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EP-4741826-A1 - ANTI-NEPHRIN AUTOANTIBODY ASSAY KIT, DETECTION METHOD THEREFOR AND PREPARATION METHOD THEREFOR

EP4741826A1EP 4741826 A1EP4741826 A1EP 4741826A1EP-4741826-A1

Abstract

The present application relates to a magnetic microparticle specifically binding to an anti-Nephrin autoantibody in a sample, including a magnetic bead coated with a polypeptide or a fragment thereof capable of specifically binding to the anti-Nephrin autoantibody in a mammal; wherein the magnetic bead is a carboxyl magnetic bead, and the particle size of the carboxyl magnetic bead is 1 µm to 5 µm.

Inventors

  • The designation of the inventor has not yet been filed

Assignees

  • Shenzhen Yhlo Biotech Co., Ltd.

Dates

Publication Date
20260513
Application Date
20251010

Claims (17)

  1. A magnetic microparticle specifically binding to an anti-Nephrin autoantibody in a sample, comprising a Nephrin antigen and a magnetic bead, the Nephrin antigen being coated on a surface of the magnetic bead; wherein the magnetic bead is a carboxyl magnetic bead, and a particle size of the carboxyl magnetic bead is about 1 µm to 5 µm; optionally, the particle size of the carboxyl magnetic bead is about 1 µm to 3 µm; optionally, a particle size of the magnetic bead is about 3 µm.
  2. The magnetic microparticle according to claim 1, wherein the Nephrin antigen is a polypeptide or a fragment thereof capable of specifically binding to an anti-Nephrin autoantibody in a mammal, and the Nephrin antigen has a sequence as set forth in SEQ ID NO: 1.
  3. A preparation method for the magnetic microparticle according to claim 1 or 2, comprising: obtaining a mixed solution comprising an activated magnetic bead, adding a Nephrin antigen to the mixed solution and mixing uniformly, the Nephrin antigen being a polypeptide or a fragment thereof capable of specifically binding to an anti-Nephrin autoantibody in a mammal; suspending and reacting the mixed solution for about 1 to 2 hours at a temperature of about 20 to 25 °C; washing a reacted product with a blocking agent, wherein the blocking agent comprises about 1% (w/v) of bovine serum albumin, about 0.5% (w/v) of casein, or about 0.5% (w/v) of fish gelatin; and obtaining a Nephrin antigen-coated magnetic bead.
  4. The preparation method according to claim 3, further comprising diluting the Nephrin antigen-coated magnetic bead in a diluent to obtain a magnetic microparticle-diluted solution; wherein a concentration of the magnetic microparticles in the magnetic microparticle-diluted solution is about 0.1 to 0.2 mg/mL.
  5. The preparation method according to any one of claims 3 to 4, wherein the diluent comprises about 0.80 to 1.30 g/L of tris(hydroxymethyl)aminomethane, about 5.00 to 8.00 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 7.00 to 11.00 g/L of sodium chloride, about 1.00 to 5.00 g/L of Triton-X 100, about 0.20 to 0.70 g/L of Tween-20, and about 0.20 to 0.70 g/L of ProClin 300; optionally, the diluent comprises about 1.12 g/L of tris(hydroxymethyl)aminomethane, about 6.42 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 9.00 g/L of sodium chloride, about 3.00 g/L of Triton-X 100, about 0.50 g/L of Tween-20, and about 0.50 g/L of ProClin 300.
  6. The preparation method according to claim 3, wherein a ratio of the magnetic bead added to the Nephrin antigen is: adding about 5 to 20 µg of the Nephrin antigen, optionally, adding about 10 to 20 µg of the Nephrin antigen, to a magnetic bead diluent containing about 1 mg of the magnetic bead.
  7. An anti-Nephrin autoantibody assay kit, comprising the magnetic microparticle according to claim 1 or 2, or the magnetic microparticle prepared by the preparation method according to any one of claims 3 to 6.
  8. The kit according to claim 7, further comprising a chemiluminescent label-labeled IgG antibody and a diluent thereof; optionally, a concentration of the chemiluminescent label-labeled IgG antibody in the diluent thereof is 0.02 to 0.04 µg/mL.
  9. The kit according to any one of claims 7 to 8, wherein the chemiluminescent label is selected from acridinium ester, acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide, or acridinium trifluoromethanesulfonamide; optionally, the chemiluminescent label is acridinium ester.
  10. The kit according to any one of claims 7 to 9, wherein the chemiluminescent label-labeled IgG antibody is diluted in a label diluent, and the label diluent comprises about 0.30 to 0.70 g/L of sodium dihydrogen phosphate, about 15.00 to 18.00 g/L of disodium hydrogen phosphate, about 35.00 to 45.00 g/L of sodium chloride, about 7.00 to 13.00 g/L of bovine serum albumin, about 3.00 to 7.00 g/L of Triton-X 405, and about 0.30 to 0.70 g/L of ProClin 300; optionally, the label diluent comprises 0.50 g/L of sodium dihydrogen phosphate, 16.75 g/L of disodium hydrogen phosphate, about 40.00 g/L of sodium chloride, about 10.00 g/L of bovine serum albumin, about 5.00 g/L of Triton-X 405, and about 0.50 g/L of ProClin 300.
  11. The kit according to any one of claims 7 to 10, further comprising a sample diluent; the sample diluent comprises about 0.50 to 1.50 g/L of tris(hydroxymethyl)aminomethane, about 5.00 to 8.00 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 7.00 to 11.00 g/L of sodium chloride, and about 17.00 to 23.00 g/L of bovine serum albumin; optionally, the sample diluent comprises about 1.00 g/L of tris(hydroxymethyl)aminomethane, about 6.58 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 9.00 g/L of sodium chloride, and about 20.00 g/L of bovine serum albumin; optionally, the sample diluent further comprises about 0.30 to 0.70 g/L of Tween 20, and about 0.30 to 0.70 g/L of ProClin 300; optionally, the sample diluent further comprises about 0.50 g/L of Tween 20 and about 0.50 g/L of ProClin 300.
  12. The kit according to any one of claims 7 to 11, further comprising a calibrator and a calibrator buffer solution, wherein the calibrator comprises the anti-Nephrin autoantibody.
  13. The kit according to any one of claims 7 to 12, wherein the calibrator buffer solution comprises about 0.10 to 0.30 g/L of tris(hydroxymethyl)aminomethane, about 1.00 to 1.60 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 7.00 to 11.00 g/L of sodium chloride, about 3.00 to 7.00 g/L of bovine serum albumin, about 0.30 to 0.70 g/L of Tween, and about 0.30 to 0.70 g/L of ProClin 300; optionally, the calibrator buffer solution comprises about 0.20 g/L of tris(hydroxymethyl)aminomethane, about 1.32 g/L of tris(hydroxymethyl)aminomethane hydrochloride, about 9.00 g/L of sodium chloride, about 5.00 g/L of bovine serum albumin, about 0.50 g/L of Tween, and about 0.50 g/L of ProClin 300.
  14. A method for detecting an anti-Nephrin autoantibody in a sample using the magnetic microparticle according to claim 1 or 2, the magnetic microparticle 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: adding about 20 to 50 µL of a sample to be tested to a sample diluent and mixing uniformly; adding a Nephrin antigen-coated magnetic bead or a magnetic microparticle-diluted solution, and reacting for 10 to 20 minutes; adding an acridinium ester-labeled mouse anti-human IgG antibody or an acridinium ester-labeled mouse anti-human IgG antibody diluted in a label diluent, and reacting for 10 minutes; adding a pre-excitation solution and an excitation solution; and detecting a relative luminescence intensity, wherein a volume ratio of the sample to be tested, the magnetic microparticle-diluted solution, the acridinium ester-labeled mouse anti-human IgG antibody diluted in the label diluent, and the sample diluent that are added is (15 to 25): (40 to 60): (80 to 120): (80 to 120), preferably 20: 50: 100: 100.
  15. The method according to claim 14, wherein the pre-excitation solution comprises 0.80 to 1.70 % (w/v) of hydrogen peroxide, and the excitation solution comprises about 0.2 to 0.5 mol/L of sodium hydroxide; optionally, the pre-excitation solution comprises about 1.32% (w/v) of hydrogen peroxide, and the excitation solution comprises about 0.35 mol/L of sodium hydroxide.
  16. The method according to any one of claims 14 to 15, further comprising: determining a calibration curve based on concentrations of a calibrator and luminescence intensity values thereof; matching the relative luminescence intensity of the sample with the calibration curve of luminescence intensity values of the calibrator; and obtaining a content of the anti-Nephrin autoantibody in the sample to be tested.
  17. The method according to any one of claims 14 to 16, wherein the sample is serum.

Description

RELATED APPLICATIONS The present application claims priority to Chinese Patent Application No. 202411462363.7, filed on October 18, 2024, titled "ANTI-NEPHRIN AUTOANTIBODY ASSAY KITSAND DETECTION METHOD AND PREPARATION METHODS", the entire content of which is incorporated herein by reference. TECHNICAL FIELD The present application relates to the field of in vitro detection technology, and particularly to an anti-Nephrin autoantibody assay kit and detection method and preparation method thereof. BACKGROUND Nephrin protein is mainly present in the slit diaphragm of podocytes in the renal glomerulus and is a membrane protein crucial for maintaining the normal function of the renal glomerular filtration barrier. As early as before 2021, we first discovered and screened podocyte autoantibodies specific for idiopathic nephrotic syndrome and identified the existence of anti-Nephrin autoantibodies1. However, the concentration of anti-Nephrin autoantibodies in serum is relatively low compared to other podocyte autoantibodies we discovered. Therefore, detections based on conventional enzyme-linked immunosorbent assay (ELISA) and immunoblotting methods result in a low detection rate due to limited sensitivity. Subsequent research found that post-transplant recurrence in patients with focal segmental glomerulosclerosis type nephrotic syndrome is related to Nephrin autoantibodies, suggesting that anti-Nephrin autoantibodies may be an important pathogenic factor2,3. Recent clinical cohort studies further revealed that anti-Nephrin autoantibodies are widely present in patients with minimal change type and idiopathic nephrotic syndrome, closely correlated with disease activity, and serve as effective biomarkers for monitoring disease changes and treatment response4,5. In summary, accumulating evidences indicate that anti-Nephrin autoantibodies hold significant clinical value. There is an urgent clinical need to establish and develop anti-Nephrin antibody detection methods and kits that have short detection time, high sensitivity, strong specificity, stable and reliable results, high uniformity, and ease of clinical promotion and application. Existing methods for detecting anti-Nephrin antibodies include enzyme-linked immunosorbent assay, radioimmunoassay, etc.. Enzyme-linked immunosorbent assay has insufficient sensitivity, involves lengthy experimental procedures, and reagents show significant batch-to-batch variations. Although radioimmunoassay has high sensitivity and specificity, the use of radioactive tracers leads to radioactive contamination and hazards. Furthermore, commonly used radionuclides have short half-lives, resulting in a limited shelf life for kits, and fail to perform automated analysis, causing significant challenges for clinical promotion and application. There is an urgent need to develop conventional, reliable, efficient, and clinically accessible quantitative detection methods and assay kits for anti-Nephrin autoantibodies. [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.[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.[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.[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.[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 To address the technical problems in the prior art, a first objective 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. A second objective of the present application is to provide a detection method for the anti-Nephrin autoantibody assay kit, which can solve the problems of complex procedures and long detection time associated with detection methods of existing kits. A third objective of the present application is to provide a preparation method for the anti-Nephrin autoantibody assay kit, which can solve the problems of low process stability, difficult operation, high production costs, and difficulty in clinical promotion of existing kits. The first and third objectives of the present application are achieved by the following technical solutions: A magnetic micro