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RU-2026107939-A - KITS FOR ANALYSIS OF AUTOANTIBODY TO NEPHRIN AND A METHOD FOR ITS DETECTION AND METHOD OF OBTAINING

RU2026107939ARU 2026107939 ARU2026107939 ARU 2026107939ARU-2026107939-A

Inventors

  • Е, Цин
  • МАО, Цзяньхуа
  • ГУ, Жуй

Assignees

  • ШЭНЬЧЖЭНЬ УайЭйчЭлОу БИОТЕХ КО., ЛТД.

Dates

Publication Date
20260504
Application Date
20251010
Priority Date
20241018

Claims (20)

  1. 1. A magnetic microparticle that specifically binds to an autoantibody to nephrin in a sample, comprising an antigen that is nephrin and a magnetic bead, wherein the antigen that is nephrin is coated onto the surface of the magnetic bead;
  2. wherein the magnetic bead is a carboxyl magnetic bead, and the particle size of the carboxyl magnetic bead is from about 1 μm to 5 μm;
  3. optionally, the particle size of the carboxyl magnetic bead is from about 1 μm to 3 μm;
  4. optionally, the particle size of the magnetic bead is approximately 3 μm.
  5. 2. The magnetic microparticle of claim 1, wherein the nephrin antigen is a polypeptide or fragment thereof capable of specifically binding to an autoantibody to nephrin in a mammal, and wherein the nephrin antigen has the sequence presented under SEQ ID NO: 1.
  6. 3. A method for producing a magnetic microparticle according to paragraph 1 or 2, comprising:
  7. obtaining a mixed solution containing an activated magnetic bead,
  8. adding a nephrin antigen to the mixed solution and mixing until homogeneous, wherein the nephrin antigen is a polypeptide or fragment thereof capable of specifically binding to an autoantibody to nephrin in a mammal;
  9. suspending the mixed solution and performing a reaction for about 1 to 2 hours at a temperature of about 20 to 25°C;
  10. washing the reaction product with a blocking agent, 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
  11. obtaining a magnetic bead coated with an antigen, which is nephrin.
  12. 4. The method of production according to claim 3, further comprising diluting the magnetic bead coated with the antigen, which is nephrin, using a diluent to obtain a dilute solution of magnetic microparticles, wherein the concentration of magnetic microparticles in the dilute solution of magnetic microparticles is from about 0.1 to 0.2 mg/ml.
  13. 5. The method of production according to claim 4, wherein the diluent contains from about 0.80 to 1.30 g/L tris(hydroxymethyl)aminomethane, from about 5.00 to 8.00 g/L tris(hydroxymethyl)aminomethane hydrochloride, from about 7.00 to 11.00 g/L sodium chloride, from about 1.00 to 5.00 g/L Triton X-100, from about 0.20 to 0.70 g/L Tween-20 and from about 0.20 to 0.70 g/L ProClin 300; optionally, the diluent contains approximately 1.12 g/L tris(hydroxymethyl)aminomethane, approximately 6.42 g/L tris(hydroxymethyl)aminomethane hydrochloride, approximately 9.00 g/L sodium chloride, approximately 3.00 g/L Triton X-100, approximately 0.50 g/L Tween-20, and approximately 0.50 g/L ProClin 300.
  14. 6. The method of production according to claim 3, wherein when adding the ratio of magnetic bead and antigen which is nephrin comprises adding from about 5 to 20 μg of antigen which is nephrin, optionally adding from about 10 to 20 μg of antigen which is nephrin, to a diluted solution of magnetic beads containing about 1 mg of magnetic beads.
  15. 7. A kit for analysis of an autoantibody to nephrin, comprising a magnetic microparticle according to claim 1 or 2 or a magnetic microparticle obtained by the method according to any of claims 3 to 6.
  16. 8. The kit of claim 7, further comprising a chemiluminescently labeled anti-IgG antibody and a diluent thereof; optionally, the concentration of the chemiluminescently labeled anti-IgG antibody in the diluent thereof is from 0.02 to 0.04 μg/ml.
  17. 9. The kit of claim 8, wherein the chemiluminescent label is selected from an acridinium ester, an acridinium sulfonamide, acridinium toluenesulfonamide, acridinium p-methylsulfonamide, or acridinium trifluoromethanesulfonamide; optionally, the chemiluminescent label is an acridinium ester.
  18. 10. The kit of any one of claims 7 to 9, wherein the chemiluminescently labeled anti-IgG antibody is diluted with a labeled reagent diluent, and wherein the labeled reagent diluent comprises from about 0.30 to 0.70 g/L sodium dihydrogen phosphate, from about 15.00 to 18.00 g/L sodium hydrogen phosphate, from about 35.00 to 45.00 g/L sodium chloride, from about 7.00 to 13.00 g/L bovine serum albumin, from about 3.00 to 7.00 g/L Triton X-405, and from about 0.30 to 0.70 g/L ProClin 300; optionally, the diluent for the labeled reagent contains 0.50 g/L sodium dihydrogen phosphate, 16.75 g/L sodium hydrogen phosphate, approximately 40.00 g/L sodium chloride, approximately 10.00 g/L bovine serum albumin, approximately 5.00 g/L Triton X-405, and approximately 0.50 g/L ProClin 300.
  19. 11. The kit according to any one of paragraphs 7-10, further comprising a sample diluent;
  20. wherein the sample diluent comprises from about 0.50 to 1.50 g/L tris(hydroxymethyl)aminomethane, from about 5.00 to 8.00 g/L tris(hydroxymethyl)aminomethane hydrochloride, from about 7.00 to 11.00 g/L sodium chloride, and from about 17.00 to 23.00 g/L bovine serum albumin;