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CN-121978236-A - Method for detecting target antigen of membranous nephropathy

CN121978236ACN 121978236 ACN121978236 ACN 121978236ACN-121978236-A

Abstract

The invention discloses a method for detecting a target antigen of membranous nephropathy, which comprises the following steps of adding a protein extract into a kidney glomerular tissue to be detected, incubating, adding a mixed enzyme of LysC enzyme and Trypsin enzyme, incubating, adding a dithiothreitol solution, incubating, adding an iodoacetamide solution, incubating at room temperature in a dark place, adding a precipitator, carrying out ultrasonic treatment, centrifuging, sampling a supernatant to a C 18 small column, eluting the small column, collecting an eluent, freezing the eluent, drying, re-dissolving by using the sample solution to obtain a sample solution, and carrying out liquid chromatography tandem mass spectrometry detection on the sample solution. The detection method can accurately detect the target antigen of the membranous nephropathy when the sampling volume is obviously reduced, thereby greatly reducing the sampling difficulty and greatly promoting the application of the laser micro-cutting combined liquid chromatography-mass spectrometry technology in detecting the target antigen of the membranous nephropathy. The method is simple in flow and short in detection time.

Inventors

  • CHEN XIURU
  • SHE XUHUI
  • WANG LIN
  • LIN YANXIU
  • LIANG JIAWEI

Assignees

  • 广州金域医学检验中心有限公司

Dates

Publication Date
20260505
Application Date
20260130

Claims (10)

  1. 1. A method for detecting a target antigen of membranous nephropathy, comprising the steps of: (1) Adding a protein extracting solution into the kidney glomerular tissue to be detected, and incubating at 90-110 ℃ for 40-50 min, wherein the protein extracting solution comprises 10.8 mmol/L-12.0 mmol/L sodium deoxycholate, 18.0 mmol/L-22.0 mmol/L tris and 1.8 mmol/L-2.2 mmol/L ethylenediamine tetraacetic acid; (2) Adding mixed enzyme of LysC enzyme and Trypsin enzyme, and incubating at 36-38 ℃ for 3-8 hours; (3) Adding dithiothreitol solution, and incubating for 20-40 minutes at 36-38 ℃; (4) Adding an iodoacetamide solution, and incubating for 20-40 minutes at room temperature in a dark place; (5) Adding a precipitator, carrying out ultrasonic treatment and centrifugation, taking supernatant, loading the supernatant to a C 18 small column, eluting the small column, and collecting eluent; (6) Freezing the eluent, drying, and re-dissolving with a sample loading solution to obtain a sample solution; (7) And performing liquid chromatography tandem mass spectrometry detection on the sample solution.
  2. 2. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the protein extract in the step (1) comprises 11.0 mmol/L to 12.0 mmol/L sodium deoxycholate, 19.0 mmol/L to 21.0 mmol/L tris and 1.9 mmol/L to 2.1 mmol/L ethylenediamine tetraacetic acid; Preferably, the protein extract comprises 11.5 mmol/L-12.0 mmol/L sodium deoxycholate, 20.0 mmol/L-21.0 mmol/L tris and 2.0 mmol/L-2.1 mmol/L ethylenediamine tetraacetic acid.
  3. 3. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the glomerular tissue to be detected is obtained in step (1) by using a laser microdissection instrument; and/or, the volume of the glomerular tissue in the step (1) is (250000 μm 2 ~300000 μm 2 ) ×7 μm-8 μm; preferably, the volume of glomerular tissue is (280000 μm 2 ~300000 μm 2 ) (7 μm to 8 μm).
  4. 4. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the ratio of the amount of the glomerular tissue to the amount of the protein extract in the step (1) is (250000 μm 2 ~300000 μm 2 ): (7 μm to 8 μm): 50 μl; and/or, the temperature of incubation in the step (1) is 95-105 ℃, and the incubation time is 44-46 min.
  5. 5. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the amount ratio of LysC enzyme to trypsinase in the mixed enzyme in the step (2) is 0.8-1.2:1.
  6. 6. The method according to claim 1, wherein the ratio of the amount of the mixed enzyme in step (2) to the amount of the glomerular tissue in step (1) is 0.02 μg to 0.04 μg (250000 μΜ 2 ~300000 μm 2 ): (7 μΜ to 8 μΜ), preferably 0.025 μg to 0.030 μg (250000 μΜ 2 ~300000 μm 2 ): (7 μΜ to 8 μΜ), and/or the incubation time in step (2) is 3h to 4h.
  7. 7. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the concentration of the dithiothreitol solution in the step (3) is 4 mmol/L to 6 mmol/L; and/or the concentration of the iodoacetamide solution in the step (4) is 14 mmol/L-16 mmol/L.
  8. 8. The method for detecting a target antigen for membranous nephropathy according to claim 1, wherein the precipitant in step (5) is 3.5% -4.5% aqueous trifluoroacetic acid or 3.5% -4.5% aqueous formic acid, preferably 3.8% -4.2% aqueous trifluoroacetic acid; And/or the dosage ratio of the precipitant in the step (5) to the protein extract in the step (1) is 4-6 mu L50 mu L, preferably 4.5-5.5 mu L50 mu L; and/or the solvent used in the step (5) is a mixed solution of acetonitrile, water and trifluoroacetic acid in a volume ratio of 55-65:35-45:0.08-0.12; And/or, the freezing temperature in the step (6) is-82 ℃ to-78 ℃, and the freezing time is 15-25 min; and/or, in the step (6), the sample loading liquid is 0.08% -0.12% trifluoroacetic acid aqueous solution.
  9. 9. The method for detecting a target antigen for membranous nephropathy according to any one of claims 1 to 8, wherein the chromatographic conditions of the liquid chromatography in step (7) comprise: The mobile phase A is 0.08-0.12% formic acid aqueous solution, the mobile phase B is 75-85% acetonitrile solution containing 0.08-0.12% formic acid, and the sample injection amount is 14-16 mu L; The liquid phase gradient program of the sample loading pump is 0 to 5min, the flow rate is 5.000 mu L/min, 5min to 6 min, the flow rate is 5.000 mu L/min to 1.000 mu L/min, 6 min to 45 min, the flow rate is 1.000, 45 min to 46 min, the flow rate is 1.000 mu L/min to 5.000 mu L/min, 46 min to 50min, the flow rate is 5.000 mu L/min, 0 to 50min and 100 percent of mobile phase A; The nano-lift pump liquid phase gradient program comprises 0-5 min,90% of mobile phase A, 5 min-35 min,90% of mobile phase A-55% of mobile phase A, 35-min-40 min,55% of mobile phase A-10% of mobile phase A, 40 min-45 min,10% of mobile phase A, 45-min-46 min,10% of mobile phase A-90% of mobile phase A, 46 min-50 min,90% of mobile phase A, 0-50 min and flow rate of 0.3 mu L/min; and/or, the parameters of the mass spectrum in step (7) comprise: resolution 70000 (MS 1), 17500 (MS 2); MS1 scanning range is 300 m/z-2000 m/z; Collision energy: 30% HCD; isolation window 2.0 m/z (DDA); Maximum injection time 50 MS (MS 1)/200 MS (MS 2).
  10. 10. The method of any one of claims 1-8, wherein the membranous nephropathy target antigen comprises PLA2R, THSD7A, NELL1, SEMA3B, VASN, HTRA1, NTNG1, EXT2, NCAM1, CNTN1, FAT1, NDNF, and/or PCSK6.

Description

Method for detecting target antigen of membranous nephropathy Technical Field The invention belongs to the technical field of disease detection, and particularly relates to a method for detecting a membranous nephropathy target antigen based on a laser micro-cutting combined liquid chromatography-mass spectrometry (LMD-LC/MS). Background Membranous Nephropathy (MN) is a glomerulopathy caused by the deposition of immune complexes along the subepithelial region of the glomerular basement membrane. Identification and typing of target antigens (MNA) of membranous nephropathy are critical for accurate diagnosis of membranous nephropathy, and clear target antigens are core preconditions for understanding pathogenesis and guiding personalized treatment. Currently, the medical community has clarified at least 14 MNAs, and new target antigens are being gradually discovered. Currently, the main (traditional) method for clinically identifying MNAs is the immunohistochemical/immunofluorescence (IHC/IF) technology, two antigens, PLA2R and THSD7A, can be detected by conventional pathological examination, but few detection mechanisms can also detect NEll1, EXT2, sema3B, PCDH antigens, but most antigens still cannot be detected by the traditional method due to limited commercial antibodies. Based on the existing proteomics method, the whole process is usually required to be carried out for 20-24 hours, and is tedious and time-consuming. The development of laser microdissection combined with liquid chromatography-mass spectrometry (LMD-LC/MS) has led to the development of a further shift in the diagnostic depth of the disease from the tissue cell level to the molecular level. However, few reports of laser microdissection combined liquid chromatography-mass spectrometry (LMD-LC/MS) applied to detection of target antigens of membranous nephropathy are available, and the guidance of a standardized method is absent, while in the few reports of detection of target antigens of membranous nephropathy by using the method, the required tissue quantity is large (250000 μm 2*10 μm~550000 μm2 x10 μm). Because kidney is an important solid organ of human body, the kidney tissue sample of children patients is extremely difficult to obtain, and the excessive material amount can increase the kidney function burden and even cause irreversible damage to kidney tissues, so that the higher sample use amount hinders the research progress of detection of target antigens of membranous nephropathy. Disclosure of Invention Based on the above, the invention aims to provide a method for detecting a target antigen of membranous nephropathy based on a laser micro-cutting combined liquid chromatography-mass spectrometry (LMD-LC/MS), which has small required tissue amount and high detection efficiency when used for detecting the target antigen of membranous nephropathy. The technical scheme for realizing the aim of the invention comprises the following steps. The invention provides a method for detecting a target antigen of membranous nephropathy, which comprises the following steps: (1) Adding a protein extracting solution into the kidney glomerular tissue to be detected, and incubating at 90-110 ℃ for 40-50 min, wherein the protein extracting solution comprises 10.8 mmol/L-12.0 mmol/L sodium deoxycholate, 18.0 mmol/L-22.0 mmol/L tris and 1.8 mmol/L-2.2 mmol/L ethylenediamine tetraacetic acid; (2) Adding mixed enzyme of LysC enzyme and Trypsin enzyme, and incubating at 36-38 ℃ for 3-8 hours; (3) Adding dithiothreitol solution, and incubating for 20-40 minutes at 36-38 ℃; (4) Adding an iodoacetamide solution, and incubating for 20-40 minutes at room temperature in a dark place; (5) Adding a precipitator, carrying out ultrasonic treatment and centrifugation, taking supernatant, loading the supernatant to a C 18 small column, eluting the small column, and collecting eluent; (6) Freezing the eluent, drying, and re-dissolving with a sample loading solution to obtain a sample solution; (7) And performing liquid chromatography tandem mass spectrometry detection on the sample solution. The inventor of the invention finds that when the sampling volume of the glomerular tissue to be detected is obviously reduced when the detection method of the target antigen of the membranous nephropathy is explored, the target antigen of the membranous nephropathy can be accurately detected by adopting a proper preparation method of a sample solution and optimized liquid chromatography tandem mass spectrometry conditions, thereby greatly reducing the sampling difficulty and greatly promoting the application of a laser micro-cutting combined liquid chromatography-mass spectrometry (LMD-LC/MS) technology in the detection of the target antigen of the membranous nephropathy. In addition, the detection method has simple flow and short detection time. Drawings FIG. 1 is a chromatogram of a test solution prepared in example 6 of the present invention at an enzyme level of 0.5. Mu.g. FI