WO2016149971A1 - Kit de réactifs pour le dépistage précoce de la néphropathie diabétique, méthode de test de biomarqueurs et applications - Google Patents

Kit de réactifs pour le dépistage précoce de la néphropathie diabétique, méthode de test de biomarqueurs et applications Download PDF

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WO2016149971A1
WO2016149971A1 PCT/CN2015/076923 CN2015076923W WO2016149971A1 WO 2016149971 A1 WO2016149971 A1 WO 2016149971A1 CN 2015076923 W CN2015076923 W CN 2015076923W WO 2016149971 A1 WO2016149971 A1 WO 2016149971A1
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detection
antibody
reagent
detection site
collagen
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张贯京
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E Techno Information Technologies Co Ltd
Bio Tech Academy China Co Ltd
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Bio Tech Academy China Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the invention relates to the field of biological detection, in particular to a test tray for early detection of diabetic nephropathy, a method for detecting biomarkers and an application thereof.
  • Diabetic nephropathy is one of the most serious chronic microvascular complications of diabetes, and eventually causes end-stage renal failure, which is the leading cause of death in diabetic patients.
  • Diabetic nephropathy is one of the most serious chronic microvascular complications of diabetes, and eventually causes end-stage renal failure, which is the leading cause of death in diabetic patients.
  • the World Health Organization's statistics in 2013, there are approximately 347 million people worldwide with diabetes, and more than 80% of deaths from diabetes occur in developing countries.
  • the World Health Organization predicts that by 2030, the number of deaths due to diabetes in the world will double, 30% to 40% of people with type 2 diabetes will develop diabetic nephropathy, and 20% to 40% of patients with type 1 diabetes will be 15 It will also develop into diabetic nephropathy after 30 years, causing a huge and heavy social and economic burden.
  • diabetic nephropathy is difficult to detect early, and the diagnosis of diabetic nephropathy, gold standard creatinine and proteinuria can only indirectly reflect the substantial lesions of the kidney (glomerulus mesangial cell expansion, basement membrane thickening, podocyte injury, renal tubules). Epithelial cells and stromal cell damage, etc., can not diagnose early diabetic nephropathy.
  • renal biopsy can assist in the early diagnosis of diabetic nephropathy, the technique is traumatic, causes complications after surgery, and has high technical difficulty. It cannot be used as a routine item for examination of patients with diabetic nephropathy.
  • the type and content of protein in urine directly reflects the health of the urinary system, especially the kidneys. Status can be used to predict the occurrence, development and prognosis of diabetic nephropathy. At the same time, a large number of studies have found that multiple protein biomarker groups are closely related to the clinical and prognosis of patients with chronic diseases, indicating that simultaneous detection of multiple protein factors can contribute to a more comprehensive understanding of disease progression.
  • An early detection test tray for diabetic nephropathy comprising a detection reagent and a capture reagent;
  • the detection reagent includes two or three of a first detection reagent, a second detection reagent, a third detection reagent, and a fourth detection reagent, and the solute of the first detection reagent is a first podoprotein antibody.
  • the solute of the second detecting reagent is the first type IV collagen antibody
  • the solute of the third detecting reagent is the first hepatic fatty acid binding protein antibody
  • the solute of the fourth detecting reagent is the first neutrophil gelatin.
  • the solute of the capture reagent comprises a second podoside protein antibody that binds a first detectable label component, a second type IV collagen antibody that binds to a second detectable label component, and a third detectable label component Two or three of a second liver type fatty acid binding protein antibody and a second neutrophil gelatinase-associated lipocalin antibody that binds a fourth detectable label component; The detection reagent and the capture reagent are matched to each other.
  • This early detection test strip for diabetic nephropathy can be used as a biomarker by two or three of podophyllin, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin. According to the actual test results, it provides auxiliary judgment for kidney injury, which provides assistance for distinguishing early diabetic nephropathy.
  • FIG. 1 is a flow chart of a method for detecting a biomarker according to an embodiment
  • Figure 2a is a verification diagram of podoside protein in the early urine of diabetic nephropathy, * indicates P ⁇ 0.05
  • Figure 2b is a verification diagram of type IV collagen in the early urine of diabetic nephropathy, * indicates P ⁇ 0.05
  • Figure 2c is a verification diagram of hepatic fatty acid binding protein in the early urine of diabetic nephropathy, * indicates P ⁇ 0.05
  • Figure 2d is a verification diagram of neutrophil gelatinase-associated lipocalin in the early urine of diabetic nephropathy, * indicates P ⁇ 0.05
  • Figure 3a is a ROC curve of podoside protein
  • Figure 3b is a ROC plot of type IV collagen
  • Figure 3c is a ROC plot of hepatic fatty acid binding protein
  • Figure 3d is a ROC plot of neutrophil gelatinase-associated lipocalin
  • Podocalyxin is an O-linked transmembrane glycoprotein located in the apical membrane region of the foot cell of the foot of the foot. It is the main component of the glomerular basement membrane charge barrier, ensuring the integrity of the glomerular filtration membrane charge barrier. It plays an important role in renal filtration function.
  • Type IV collagen (Collagen IV) is an important component of the glomerular basement membrane and mesangial matrix, and plays an important role in the glomerular basement membrane filtration integrity. Hyperglycemia in diabetics produces IVcollagen.
  • L-FABP Hepatic fatty acid binding protein
  • Neutrophil gelatinase-associated lipocalin is an iron-binding protein expressed in renal tubular endothelial cells and involved in various biological functions such as apoptosis, innate immunity, kidney growth, etc. .
  • NGAL neutrophil gelatinase-associated lipocalin
  • NGAL is highly expressed in urine, and the increase of NGAL expression is directly proportional to the injury of proximal tubules in the renal tubules, indicating that NGAL in urine is a biomarker of tubulointerstitial cell injury.
  • podocalyxin and type IV collagen Can be used as a biomarker for glomerular injury
  • liver-type fatty acid binding protein L-FABP
  • neutrophil gelatinase-associated lipocalin NGAL
  • the above four biomarkers are selected for early diagnosis of diabetic nephropathy, and the four biomarkers are detected, and according to the content of the four biomarkers, an auxiliary judgment is provided for the kidney injury, thereby providing assistance for distinguishing early diabetic nephropathy. .
  • An early detection test strip for diabetic nephropathy includes a substrate, a detection reagent, and a capture reagent.
  • the detection reagent includes two or three of the first detection reagent, the second detection reagent, the third detection reagent, and the fourth detection reagent.
  • the solute of the first detecting reagent is the first podoprotein
  • the solute of the second detecting reagent is the first type IV collagen antibody
  • the solute of the third detecting reagent is the first hepatic fatty acid binding protein antibody
  • the fourth detecting reagent The solute is a first neutrophil gelatinase-associated lipocalin antibody.
  • the solute of the capture reagent comprises a second podoside protein antibody that binds to the first detectable label component, a second type IV collagen antibody that binds to the second detectable label component, and a second that binds the third detectable label component Two or three of a liver type fatty acid binding protein antibody and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • the detection reagent and the capture reagent match each other.
  • the meaning that the detection reagent and the capture reagent match each other is specifically: when the detection reagent includes the first detection reagent (or the second detection reagent, or the third detection reagent, or the fourth detection reagent), the solute of the capture reagent includes the combination a second glycoprotein antibody (or a second type IV collagen antibody that binds to a second detectable label component, or a second liver type fatty acid binding protein antibody that binds a third detectable label component) Or a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable label component.
  • the solute of the capture reagent includes the combination a second glycoprotein antibody (or a second type IV collagen antibody that binds to a second detectable label component, or a second liver type fatty acid binding protein antibody that binds a third detectable label component) Or a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable label component.
  • the detection reagent comprises a first detection reagent and a fourth detection reagent
  • the solute of the capture reagent comprises a second podoprotein antibody combined with the first detectable label component and a second medium combined with the fourth detectable label component A neutrophil gelatinase-associated lipocalin antibody.
  • the detecting reagent comprises a second detecting reagent, a third detecting reagent and a fourth detecting reagent, wherein the solute of the capturing reagent comprises a second type IV collagen antibody combined with the second detectable labeling component, and the third detectable label is combined a second hepatic fatty acid binding protein antibody of the composition and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • a detection site is provided on the substrate, and the substrate may be a nitrocellulose membrane, a nylon membrane or a glass substrate.
  • the substrate is selected from a nitrocellulose membrane.
  • the substrate can be omitted, and it can be used in conjunction with a substrate sold by another company when the test strip for early detection of diabetic nephropathy is used.
  • the detection site includes a first detection site, a second detection site, a third detection site, and a fourth detection site, where the first detection site corresponds to the first detection reagent, and the second detection site corresponds to The second detecting reagent, the third detecting site corresponds to the third detecting reagent, and the fourth detecting site corresponds to the fourth detecting reagent.
  • the first detection site, the second detection site, the third detection site, and the fourth detection site are all four, and the error is avoided by repeating the test.
  • This early detection test box for diabetic nephropathy can provide an auxiliary judgment for kidney damage based on actual test results, thereby providing assistance for distinguishing early diabetic nephropathy.
  • Diabetic nephropathy early test kit also includes candied glycoprotein standard, type IV collagen standard, liver type fatty acid binding protein standard and neutrophil gelatinase related lipocalin standard, these four standards All purchased from Beijing Ao Rui Dongyuan Biotechnology Co., Ltd.
  • the first positive control site, the second positive control site, the third positive control site and the fourth positive control site are also required on the substrate, and the foot glycoprotein standard product corresponds to the first positive control site.
  • the type IV collagen standard corresponds to the second positive control site
  • the liver type fatty acid binding protein standard corresponds to the third positive control site
  • the neutrophil gelatinase-associated lipocalin standard corresponds to the fourth positive control site.
  • the first detectable label component can be an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, or a radioactive substance.
  • the second detectable labeling component can be an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, or a radioactive substance.
  • the third detectable labeling component can be an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, or a radioactive substance.
  • the fourth detectable labeling component can be an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, or a radioactive substance.
  • the first detectable label component, the second detectable label component, the third detectable label component, and the fourth detectable label component are all horseradish peroxidase (HRP).
  • the test strip for early detection of diabetic nephropathy further includes a test solution.
  • the solute of the test solution is TMB (3,3',5,5'-tetramethylbenzidine).
  • the first podoprotein antibody (purchased from Sigma, product number: AMAB90667) is a monoclonal antibody
  • the second podoside protein antibody (purchased from Sigma, Cat. No. SAB2500809) is a polyclonal antibody
  • the first type IV collagen antibody (purchased from Sigma, product number: C1926) is a monoclonal antibody
  • the second type IV collagen antibody (purchased from Abcam, product number: ab6586) is a polyclonal antibody.
  • the first hepatic fatty acid-binding protein antibody (purchased from Sigma, Inc., product number: WH0002167M1) is a monoclonal antibody
  • the second hepatic fatty acid-binding protein antibody (Abcam, Inc.: ab101837) is a polyclonal antibody
  • the first neutrophil gelatinase-associated lipocalin antibody (purchased from Abcam, Cat. No.: ab23477) is a monoclonal antibody
  • the second neutrophil gelatinase-associated lipocalin antibody (Abeam) Company, shipment No.: ab166677) is a polyclonal antibody.
  • This early detection test box for diabetic nephropathy uses the glycoprotein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarkers, which can be based on actual test results.
  • Injury provides an adjunct to provide assistance in distinguishing early diabetic nephropathy.
  • the method for detecting a biomarker according to an embodiment shown in FIG. 1 adopts the above-mentioned early test strip for diabetic nephropathy, comprising the following steps:
  • the detection reagent includes two or three of the first detection reagent, the second detection reagent, the third detection reagent, and the fourth detection reagent.
  • the solute of the first detecting reagent is the first podoprotein
  • the solute of the second detecting reagent is the first type IV collagen antibody
  • the solute of the third detecting reagent is the first hepatic fatty acid binding protein antibody
  • the fourth detecting reagent The solute is a first neutrophil gelatinase-associated lipocalin antibody.
  • the solute of the capture reagent comprises a second podoside protein antibody that binds to the first detectable label component, a second type IV collagen antibody that binds to the second detectable label component, and a second that binds the third detectable label component Two or three of a liver type fatty acid binding protein antibody and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • the detection reagent and the capture reagent match each other.
  • the meaning that the detection reagent and the capture reagent match each other is specifically: when the detection reagent includes the first detection reagent (or the second detection reagent, or the third detection reagent, or the fourth detection reagent), the solute of the capture reagent includes the combination a second glycoprotein antibody (or a second type IV collagen antibody that binds to a second detectable label component, or a second liver type fatty acid binding protein antibody that binds a third detectable label component) Or a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable label component.
  • the solute of the capture reagent includes the combination a second glycoprotein antibody (or a second type IV collagen antibody that binds to a second detectable label component, or a second liver type fatty acid binding protein antibody that binds a third detectable label component) Or a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable label component.
  • the detection reagent comprises a first detection reagent and a fourth detection reagent
  • the solute of the capture reagent comprises a second podoprotein antibody combined with the first detectable label component and a second medium combined with the fourth detectable label component A neutrophil gelatinase-associated lipocalin antibody.
  • the detecting reagent comprises a second detecting reagent, a third detecting reagent and a fourth detecting reagent, wherein the solute of the capturing reagent comprises a second type IV collagen antibody combined with the second detectable labeling component, and the third detectable label is combined a second hepatic fatty acid binding protein antibody of the composition and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • the detection site includes a first detection site, a second detection site, a third detection site, and a fourth detection site, where the first detection site corresponds to the first detection reagent, and the second detection site corresponds to The second detecting reagent, the third detecting site corresponds to the third detecting reagent, and the fourth detecting site corresponds to the fourth detecting reagent.
  • the detection reagent includes a second detection reagent, a third detection reagent and a fourth detection reagent, and the solute of the capture reagent comprises a second type IV collagen antibody combined with a second detectable label component, and a third detectable label component
  • the second liver type fatty acid binding protein antibody and the second neutrophil gelatinase-associated lipocalin antibody which binds the fourth detectable label component are as an example, and S10 is specifically: adding the second detection reagent to the second detection a third detection reagent is added to the third detection site, and the fourth detection reagent is added to the fourth detection site, and the first detection site, the second detection site, and the third are respectively fully reacted.
  • the detection site and the fourth detection site are washed clean.
  • the detection reagent includes a second detection reagent, a third detection reagent and a fourth detection reagent, and the solute of the capture reagent comprises a second type IV collagen antibody combined with a second detectable label component, and a third detectable label component
  • the second liver type fatty acid binding protein antibody and the second neutrophil gelatinase-associated lipocalin antibody which binds the fourth detectable labeling component are exemplified, and the S20 is specifically: the second detection site after washing respectively The third detection site and the fourth detection site are closed, and the second detection site, the third detection site, and the fourth detection site are respectively washed after the closure is completed.
  • the detection reagent includes a second detection reagent, a third detection reagent and a fourth detection reagent, and the solute of the capture reagent comprises a second type IV collagen antibody combined with a second detectable label component, and a third detectable label component
  • the second liver type fatty acid binding protein antibody and the second neutrophil gelatinase-associated lipocalin antibody which binds the fourth detectable labeling component are as an example, and S30 is specifically: the sample to be detected is separately added to the closed and The second detection site, the third detection site, and the fourth detection site are washed cleanly, and the second detection site, the third detection site, and the fourth detection site are respectively washed after being fully reacted.
  • the sample to be tested selects a urine sample.
  • urine samples need to be diluted before being tested, and the dilution factor can be 100 times.
  • the capture reagent is added dropwise to the detection site, and the detection site is washed clean after sufficient reaction.
  • the detection reagent includes a second detection reagent, a third detection reagent and a fourth detection reagent, and the solute of the capture reagent comprises a second type IV collagen antibody combined with a second detectable label component, and a third detectable label component
  • the second liver type fatty acid binding protein antibody and the second neutrophil gelatinase-associated lipocalin antibody which binds the fourth detectable labeling component are as an example, and the S40 is specifically: the capture reagent is separately added to the second detection position.
  • the third detection site and the fourth detection site, the second detection site, the third detection site and the fourth detection site are respectively washed clean after sufficient reaction.
  • the detection site contains a first detectable label component, a second detectable label component, a third detectable label component, and a fourth detectable label component, and according to the detection result, the podoside protein, IV is obtained from the sample to be detected.
  • the detection reagent includes a second detection reagent, a third detection reagent and a fourth detection reagent, and the solute of the capture reagent comprises a second type IV collagen antibody combined with a second detectable label component, and a third detectable label component
  • the second liver type fatty acid binding protein antibody and the second neutrophil gelatinase-associated lipocalin antibody which binds the fourth detectable label component are as an example, and the S50 is specifically: detecting the second detection site and the third detection, respectively.
  • the second detectable marker component, the third detectable marker component and the fourth detectable marker component are contained in the site and the fourth detection site, and determining whether the sample to be detected contains type IV collagen or liver type according to the detection result Fatty acid binding protein and neutrophil gelatinase-associated lipocalin.
  • This method of detecting kidney damage can provide an auxiliary judgment for kidney damage, thereby providing assistance for distinguishing early diabetic nephropathy.
  • the cutoff value can be set first, and then the content of the podophyllotoxin, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin in the obtained sample to be tested can be cut off and truncated. Values are compared.
  • the concentration cutoff of Podocalyxin is 67.15 ng/g.
  • the concentration cutoff of Cr, Collagen IV is 2.945 ⁇ g/g Cr
  • the cutoff value of L-FABP is 4.75 ⁇ g/g Cr
  • the cutoff value of NGAL is 56.8 ⁇ g/g. Cr.
  • the biomarker detection method can also be used to evaluate the effectiveness of the method for treating kidney injury, specifically: the sample to be tested includes the pre-treatment sample and the post-treatment sample; the pre-treatment sample will be obtained from the foot glycoprotein, type IV collagen. Protein, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin are associated with podoside protein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase in the treated samples. The levels of lipocalin were compared and the effectiveness of the treatment was evaluated based on the alignment results.
  • the specific evaluation process for the effectiveness of the above treatment methods is: if the concentration of the biomarker in the sample after treatment is decreased or remains unchanged compared to the pre-treatment sample, it indicates that the treatment is effective; if it is found to be elevated, and has statistics Significant meaning, it means invalid.
  • the biomarker detection method using the above-mentioned early detection test strip for diabetic nephropathy uses a glycoprotein, type IV collagen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarkers. According to the actual test results, it can provide auxiliary judgment for kidney injury, thus providing assistance for distinguishing early diabetic nephropathy.
  • the inventors of the present application have found that podocalyxin and type IV collagen (Collagen) have been combined with literature reports and research experiments. IV), hepatic fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) are closely related to the development of early stage disease of diabetic nephropathy, and the use of protein chip detection found in patients with diabetic nephropathy The levels of these four markers in the urine were significantly higher than the normal controls (p ⁇ 0.05) and increased with the progression of the disease.
  • L-FABP hepatic fatty acid binding protein
  • NGAL neutrophil gelatinase-associated lipocalin
  • Foot glycoprotein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin can also be used as biomarkers in the preparation of renal injury diagnostic reagents or renal injury diagnostic devices.
  • Simultaneous detection of type IV collagen (Collagen IV), liver-type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) have the following advantages: 1) From the perspective of renal structural function, early damage of diabetic nephropathy may occur in renal small The ball may also occur in the renal tubules.
  • the three biomarkers used in this kit contain both glomerular damage-specific biomarkers (type IV collagen) and tubular specific markers of liver damage (hepatic fatty acid binding protein).
  • diabetic kidney injury helps to avoid missed diagnosis, early detection and early diagnosis of diabetic kidney injury; 2) from the molecular pathway of diabetic nephropathy It is noted that the mechanism of diabetic nephropathy injury involves multiple molecular pathways such as renal fibrosis (type IV collagen), inflammatory response (neutrophil gelatinase-associated lipocalin), oxidative stress (hepatic fatty acid binding protein) Etc.
  • renal fibrosis type IV collagen
  • inflammatory response neutrophil gelatinase-associated lipocalin
  • oxidative stress hepatic fatty acid binding protein
  • Predicting disease from multiple molecular pathways helps early detection of diabetes; therefore, simultaneous detection of type IV collagen, liver type fat Binding protein and neutrophil gelatinase-associated lipocalin three biomarkers, and it is determined by selecting a reasonable method, greatly enhance the sensitivity and specificity of detecting renal injury.
  • Example 1 Screening and verification of biomarkers for early diagnosis of diabetic nephropathy
  • pathological screening A total of 205 subjects with known physical condition were selected, including 50 healthy controls and 155 patients with type 2 diabetes (all of whom had diabetes for more than 2 years); and 155 diabetic patients according to proteinuria Divided, including 61 patients with normal proteinuria, 52 patients with microalbuminuria, and 42 patients with massive proteinuria.
  • MDRD The GRF formula estimates glomerular filtration rate (eGFR). Note: This study has been approved by an ethics review agency and all subjects have signed informed consent.
  • iTRAQ Quantitative Proteomics Using ProteoMinerTM Protein Enrichment Kit (Bio-Rad, Cat. No.: 163-3006) High abundance protein was removed, total urine protein was extracted by cold acetone/trichloroacetic acid method, and protein concentration was determined by Bradford method.
  • ELISA method to verify differentially expressed protein The ELISA technique was used to confirm the expression of differential proteins (full glycoprotein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin) in urine of normal controls and disease groups. The aim was to verify the accuracy of the results of mass spectrometry and the accuracy of these differential proteins as early biomarkers for diabetic nephropathy. Specifically, the urine was diluted 1:100 with a dilution buffer, and then the content of biomarkers in urine of different disease groups was measured using an ELISA plate for different protein biomarkers, and each sample was repeatedly measured three times.
  • differential proteins full glycoprotein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin
  • Diabetic nephropathy is a complex complication that can lead to tubular damage or glomerular damage in the early stages of diabetic nephropathy.
  • the four biomarkers in the present application wherein podophyllotoxin and type IV collagen mainly reflect glomerular damage; hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin mainly reflect renal tubular damage.
  • two or more biomarkers are used in this experiment, and the judgment methods of (1) and (2) are used for comprehensive judgment.
  • biomarker combinations lead to different diagnostic sensitivity and specificity. Therefore, the best combination of biomarkers can be selected to diagnose early diabetic nephropathy.
  • ND No Difference
  • *p ⁇ 0.001 vs. healthy control #p ⁇ 0.001 Vs patients with normal proteinuria
  • ⁇ p ⁇ 0.001 vs patients with microalbuminuria HbA1C for glycated hemoglobin
  • Serum Cr for serum creatinine
  • UACR urinary albumin to creatinine ratio.
  • the differentially expressed proteins screened by the urine proteomics experiment were verified by ELISA: podoside protein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin, and the following Table 3 was obtained.
  • ND No Difference
  • *p ⁇ 0.001 vs. healthy control ⁇ p ⁇ 0.05 Vs healthy controls
  • #p ⁇ 0.001 vs normal proteinuria ⁇ p ⁇ 0.001 vs microalbuminuria.
  • Sensitivity and specificity analysis showed that when the highest "youden" index was selected, the sensitivity, specificity and concentration cutoff values of the four biomarkers in urine were: podocalyxin (sensitivity 89.8%; specificity 76.2%; concentration) The cutoff value is 67.15ng/g Creatinine), Collagen IV (sensitivity 71.2%; specificity 90.5%; concentration cutoff value 2.945 ⁇ g/g Creatinine), L-FABP (sensitivity 79.6%; specificity 95.2%; concentration cutoff value 4.75 ⁇ g/g) Creatinine) and NGAL (sensitivity 84.7%; specificity 85.8%; concentration cutoff value 49.85 ng/mL). These results indicate that these four protein molecules may be involved in the pathogenesis of diabetic nephropathy and are believable biomarkers of early diabetic nephropathy.
  • the relative content of the four protein markers can be determined by reference to the normal range by the results of the healthy control and the cutoff values of the four protein molecules, and the results of the four biomarkers can be combined with any of the proteins.
  • concentration exceeds the normal range, it can be initially diagnosed as early diabetic nephropathy; combined with GFR, proteinuria and other indicators for comprehensive assessment of early diabetic nephropathy.
  • the sensitivity and specificity of early diabetic nephropathy were diagnosed by combining four biomarkers in urine.
  • the NGAL measurement is corrected by Cr, and the cutoff value is analyzed by the ROC curve.
  • Urine NGAL concentration cutoff is 56.8 ⁇ g/g Cr; the cutoff value of L-FABP in urine is 4.75 ⁇ g/g Cr; the cutoff value of Collagen IV in urine is 2.945 ⁇ g/g Cr; the cutoff value of podocalyxin concentration in urine was 67.15 ng/g Cr.
  • the various combinations were statistically processed to compare the sensitivity and specificity of the different combinations, and the following Table 6 was obtained.
  • the sensitivity and specificity also vary depending on the type and amount of the combined biomarker. This means that a combination of biomarkers can be selected according to the purpose of the experiment, and can be used for disease diagnosis efficiently and specifically.
  • High sensitivity combination IV Collagen ⁇ NGAL; IV Collagen ⁇ Podocalyxin; NGAL ⁇ Podocalyxin; L-FABP ⁇ Podocalyxin; IV Collagen ⁇ NGAL ⁇ L-FABP
  • high specificity combination IV Collagen ⁇ NGAL; IV Collagen ⁇ Podocalyxin; NGAL ⁇ Podocalyxin; L-FABP ⁇ Podocalyxin; IV Collagen ⁇ NGAL ⁇ L-FABP
  • IV Collagen ⁇ NGAL ⁇ L-FABP is suitable for secondary inspection or tertiary inspection, etc., and requires more reliable judgment.
  • the initial examination result is secondarily determined by a combination method with high specificity, and finally an effective and reliable diagnosis result of early diabetic nephropathy is obtained.
  • NGAL and Podocalyxin "IV Collagen, NGAL and L-FABP"
  • these two combinations can obtain higher sensitivity and specificity by selecting appropriate judgment methods.

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  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne un kit de réactifs comprenant des réactifs d'essai pour le dépistage précoce de la néphropathie diabétique. Les réactifs d'essai comprennent soit deux, soit trois réactifs parmi un premier réactif d'essai, un deuxième réactif d'essai, un troisième réactif d'essai, et un quatrième réactif d'essai. Un soluté pour le premier réactif d'essai est un premier anticorps anti-podocalyxine. Un soluté pour le deuxième réactif d'essai est un premier anticorps anti-collagène IV. Un soluté pour le troisième réactif d'essai est un premier anticorps anti-protéine se liant aux acides gras hépatiques. Un soluté pour le quatrième réactif d'essai est un premier anticorps anti-lipocaline associée à la gélatinase des polynucléaires neutrophiles. En utilisant soit deux, soit trois composés parmi la podocalyxine, le collagène IV, la protéine se liant aux acides gras hépatiques, et la lipocaline associée à la gélatinase des polynucléaires neutrophiles comme biomarqueurs, le kit de réactifs pour le dépistage précoce de la néphropathie diabétique peut contribuer à déterminer des lésions rénales sur la base d'un résultat d'essai réel, contribuant ainsi à distinguer la néphropathie diabétique à un stade précoce.
PCT/CN2015/076923 2015-03-24 2015-04-18 Kit de réactifs pour le dépistage précoce de la néphropathie diabétique, méthode de test de biomarqueurs et applications Ceased WO2016149971A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112946303A (zh) * 2021-02-23 2021-06-11 江苏省中医院 Tag54:2-fa18:1及其组合物在糖尿病、糖尿病肾病诊断方面的应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104764885B (zh) * 2015-03-24 2016-08-24 深圳市贝沃德克生物技术研究院有限公司 糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用
CN105092844A (zh) * 2015-07-10 2015-11-25 深圳市贝沃德克生物技术研究院有限公司 胰腺癌蛋白生物标记物的检测试剂盒及检测系统
CN105092845A (zh) * 2015-07-10 2015-11-25 深圳市贝沃德克生物技术研究院有限公司 胰腺癌蛋白生物标记物、用途及其检测芯片和检测装置
CN205317786U (zh) * 2016-01-16 2016-06-15 深圳市易特科信息技术有限公司 用于早期糖尿病肾病检测的金标抗体试纸条
CN108334747B (zh) * 2017-01-20 2020-09-04 北京松果天目健康管理有限公司 获取肿瘤尿蛋白标志物的方法及得到的肿瘤相关离群尿蛋白库

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010143423A1 (fr) * 2009-06-10 2010-12-16 国立大学法人 新潟大学 Méthode de détection d'une néphropathie diabétique
WO2013183596A1 (fr) * 2012-06-06 2013-12-12 国立大学法人名古屋大学 Biomarqueur pour les maladies rénales et son utilisation
CN104764885A (zh) * 2015-03-24 2015-07-08 深圳市贝沃德克生物技术研究院有限公司 糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用
CN204479594U (zh) * 2015-03-24 2015-07-15 深圳市易特科信息技术有限公司 基于生物标志物的糖尿病肾病早期检测试剂盒
CN204479593U (zh) * 2015-03-24 2015-07-15 深圳市易特科信息技术有限公司 基于生物标志物的肾损伤检测试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010143423A1 (fr) * 2009-06-10 2010-12-16 国立大学法人 新潟大学 Méthode de détection d'une néphropathie diabétique
WO2013183596A1 (fr) * 2012-06-06 2013-12-12 国立大学法人名古屋大学 Biomarqueur pour les maladies rénales et son utilisation
CN104764885A (zh) * 2015-03-24 2015-07-08 深圳市贝沃德克生物技术研究院有限公司 糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用
CN204479594U (zh) * 2015-03-24 2015-07-15 深圳市易特科信息技术有限公司 基于生物标志物的糖尿病肾病早期检测试剂盒
CN204479593U (zh) * 2015-03-24 2015-07-15 深圳市易特科信息技术有限公司 基于生物标志物的肾损伤检测试剂盒

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MEHTA, S. ET AL.: "Urinary Biomarkers of Diabetic Nephropathy", CURRENT BIOMARKER FINDINGS, vol. 3, 22 November 2013 (2013-11-22), pages 67 - 78 *
QIAN, XIAOLING ET AL.: "The Predictive Value of Combined Detecting Urine PCX, Nephrin and MCP-1 for Diabetic Nephropathy in Newly Diagnosed T2DM Patients", PROCEEDING OF CLINICAL MEDICINE, vol. 21, no. 12, 31 December 2012 (2012-12-31), pages 933 - 938 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112946303A (zh) * 2021-02-23 2021-06-11 江苏省中医院 Tag54:2-fa18:1及其组合物在糖尿病、糖尿病肾病诊断方面的应用
CN112946303B (zh) * 2021-02-23 2023-10-20 江苏省中医院 Tag54:2-fa18:1及其组合物在糖尿病、糖尿病肾病诊断方面的应用

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