent batch difference is large. Although the radioimmunoassay has high sensitivity and specificity, the use of radioactive tracers will cause radioactive pollution and harm, and the commonly used radionuclides have short half-lives, the effective period of the reagent kit is limited, and automatic analysis cannot be performed, which makes it difficult to promote and apply to the clinic. There is an urgent need to find a conventional, reliable, efficient, and clinically accessible anti-Nephrin autoantibody quantitative detection method and detection kit.

[0006] [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,Xia Y,Lu Z,Shu Q,Mao J.The important roles and molecular mechanisms of annexin A2 autoantibody in children with nephrotic syndrome.Ann Transl Med.2021 Sep;9(18):1452.

[0007] [2]Hattori M.Anti-nephrin autoantibodies:novel predictors of post-transplant recurrence of focal segmental glomerular sclerosis.Kidney Int.2024 Oct;106(4):570-572;

[0008] [3]Hattori M,Shirai Y,Kanda S,et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant.2022 Oct;22(10):2478-2480;

[0009] [4] Watts AJB, Keller KH, Lerner G, et al. Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology. J Am Soc Nephrol. 2022 Jan;33(1):238-252. doi:10.1681 / ASN.2021060794;

[0010] [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

[0011] In view of the technical problems in the prior art, the first object of the present application is to provide an anti-Nephrin autoantibody assay kit, which can solve the problems of low accuracy, low sensitivity and low signal-to-noise ratio of existing kits.

[0012] The second object of the present application is to provide a detection method for an anti-Nephrin autoantibody assay kit, which can solve the problems of complex detection method and long detection time of existing kits.

[0013] The third object of the present application is to provide a preparation method for 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.

[0014] The first and third objects of the present application are achieved by the following technical solutions:

[0015] A magnetic micro-particle specifically combined with an anti-Nephrin antibody in a sample, the magnetic micro-particle comprising a Nephrin antigen and a magnetic bead, the Nephrin antigen 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; optionally, the particle size of the carboxyl magnetic bead is 1-3 μm; optionally, the particle size of the magnetic bead is 3 μm.

[0016] The magnetic microparticle as described above, wherein the Nephrin antigen is a polypeptide or a fragment thereof capable of specifically binding to anti-Nephrin autoantibody of a mammal, and the sequence of the polypeptide is shown as SEQ ID NO. 1.

[0017] The magnetic microparticle as described above, wherein the magnetic microparticle is blocked by a blocking agent; and the blocking agent comprises 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin.

[0018] A method for preparing a magnetic microparticle capable of specifically binding to anti-Nephrin antibody in a sample, comprising: obtaining a mixture comprising activated magnetic beads; adding a Nephrin antigen to the mixture and mixing uniformly; wherein the Nephrin antigen is a polypeptide or a fragment thereof capable of specifically binding to anti-Nephrin autoantibody of a mammal; suspending the reaction in an environment with a temperature of 20-25°C for 1-2 hours; washing the product after the reaction in a blocking agent; 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 the Nephrin antigen-coated magnetic beads.

[0019] The method as described above, further comprising: diluting the Nephrin antigen-coated magnetic beads in a diluent to obtain a magnetic microparticle diluent; wherein the concentration of the magnetic microparticles in the magnetic microparticle diluent is 0.1-0.2 mg / mL.

[0020] The method as described above, wherein the diluent comprises: (0.80-1.30) g / L Tris; (5.00-8.00) g / L Tris-HCl; (7.00-11.00) g / L NaCl; (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; and optionally, the diluent comprises: 1.12 g / L Tris; 6.42 g / L Tris-HCl; 9.00 g / L NaCl; 3.00 g / L Triton-X 100; 0.50 g / L Tween-20; and 0.50 g / L ProClin 300.

[0021] The method as described above, wherein the blocking agent comprises fish gelatin.

[0022] The method as described above, wherein the magnetic beads and the Nephrin antigen are added in a ratio of about 1 mg of magnetic beads to 5-20 μg of Nephrin antigen; and optionally, 10-20 μg of Nephrin antigen is added.

[0023] The method as claimed in any one of the above, wherein the magnetic beads are carboxyl magnetic beads.

[0024] The method as claimed in any one of the above, wherein the magnetic beads have a particle size of 1-5 μm; or a particle size of 1-3 μm; or a particle size of 3 μm.

[0025] An anti-Nephrin autoantibody assay kit, comprising: the magnetic microparticles as claimed in any one of the above or prepared by the preparation method as claimed in any one of the above.

[0026] The kit as claimed in any one of the above, further comprising: a chemiluminescent marker-labeled IgG antibody; or the chemiluminescent marker-labeled IgG antibody has a concentration of 0.02-0.04 μg / mL.

[0027] The kit as claimed in any one of the above, wherein the chemiluminescent marker is selected from acridan ester, acridan sulfonamide, acridan toluenesulfonamide, acridan p-toluenesulfonamide or acridan triflate; or the chemiluminescent marker is acridan ester.

[0028] The kit as claimed in any one of the above, wherein the chemiluminescent marker-labeled IgG antibody is diluted in a labeling diluent, and the labeling diluent comprises: (0.30-0.70) g / L sodium dihydrogen phosphate; (15.00-18.00) g / L disodium hydrogen 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; or the labeling diluent comprises: 0.50 g / L sodium dihydrogen phosphate; 16.75 g / L disodium hydrogen 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.

[0029] The kit as claimed in any one of the above, further comprising: a sample diluent, and 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.

[0030] 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.

[0031] Optionally, the sample diluent further comprises (0.30-0.70) g / L Tween 20; and (0.30-0.70) g / L ProClin 300; optionally, the sample diluent further comprises 0.50 g / L Tween 20; and 0.50 g / L ProClin 300.

[0032] The kit as described above, further comprising: a calibrator comprising anti-Nephrin autoantibody; and a calibrator buffer; optionally, the calibrator buffer comprises: (0.10-0.30) g / L Tris; (1.00-1.60) g / L Tris-HCl; (7.00-11.00) g / L NaCl; (3.00-7.00) g / L BSA; (0.30-0.70) g / L Tween; and (0.30-0.70) g / L ProClin 300; further optionally, the calibrator buffer comprises: 0.20 g / L Tris; 1.32 g / L Tris-HCl; 9.00 g / L NaCl; 5.00 g / L BSA; 0.50 g / L Tween; and 0.50 g / L ProClin 300.

[0033] The second object of the present application is achieved by the following technical solution:

[0034] A method for detecting anti-Nephrin autoantibody in a sample according to the method as described in any one of the above, the Nephrin antigen-coated magnetic beads as described above, or the kit as described in any one of the above, comprising: adding the sample to be tested into a sample diluent and mixing uniformly; adding the Nephrin antigen-coated magnetic beads or magnetic microparticle diluent, and reacting for a certain period of time; adding acridan ester-labeled mouse anti-human IgG antibody or acridan ester-labeled mouse anti-human IgG antibody diluted in a labeling diluent, and reacting for a certain period of time; adding pre-priming solution and priming solution; and detecting the relative luminescence intensity.

[0035] The method as described above, the pre-priming solution comprises (0.80-1.70) % (W / V) hydrogen peroxide; and the priming solution comprises (0.2-0.5) mol / L sodium hydroxide; optionally, the pre-priming solution comprises 1.32 % (W / V) hydrogen peroxide; and the priming solution comprises 0.35 mol / L sodium hydroxide.

[0036] The method as described above further comprises obtaining the concentration and the luminescence intensity value of the calibrators, and determining the calibration curve, see Fig. 21, wherein the calibrator concentrations are CAL1: 0.42 AU / mL, CAL2: 39.3 AU / mL; matching the luminescence intensity value of the sample with the standard curve of the luminescence intensity value of the calibrators, to obtain the content of the anti-Nephrin antibody in the sample to be tested.

[0037] The method as described above, the adding 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.

[0038] The method as described above, the reaction time of adding the Nephrin antigen coated magnetic beads or the magnetic particle diluent is 10-20 min.

[0039] The method as described above, the sample is serum.

[0040] The details of one or more embodiments of the application are set forth in the description below, and other features, objects, and advantages of the application will be apparent from the description and the claims. BRIEF DESCRIPTION OF DRAWINGS

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

[0042] Figs. 1-10 are indirect immunofluorescence images of positive samples according to an embodiment of the present application;

[0043] Figs. 2-20 are indirect immunofluorescence images of negative samples according to an embodiment of the present application;

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

[0045] Fig. 22 is a ROC curve of the detection results of the anti-Nephrin autoantibody kit of the sample according to the embodiment. DETAILED DESCRIPTION

[0046] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0047] In the present application, "anti-Nephrin autoantibody" is a key protein of the complex podocyte slit diaphragm structure, which has a wide range of signal functions; when the gene is mutated or experimental Nephrin is knocked out, severe podocyte injury occurs. Anti-Nephrin autoantibody is widely present in patients with nephrotic syndrome in all age groups, which induces nephrotic syndrome, Nephrin phosphorylation and cell rearrangement at the proteomics level, resulting in typical ultrastructural changes 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 autoantibody in mammals including humans. In some embodiments, the Nephrin antigen refers to a human recombinant Nephrin protein extracellular segment polypeptide. Further, in some embodiments, the sequence of the Nephrin antigen is as shown in SEQ ID NO. 1 or SEQ ID NO. 1:

[0049] SEQ ID NO. 1:

[0050] SEQ ID NO. 2:

[0051] In the present application, "magnetic bead coating amount" refers to the addition ratio of activated magnetic beads and human recombinant Nephrin protein extracellular segment (Nephrin antigen) when preparing magnetic microparticles. For example, 10 μg of Nephrin antigen is added per milliliter of activated magnetic beads, which can be considered as a magnetic bead coating amount of 10 μg / ml.

[0052] In the present application, "sample" refers to the serum of a subject.

[0053] The present application relates to a magnetic microparticle that specifically binds to anti-Nephrin antibody in a sample, the magnetic microparticle comprising a Nephrin antigen and a magnetic bead, the Nephrin antigen being 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; optionally, the particle size of the carboxyl magnetic bead is 1-3 μm; optionally, the particle size of the magnetic bead is 3 μm.

[0054] In some embodiments, the Nephrin antigen is a polypeptide or fragment thereof that can specifically bind to anti-Nephrin autoantibody in mammals, and the sequence thereof is as shown in SEQ ID NO. 1.

[0055] In some embodiments, the magnetic microparticle is blocked by a blocking agent; optionally, the blocking agent comprises 1% (w / v) bovine serum albumin, 0.5% (w / v) casein or 0.5% (w / v) fish gelatin.

[0056] The application relates to a preparation method of magnetic particles specifically combined with anti-Nephrin antibodies in a sample, comprising the following steps: obtaining a mixed solution containing 1 mg of activated magnetic beads; adding 5-20 ug of Nephrin antigens into the mixed solution, and adding 10-20 ug of Nephrin antigens optionally, and mixing uniformly; wherein the Nephrin antigens are polypeptides or fragments thereof which can be specifically combined with anti-Nephrin autoantibodies of mammals; suspending and reacting for 1-2 hours in an environment with a temperature of 20-25 DEG C; adding the product after the reaction into a blocking agent for cleaning; wherein the blocking agent comprises 2% (w / v) of bovine serum albumin, 2% (w / v) of casein or 2% (w / v) of fish gelatin; optionally, the blocking agent comprises fish gelatin; and obtaining the magnetic beads coated with the Nephrin antigens.

[0057] In some embodiments, the method for coating the magnetic beads with the Nephrin antigens further comprises: diluting the magnetic beads coated with the Nephrin antigens 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.

[0058] In some embodiments, the diluent comprises: (0.80-1.30) g / L of Tris; (5.00-8.00) g / L of Tris-HCl; (7.00-11.00) g / L of NaCl; (1.00-5.00) g / L of Triton-X 100; (0.20-0.70) g / L of Tween-20; and (0.20-0.70) g / L of ProClin 300; optionally, the diluent comprises: 1.12 g / L of Tris; 6.42 g / L of Tris-HCl; 9.00 g / L of NaCl; 3.00 g / L of Triton-X 100; 0.50 g / L of Tween-20; and 0.50 g / L of ProClin 300.

[0059] In some embodiments, the magnetic beads are carboxyl magnetic beads. In some embodiments, the particle size of the magnetic beads is 1-5 um; optionally, the particle size of the magnetic beads is 1-3 um; more optionally, the particle size of the magnetic beads is 3 um.

[0060] The application relates to an anti-Nephrin autoantibody determination kit, comprising: the magnetic particles as described in any one of the above or prepared by the preparation method as described in any one of the above.

[0061] In some embodiments, the kit further comprises: IgG antibodies labeled with a chemiluminescent marker; optionally, the concentration of the IgG antibodies labeled with the chemiluminescent marker is 0.02-0.04 ug / mL.

[0062] In some embodiments, the chemiluminescent label is selected from an acridinium ester, an acridinium sulfonamide, an acridinium toluenesulfonamide, an acridinium p-toluenesulfonamide, or an acridinium trifluoromethylsulfonamide; optionally, the chemiluminescent label is an acridinium ester.

[0063] In some embodiments, the chemiluminescent label labeled IgG antibody is diluted in a label diluent, wherein: the label diluent comprises: (0.30-0.70) g / L sodium phosphate monobasic; (15.00-18.00) g / L sodium phosphate dibasic; (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; optionally, the label diluent comprises: 0.50 g / L sodium phosphate monobasic; 16.75 g / L sodium phosphate dibasic; 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.

[0064] In some embodiments, the kit further comprises: a sample diluent; the sample diluent comprises: (0.50-1.50) g / L Tris; (5.00-8.00) g / L Tris-HCl; (7.00-11.00) g / L sodium chloride; and (17.00-23.00) g / L bovine serum albumin; optionally, the sample diluent comprises: 1.00 g / L Tris; 6.58 g / L Tris-HCl; 9.00 g / L sodium chloride; and 20.00 g / L bovine serum albumin;

[0065] optionally, the sample diluent further comprises (0.30-0.70) g / L Tween 20; and (0.30-0.70) g / L ProClin 300; optionally, the sample diluent further comprises 0.50 g / L Tween 20; and 0.50 g / L ProClin 300.

[0066] In some embodiments, the kit further comprises: a calibrator comprising anti-Nephrin autoantibody; and a calibrator buffer; optionally, the calibrator buffer comprises: (0.10-0.30) g / L Tris; (1.00-1.60) g / L Tris-HCl; (7.00-11.00) g / L NaCl; (3.00-7.00) g / L BSA; (0.30-0.70) g / L Tween; and (0.30-0.70) g / L ProClin300; further optionally, the calibrator buffer comprises: 0.20 g / L Tris; 1.32 g / L Tris-HCl; 9.00 g / L NaCl; 5.00 g / L BSA; 0.50 g / L Tween; and 0.50 g / L ProClin300.

[0067] In some embodiments, the calibrator comprises anti-Nephrin autoantibody. In some embodiments, the calibrator comprises human anti-Nephrin autoantibody. In some embodiments, the anti-Nephrin autoantibody in the calibrator is obtained from a clinical anti-Nephrin autoantibody positive patient. In some embodiments, the anti-Nephrin autoantibody in the calibrator is obtained from a clinical anti-Nephrin autoantibody positive patient sample. In some embodiments, the anti-Nephrin autoantibody in the calibrator is obtained from a clinical anti-Nephrin autoantibody positive patient sample, and the anti-Nephrin autoantibody in the calibrator is the same as the anti-Nephrin autoantibody in the patient sample.

[0068] The present application relates to a method for detecting anti-Nephrin autoantibody in a sample according to any of the above methods, the Nephrin antigen-coated magnetic beads as described above, or the kit as any of the above, comprising: adding the sample to be tested to the sample diluent and mixing uniformly; adding the Nephrin antigen-coated magnetic beads or magnetic microparticle diluent, and reacting for a certain period of time; adding the acridan ester-labeled mouse anti-human IgG antibody or the acridan ester-labeled mouse anti-human IgG antibody diluted in the labeling diluent, and reacting for a certain period of time; adding the pre-priming solution and the priming solution; and detecting the relative luminescence intensity.

[0069] In some embodiments, the pre-priming solution comprises (0.80-1.70)% (W / V) hydrogen peroxide; the priming solution comprises (0.2-0.5) mol / L sodium hydroxide; optionally, the pre-priming solution comprises 1.32% (W / V) hydrogen peroxide; the priming solution comprises 0.35 mol / L sodium hydroxide.

[0070] In some embodiments, the method for detecting anti-Nephrin autoantibodies in a sample further comprises: obtaining the concentration and luminescence intensity value of the calibrators, and determining the calibration curve, see Figure 22, wherein the calibrator concentrations are CAL1: 0.42 AU / mL, CAL2: 39.3 AU / mL; matching the luminescence intensity value of the sample with the standard curve of the luminescence intensity value of the calibrators, to obtain the content of anti-Nephrin antibodies in the sample to be tested.

[0071] In some embodiments, the volume ratio of the sample to be tested, the magnetic particle diluent, the arycrid 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.

[0072] In some embodiments, the reaction time of adding the Nephrin antigen-coated magnetic beads or the magnetic particle diluent is 10-20 min.

[0073] In the following, the preferred embodiments of the present application will be further described in detail in conjunction with the accompanying drawings. The detection standard of the embodiments of the present application is YY / T 1789.4-2022 Performance Evaluation Method for In Vitro Diagnostic Test Systems.

[0074] In the following embodiments, samples of subjects determined to be positive for anti-Nephrin autoantibodies in the clinic are selected as the experimental group, and samples of subjects determined to be negative for anti-Nephrin autoantibodies in the clinic are selected as the control group. The experimental group and the control group are both children aged 3-18 years old, and both have different degrees of nephrotic syndrome.

[0075] Further, the present application relates to a method for determining anti-Nephrin autoantibodies in a sample from a subject ex vivo, which method uses a complex of a polypeptide or fragment thereof capable of specifically binding to anti-Nephrin autoantibodies coated magnetic beads (hereinafter referred to as magnetic particles), and a chemiluminescent-labeled IgG antibody (hereinafter referred to as labeled antigen). When the magnetic particles and the labeled antigen form an immune reaction to form a magnetic particle-labeled antigen complex, in the presence of an oxidizing agent, hydrogen peroxide and sodium hydroxide to form an alkaline environment, arycrid ester decomposes to emit light, thereby obtaining the detection result of anti-Nephrin autoantibodies.

[0076] Compared with the traditional technology, the present application has the following advantages:

[0077] The proportion of the R1 component Nephrin antigen coated magnetic microparticle working solution and the R2 component mouse anti-human IgG labeled acridinium ester working solution in the kit of the present application is such that the magnetic beads can bind more antigens under the same proportion, thus the capturing effect of the antibody in the sample to be measured is better, thereby significantly improving the signal-to-noise ratio and sensitivity of the detection, and the anti-Nephrin antibody concentration in the sample can be quantitatively detected.

[0078] The detection method of the kit of the present application can selectively capture target molecules by magnetic beads, and has simple and fast operation process, and is easy to automate and clinically popularize and apply. The uniformity and accuracy of the results can be obtained within 30 min, the processing efficiency of the sample is greatly improved, and the use cost is reduced by performing multiple dilutions, and the reportable interval is expanded.

[0079] The preparation method of the kit of the present application has simple process flow, and the prepared product has good sensitivity, specificity and stability.

[0080] Example 1: Preparation method of anti-Nephrin autoantibody assay kit

[0081] 1. Preparation method of R1 reagent-magnetic microparticles

[0082] In the present embodiment, the magnetic beads can be Toysl magnetic beads or carboxyl magnetic beads with different particle sizes. The adjustment of the particle size of the magnetic beads and the adjustment of the coating amount of the magnetic beads are referred to Example 3, which will not be repeated in the present embodiment. In the present embodiment, the Toysl magnetic beads used are purchased from JSR Company, and the product batch number is MS300 / Tosyl; the carboxyl magnetic beads used are purchased from JSR Company, and the product batch number is MS300 / Carboxyl. The polypeptide or fragment thereof (antigen) capable of specifically binding to the anti-Nephrin autoantibody involved in the present embodiment is human recombinant Nephrin protein extracellular segment.

[0083] Further, in the screening and optimization process of the magnetic beads in Example 3, only the corresponding parameters in the present embodiment are replaced, and the remaining parameters remain unchanged.

[0084] In the present embodiment, the preparation method of the magnetic microparticles comprises:

[0085] The magnetic beads are added into chemical cross-linking agents EDC and Sulfo-NHS to activate the functional groups on the surface thereof.

[0086] 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) are coated and combined so that the coating amount of the magnetic beads is 5 μg / mg, 10 μg / mg, 15 μg / mg or 20 μg / mg respectively. The reaction conditions for the coating and combination are as follows: suspension reaction for 1-2 hours in an environment of 20-25 °C;

[0087] The Nephrin antigen coated magnetic beads are obtained after the blocking and washing; the components of the blocking agent can refer to Example 3;

[0088] Then, diluent (1 L of which contains 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-20 and 0.50 g of ProClin 300) is added to dilute the Nephrin antigen coated magnetic beads working solution. According to an embodiment of the present application, the concentration of the magnetic particle working solution is 0.1 mg / mL.

[0089] 2. Preparation method of R2 reagent-labeled antibody

[0090] In this embodiment, the IgG antibody is labeled with a chemiluminescent label. The IgG antibody used in this embodiment is a mouse anti-human IgG antibody purchased from GenScript Company, and the product batch number is A01855. In this application, the selection of the chemiluminescent label is not limited, such as acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-toluenesulfonamide, acridinium trifluoromethanesulfonamide, etc. can be used to label the IgG antibody. Further, in this embodiment, the chemiluminescent label for labeling the IgG antibody is acridinium ester, which is purchased from Seebio Company, and the product batch number is EKY0980A.

[0091] In this embodiment, the method for labeling the mouse anti-human IgG antibody with acridinium ester comprises:

[0092] 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 of acridinium ester per 100 μg of antibody;

[0093] The coupling conditions are as follows: the acridinium ester and the antibody are reacted at room temperature in the dark according to the above ratio for 2 hours, then a reaction termination solution is added, which includes 0.03 Mol lysine. Reaction for 1 hour at room temperature in the dark, and after the reaction is completed, the acridinium ester label is purified using a desalting column;

[0094] After purification, add acridinium ester dilution solution (1 L contains 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-X 405, and 0.50 g of ProClin 300) to obtain the 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.

[0095] 3. Preparation method of R3 reagent-sample dilution solution

[0096] In this embodiment, the sample dilution solution comprises: 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 ProClin 300 per 1 L of sample dilution solution. The concentrations of the components in the sample dilution solution are selected according to conventional concentrations.

[0097] 4. Preparation method of R4 reagent-calibrator buffer

[0098] In this embodiment, the high-value serum sample is diluted 5 times with the calibrator buffer. The preparation method of the calibrator 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 ProClin 300 per 1 L of use. It is stored at 2-8°C for standby use.

[0099] Method for determining anti-Nephrin autoantibodies in samples of Example 2

[0100] 1. Chemiluminescence method

[0101] In this embodiment, the sample refers to the serum of a subject.

[0102] The method for detecting the sample of a subject using the anti-Nephrin autoantibody determination kit of Example 1 comprises:

[0103] 20 μL of the sample to be tested is added to 100 μL of the sample dilution solution of the R3 reagent for dilution;

[0104] The diluted sample to be tested is incubated with 50 μL of the R1 reagent magnetic microparticle working solution at room temperature, the incubation time is 5-20 min, and the first washing is performed 4 times;

[0105] Then, 100 μL of the R2 reagent labeled antibody working solution is added for incubation at room temperature, the incubation time is 10 min, and the second washing is performed 4 times;

[0106] The pre-priming solution and the priming solution were added at last, and the maximum luminescence intensity was reached at 0.4 s; the pre-priming solution included 1.32% (W / V) hydrogen peroxide, and the priming solution included 0.35 mol / L sodium hydroxide;

[0107] The luminescence value (RLU) of the final reaction product was detected at a wavelength of 430 nm.

[0108] 3. Indirect immunofluorescence method

[0109] The indirect immunofluorescence method for detecting the antibody in the sample is the detection gold standard, and the detection result can be used as a control for the detection result of the chemiluminescence method of the present application.

[0110] The specific experimental steps of the indirect immunofluorescence method include:

[0111] 1. Normal human foot cell culture and preparation: normal human foot cells were planted on a cover glass, cultured to a monolayer in a suitable culture medium, and washed with PBS for 3 times, each for 5 minutes; wherein the normal human foot cells were obtained from an immortalized human foot cell line.

[0112] 2. Cell fixation: the cells were fixed with 4% paraformaldehyde at room temperature for 10-15 minutes, and after fixation, the cells were washed with PBS for 3 times, each for 5 minutes;

[0113] 3. Blocking: non-specific binding sites were blocked with PBS containing 5-10% normal goat serum or BSA, and incubated at room temperature for 30 minutes;

[0114] 4. Incubation of the first antibody: the cover glass was placed in the sample serum and incubated at 4°C overnight;

[0115] 5. Washing: after incubation, the cells were washed with PBS for 3 times, each for 5 minutes, to remove the unbound antibodies;

[0116] 6. Incubation of the second antibody: a fluorescently labeled anti-human IgG secondary antibody was used, and the cover glass was incubated at room temperature in the dark for 1 hour;

[0117] 7. Washing: after incubation, the cells were washed with PBS for 3 times, each for 5 minutes, to remove the unbound antibodies;

[0118] 8. Mounting and observation: the cover glass was mounted with an anti-fluorescence quenching mounting agent, and the fluorescent images of the cells were observed and photographed under a laser confocal microscope.

[0119] Example 3: Screening and optimization of magnetic beads

[0120] In this embodiment, the parameters for preparing the magnetic microparticles were optimized to obtain the optimal Nephrin antigen-coated magnetic beads.

[0121] 1. Effect of different types of magnetic beads on sample reactivity

[0122] In this embodiment, 10 samples of anti-Nephrin autoantibody clinically positive and clinically negative were selected, and the Nephrin antigen was combined with Toysl magnetic beads and carboxyl magnetic beads, respectively. 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.

[0123] In this embodiment, the magnetic bead coating amount was 5 μg / mg when preparing the magnetic microparticles, the particle size of the carboxyl magnetic beads was 1.5 μm, and the blocking agent was bovine serum albumin;

[0124] Further, when detecting anti-Nephrin autoantibodies in the sample, the working concentration of the magnetic microparticles was 0.2 mg / ml, the working concentration of the labeled antibody was 0.04 μg / mL, the sample addition amount was 5 μL, and the reaction time of the first step was about 10 min.

[0125] Table 1 shows the effect of different types of magnetic beads on the luminescence value of anti-Nephrin autoantibody detection in the sample according to an embodiment of the present application.

[0126] Table 1 Different types of magnetic beads optimization

[0127] In this embodiment, S represents the average value of the luminescence value detection results of the experimental group samples, N represents the average value of the luminescence value detection results of the control samples, and S / N refers to the signal-to-noise ratio. The larger the value of S / N, the greater the discrimination between the positive sample detection results and the negative sample detection results when using the kit to detect the sample, the less likely it is to appear false (such as false positive or false negative) results, the better the sensitivity and accuracy of the kit. In the following examples, the same letters have similar meanings as in this embodiment, which will not be described again. As can be seen from Table 1, the S / N value of the carboxyl magnetic beads reaches 1.34, which is much larger than that of the Toysl magnetic beads, and the signal-to-noise ratio is better, which can be used as a preferred type of magnetic beads.

[0128] 2. Effect of different antigen coating amounts on sample reactivity

[0129] In this embodiment, 4 samples of anti-Nephrin autoantibody clinically positive and clinically negative were selected, and magnetic microparticles with different magnetic bead coating amounts of carboxyl magnetic beads of the Nephrin antigen were obtained. 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.

[0130] In this embodiment, the particle size of the carboxyl magnetic beads was 1.5 μm, and the blocking agent was bovine serum albumin;

[0131] Further, when detecting anti-Nephrin autoantibodies in a sample, the concentration of the magnetic particles is 0.2 mg / ml, the concentration of the labeled antibody is 0.04 μg / mL, the sample addition amount is 5 μL, and the reaction time in the first step is about 10 min.

[0132] Table 2 shows the influence of different magnetic bead coating amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to an embodiment of the present application.

[0133] Table 2 shows the influence of different magnetic bead coating amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to an embodiment of the present application.

[0134] As shown in Table 2, the signal-to-noise ratio is best when the magnetic bead coating amount is 10 μg / mg, and thus the preferred magnetic bead coating amount in this embodiment is determined to be 10 μg / mg.

[0135] 3. Influence of different magnetic bead particle sizes on sample reactivity

[0136] In this embodiment, three samples of anti-Nephrin autoantibody clinical positives and clinical negatives are selected, magnetic particles of different particle sizes of Nephrin antigen-coated carboxyl magnetic beads are obtained, other reagents in the kit of Embodiment 1 are used, and the anti-Nephrin autoantibody in the sample is detected by the chemiluminescence method of Embodiment 2 to obtain the luminescence value RLU.

[0137] In this embodiment, the magnetic bead coating amount is 10 μg / mg when preparing the magnetic particles, and the blocking agent is bovine serum albumin.

[0138] Further, when detecting anti-Nephrin autoantibodies in a sample, the concentration of the magnetic particles is 0.2 mg / ml, the concentration of the labeled antibody is 0.04 μg / mL, the sample addition amount is 5 μL, and the reaction time in the first step is about 10 min.

[0139] Table 3 shows the influence of different magnetic bead particle sizes on the luminescence value of anti-Nephrin autoantibody detection in a sample according to an embodiment of the present application.

[0140] Table 3 shows the influence of different magnetic bead particle sizes on the luminescence value of anti-Nephrin autoantibody detection in a sample according to an embodiment of the present application.

[0141] As shown in Table 3, the signal-to-noise ratio is best when the magnetic bead particle size is 3.0 μm, and thus the preferred magnetic bead particle size in this embodiment is determined to be 3.0 μm.

[0142] 4. Influence of different blocking processes on sample reactivity

[0143] In the present embodiment, two samples of anti-Nephrin autoantibody clinically positive and clinically negative were selected, and carboxyl magnetic microparticles obtained under different blocking processes were obtained. The other reagents in the kit of the present embodiment 1 and the chemiluminescence method of embodiment 2 were used to detect anti-Nephrin autoantibody in the samples, and the luminescence value RLU was obtained.

[0144] In the present embodiment, the influence of magnetic microparticles prepared under three blocking agents on the detection results of anti-Nephrin antibody in the sample was tested. Among them:

[0145] Blocking agent 1 includes 1% (w / v) bovine serum albumin;

[0146] Blocking agent 2 includes 0.5% (w / v) casein;

[0147] Blocking agent 3 includes 0.5% (w / v) fish gelatin.

[0148] Further, in the present embodiment, the magnetic bead coating amount is 10 μg / mg when the magnetic microparticles are prepared, the magnetic bead particle size is 3 μm, and the blocking agent is bovine serum albumin;

[0149] Further, when detecting anti-Nephrin autoantibody in the sample, the working concentration of the magnetic microparticles is 0.2 mg / ml, the working concentration of the labeled antibody is 0.04 μg / mL, the sample addition amount is 5 μL, and the reaction time of the first step is about 10 min.

[0150] Table 4 shows the influence of magnetic microparticles under different blocking processes on the luminescence value of anti-Nephrin autoantibody in the sample according to one embodiment of the present application.

[0151] Table 4 Different blocking process optimization

[0152] From Table 4, it can be seen that the signal-to-noise ratio of blocking agent 3 is the best, so the preferred magnetic bead blocking agent of the present embodiment is fish gelatin.

[0153] Embodiment 4: Reagent working concentration optimization

[0154] In the present embodiment, the magnetic microparticles used are the optimal magnetic microparticles obtained under the optimized conditions of embodiment 3. The remaining reagents are obtained with reference to embodiment 1.

[0155] When detecting anti-Nephrin autoantibody in the sample using the kit as in embodiment 1, the mass ratio of magnetic microparticles to labeled antibody is 250:1, the working concentration of R1 reagent magnetic microparticles is set to 0.1, 0.2 mg / mL, the working concentration of R2 reagent labeled antibody is set to 0.02, 0.04 μg / mL, and the optimal dilution concentration is selected from the signal-to-noise ratio of the luminescence value.

[0156] Further, when detecting anti-Nephrin autoantibody in a sample, the sample addition amount is 5 μL, and the reaction time in the first step is about 10 min.

[0157] Table 5 shows the effect of different magnetic particles and labeled antibody addition amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0158] Table 5 shows the effect of different magnetic particles and labeled antibody addition amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0159] From Table 5, it can be seen that when the working solution concentration of the Rl reagent magnetic particles is 0.1 mg / mL and the working solution concentration of the R2 reagent labeled antibody is 0.02 μg / mL, the kit has the highest signal-to-noise ratio.

[0160] Example 5: Effect of sample addition amount on sample reactivity

[0161] In this example, the magnetic particles used are the optimal magnetic particles obtained under the optimized conditions of Example 3. The other reagents are obtained as in Example 1.

[0162] When the kit is used to detect anti-Nephrin autoantibody in a sample as in Example 1, different sample addition amounts are set, and the optimal dilution concentration is selected based on the signal-to-noise ratio of the luminescence value.

[0163] Further, when detecting anti-Nephrin autoantibody in a sample, the magnetic particle working solution concentration is 0.1 mg / mL, the labeled antibody working solution concentration is 0.02 μg / mL, and the reaction time in the first step is about 10 min.

[0164] Table 6 shows the effect of different sample addition amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0165] Table 6 shows the effect of different sample addition amounts on the luminescence value of anti-Nephrin autoantibody detection in a sample according to one embodiment of the present application.

[0166] From Table 6, it can be seen that after dilution, the preferred sample addition amount is 20 μL, and in this case, the kit has the highest signal-to-noise ratio.

[0167] Example 6: Reaction condition optimization

[0168] In this example, the magnetic particles used are the optimal magnetic particles obtained under the optimized conditions of Example 3. The other reagents are obtained as in Example 1.

[0169] The sample was detected by the kit as in Example 1, different first step reaction time was set, and the second step reaction time was kept at 10 min. The optimal reaction time was selected from the signal-to-noise ratio of the luminescence value.

[0170] Further, when detecting the anti-Nephrin autoantibody in the sample, the magnetic particle working concentration was 0.1 mg / ml, the labeled antibody working solution concentration was 0.02 μg / mL, and the sample addition amount was 20 μl.

[0171] Table 7 shows the effect of different reaction times on the luminescence value of the anti-Nephrin autoantibody in the sample according to one embodiment of the application.

[0172] Table 7 Different reaction time optimization

[0173] From Table 7, it can be seen that when the second step reaction time is kept at 10 min, the signal-to-noise ratio difference of the first step reaction time of 10 min and 20 min is not obvious. Considering comprehensively, the first step reaction time is selected as 10 min, which can shorten the reaction time and improve the working efficiency of the kit.

[0174] Example 7: Performance test of anti-Nephrin autoantibody kit

[0175] The anti-Nephrin antibody assay kit was used to detect the serum of 100 healthy children, and the maximum signal value was less than 15 AU / mL. In addition, the serum of 100 children with kidney disease was detected, and the minimum signal value was greater than 20 AU / mL. Therefore, the cut-off value of the detection kit was selected as 20 AU / mL. If the sample detection result is greater than or equal to 20 AU / mL, it is determined to be reactive, i.e. positive. If the sample detection result is less than 20 AU / mL, it is determined to be non-reactive, i.e. negative.

[0176] In this embodiment, 20 samples were detected by the indirect immunofluorescence method (gold standard) in Example 2, of which 10 samples were positive (as shown in Figures 1-10) and 10 samples were negative (as shown in Figures 11-20). The anti-Nephrin antibody content was detected by the kit prepared in Example 1 and the chemiluminescence method in Example 2, and the results are shown in Table 8.

[0177] Table 8 Anti-Nephrin autoantibody detection results

[0178] As shown in Table 8, the positive accuracy and negative accuracy of the kit detected by the chemiluminescence method were both 80%.

[0179] Further, FIG. 21 is a calibration curve of the anti-Nephrin autoantibody kit according to an embodiment of the present application. Serum samples with high luminescence intensity of anti-Nephrin antibodies were selected as calibrators and were diluted by a factor of two (luminescence values RLU were 2000, 4000, 6000, 8000, and 10000, respectively) to obtain the corresponding signal values of the calibrators. A scatter plot of the standard RLU (x-axis) and the signal (y-axis) was plotted. According to the scatter plot, a linear trend line (least squares method) was added, and a linear equation and a determination coefficient (R2) were plotted, R2=1, and the linear relationship was good. The concentrations of the calibrators were CAL1: 0.42 AU / mL and CAL2: 39.3 AU / mL. The luminescence intensity values of the samples were matched with the standard curve of the luminescence intensity values of the calibrators to obtain the content (AU value) of the anti-Nephrin antibodies in the samples to be tested.

[0180] FIG. 22 is a ROC curve of the anti-Nephrin autoantibody kit detection results of the samples according to the present embodiment. According to the ROC curve, the AUC is 0.997.

[0181] Example 8: Sensitivity and specificity of the anti-Nephrin autoantibody kit detection

[0182] In the present embodiment, the inventors detected more than 1000 clinically determined positive samples and negative samples and obtained their detection results. The cut-off value was set to be greater than or equal to 20 AU / mL for positive and less than 20 AU / mL for negative. In the present embodiment, the detection accuracy of more than 1000 clinically positive samples and negative samples can reach more than 99%. The detection results of 50 positive samples and 50 negative samples in the present embodiment are listed in Table 9.

[0183] Table 9: Anti-Nephrin autoantibody detection results

[0184] In the present embodiment, the detection rates of the negative samples and the positive samples were close to 100%. The inventors further counted the number of samples that were still positive after detection by the kit as TP, and the number of samples that were negative after detection as FN in the positive samples. The number of samples that were positive after detection by the kit as FP, and the number of samples that were still negative after detection as TN in the negative samples.

[0185] The formula for the sensitivity of the kit detection is: sensitivity = TP / (TP+FN)*100%.

[0186] The formula for the specificity of the kit detection is: specificity = TN / (TN+FP)*100%.

[0187] The statistics and calculation show that the sensitivity and specificity of the kit are close to 100%.

[0188] The above examples are only for illustrating the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the scope of the present application. Therefore, all equivalent technical solutions shall belong to the scope of the present application.

Claims

1. A magnetic particle specifically binding to an anti-Nephrin antibody in a sample, comprising a Nephrin antigen and a magnetic bead, wherein the Nephrin antigen is coated on the surface of the magnetic bead; the magnetic bead is a carboxyl magnetic bead; the carboxyl magnetic bead has a particle size of about 1-5 μm; optionally, the carboxyl magnetic bead has a particle size of about 1-3 μm; and optionally, the magnetic bead has a particle size of about 3 μm. wherein 2.The magnetic particle of claim 1, wherein the Nephrin antigen is a polypeptide or a fragment thereof capable of specifically binding to an anti-Nephrin autoantibody of a mammal, and has a sequence as shown in SEQ ID NO.

1. 3.A method for preparing the magnetic particle of claim 1 or 2, comprising: obtaining a mixed solution comprising activated magnetic beads; adding the Nephrin antigen to the mixed solution and mixing uniformly; wherein the Nephrin antigen is a polypeptide or a fragment thereof capable of specifically binding to an anti-Nephrin autoantibody of a mammal; suspending the reaction in an environment with a temperature of about 20-25 ℃ for about 1-2 hours; adding the product after the reaction to a blocking agent for cleaning; wherein the blocking agent comprises about 1% (w / v) bovine serum albumin, about 0.5% (w / v) casein or about 0.5% (w / v) fish gelatin; and obtaining the magnetic bead coated with the Nephrin antigen. 4.The method of claim 3, wherein the magnetic bead coated with the Nephrin antigen is diluted in a diluent to obtain a magnetic particle diluent; and the concentration of the magnetic particle in the magnetic particle diluent is about 0.1-0.2 mg / mL. 5.The method of any one of claims 3-4, wherein the diluent comprises: about (0.80-1.30) g / L Tris; about (5.00-8.00) g / L Tris-HCl; about (7.00-11.00) g / L NaCl; about (1.00-5.00) g / L Triton-X 100; about (0.20-0.70) g / L Tween-20; and about (0.20-0.70) g / L ProClin 300; optionally, the diluent comprises: about 1.12 g / L Tris; about 6.42 g / L Tris-HCl; about 9.00 g / L NaCl; about 3.00 g / L Triton-X 100; about 0.50 g / L Tween-20; and about 0.50 g / L ProClin 300. 6.The method of claim 3, wherein the ratio of the magnetic bead and the Nephrin antigen added is: about 5-20 μg of the Nephrin antigen is added to about 1 mg of the magnetic bead diluent; and optionally, about 10-20 μg of the Nephrin antigen is added. 7.An anti-Nephrin autoantibody assay kit, comprising: the magnetic particle of claim 1 or 2 or the magnetic particle prepared by the method of any one of claims 3-6; an IgG antibody labeled with a chemiluminescent marker and a diluent thereof. ​ ​ ​ ​ ​ 4. The method of claim 3, further comprising: ​ ​ ​ ​ ​ ​ 8. The kit of claim 7, further comprising: ​ Optionally, the concentration of the chemiluminescent marker labeled IgG antibody in its diluent is 0.02-0.04 μg / mL.

9. The kit according to any one of claims 7-8, wherein the chemiluminescent marker is selected from acridinium ester, acridinium sulfonamide, acridinium tolunesulfonamide, acridinium p-toluenesulfonamide or acridinium triflate. Optionally, the chemiluminescent marker is acridinium ester.

10. The kit according to any one of claims 7-9, wherein the chemiluminescent marker labeled IgG antibody is diluted in a labeling diluent, wherein: the labeling diluent comprises: about (0.30-0.70) g / L sodium phosphate monobasic; about (15.00-18.00) g / L sodium phosphate dibasic; about (35.00-45.00) g / L sodium chloride; about (7.00-13.00) g / L bovine serum albumin; about (3.00-7.00) g / L Triton-X 405; and about (0.30-0.70) g / L ProClin 300; Optionally, the labeling diluent comprises: 0.50 g / L sodium phosphate monobasic; 16.75 g / L sodium phosphate dibasic; about 40.00 g / L sodium chloride; about 10.00 g / L bovine serum albumin; about 5.00 g / L Triton-X 405; and about 0.50 g / L ProClin 300.

11. The kit of any one of claims 7-10, further comprising: a sample diluent; the sample diluent comprises: about (0.50-1.50) g / L Tris; about (5.00-8.00) g / L Tris-HCl; about (7.00-11.00) g / L sodium chloride; and about (17.00-23.00) g / L bovine serum albumin; Optionally, the sample diluent comprises: about 1.00 g / L Tris; about 6.58 g / L Tris-HCl; about 9.00 g / L sodium chloride; and about 20.00 g / L bovine serum albumin; Optionally, the sample diluent further comprises about (0.30-0.70) g / L Tween 20; and about (0.30-0.70) g / L ProClin 300; Optionally, the sample diluent further comprises about 0.50 g / L Tween 20; and about 0.50 g / L ProClin 300.

12. The kit according to any one of claims 7-11, further comprising: a calibrator comprising anti-Nephrin autoantibody; and a calibrator buffer.

13. The kit according to any one of claims 7-12, wherein the calibrator buffer comprises in the calibrator buffer: about (0.10-0.30) g / L Tris; about (1.00-1.60) g / L Tris-HCl; about (7.00-11.00) g / L sodium chloride; about (3.00-7.00) g / L bovine serum albumin; about (0.30-0.70) g / L Tween; and about (0.30-0.70) g / L ProClin 300; Optionally, the calibrator buffer comprises about 0.20 g / L Tris; about 1.32 g / L Tris-HCl; about 9.00 g / L NaCl; about 5.00 g / L BSA; about 0.50 g / L Tween; and about 0.50 g / L ProClin 300.

14. A method for detecting anti-Nephrin autoantibodies in a sample using the magnetic particles of claim 1 or 2, or the magnetic particles prepared by the method of any one of claims 3-6, or the kit of any one of claims 7-13, comprising: adding about 20-50 μL of the sample to be tested to a sample diluent and mixing well; adding a dilution of the Nephrin antigen-coated magnetic beads or magnetic particles and reacting for 10-20 minutes; adding acridinium ester-labeled mouse anti-human IgG antibody or a dilution of the acridinium ester-labeled mouse anti-human IgG antibody in label diluent and reacting for 10 minutes; adding a pre-priming solution and a priming solution; and detecting the relative light intensity; wherein, the sample to be tested, the dilution of the magnetic particles, the dilution of the acridinium ester-labeled mouse anti-human IgG antibody in label diluent, and the sample diluent are added in a volume ratio of (15-25) : (40-60) : (80-120) : (80-120); preferably 20:50:100:

100.

15. The method of claim 14, wherein the pre-priming solution comprises (0.80-1.70) % (W / V) hydrogen peroxide; and the priming solution comprises about (0.2-0.5) mol / L sodium hydroxide; optionally, the pre-priming solution comprises about 1.32 % (W / V) hydrogen peroxide; and the priming solution comprises about 0.35 mol / L sodium hydroxide.

16. The method of any one of claims 14-15, further comprising: determining a calibration curve based on the concentrations and light intensity values of the calibrators; matching the light intensity value of the sample to the calibration curve of the light intensity values of the calibrators; and obtaining the content of anti-Nephrin autoantibodies in the sample to be tested.

17. The method of any one of claims 14-16, wherein the sample is serum. ​

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