WO2016149972A1 - 糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用 - Google Patents

糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用 Download PDF

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WO2016149972A1
WO2016149972A1 PCT/CN2015/076924 CN2015076924W WO2016149972A1 WO 2016149972 A1 WO2016149972 A1 WO 2016149972A1 CN 2015076924 W CN2015076924 W CN 2015076924W WO 2016149972 A1 WO2016149972 A1 WO 2016149972A1
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detection site
antibody
detection
reagent
collagen
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French (fr)
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张贯京
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Shenzhen Qianhai AnyCheck Information Technology Co Ltd
E Techno Information Technologies Co Ltd
Bio Tech Academy China Co Ltd
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Shenzhen Qianhai AnyCheck Information Technology Co Ltd
E Techno Information Technologies Co Ltd
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
    • 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 an early screening test kit for 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.
  • patients with diabetic nephropathy miss the best treatment opportunity, causing the disease to deteriorate rapidly and irreversibly. Therefore, it is of great practical significance to actively search for early diagnosis markers of diabetic nephropathy and its detection kits, and to conduct early intervention.
  • Protein biomarkers are a measurable indicator of the presence and state of disease. In the process of disease, stress and rehabilitation, any physiological changes can finally be reflected at the protein level, which is the most intuitive means of disease research.
  • the detection of current protein molecular markers has become the most effective method for early diagnosis, disease typing, drug target screening and drug development.
  • Urine is the metabolic end product produced by blood filtration through the glomerulus, reabsorption, excretion and secretion of renal tubules and collecting tubes.
  • 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.
  • 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 screening kit for diabetic nephropathy comprising a detection reagent and a capture reagent;
  • the detecting reagent includes a first detecting reagent, a second detecting reagent, a third detecting reagent and a fourth detecting reagent, wherein the solute of the first detecting reagent is a first podoprotein, and the solute of the second detecting reagent Is a first type IV collagen antibody, the solute of the third detection reagent is a first liver type fatty acid binding protein antibody, and the solute of the fourth detection reagent is a first neutrophil gelatinase-associated lipocalin antibody ;
  • 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 A second liver type fatty acid binding protein antibody and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • podophyllotoxin type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarkers in the preparation of a renal injury diagnostic reagent or a renal injury diagnostic device.
  • This early screening kit 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.
  • 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 graph of podoside protein
  • Figure 3b is a ROC graph of type IV collagen
  • Figure 3c is a ROC graph 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 screening test kit for diabetic nephropathy comprising a substrate, a detection reagent, and a capture reagent.
  • the detection reagent includes a first detection reagent, a second detection reagent, a third detection reagent, and a 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 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 A hepatic fatty acid binding protein antibody and a second neutrophil gelatinase-associated lipocalin antibody that binds to a fourth detectable labeling component.
  • 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 when used in the early screening test kit for diabetic kidney disease, it can be used with a substrate sold by another company.
  • 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 screening kit for diabetic nephropathy can provide an auxiliary judgment for kidney damage based on actual test results, thereby providing assistance for distinguishing early diabetic nephropathy.
  • the Early Diabetes Nephrology Screening Kit also includes candied glycoprotein standards, type IV collagen standards, hepatic fatty acid binding protein standards, and neutrophil gelatinase-associated lipocalin standards, these four standards. All purchased from Beijing Ao Rui Dongyuan Biotechnology Co., Ltd. Correspondingly, 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 diabetic nephropathy early screening kit further includes a detection 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 screening kit 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 biomarker detecting method of an embodiment shown in FIG. 1 adopts the above-mentioned diabetic nephropathy early screening kit, and includes the following steps:
  • the detection reagent includes a first detection reagent, a second detection reagent, a third detection reagent, and a 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 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 A hepatic fatty acid binding protein antibody 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.
  • S10 is specifically: adding the first detection reagent to the first detection site, adding the second detection reagent to the second detection site, adding the third detection reagent to the third detection site, and performing the fourth detection
  • the reagent is added to the fourth detection site, and the first detection site, the second detection site, the third detection site, and the fourth detection site are washed separately after sufficient reaction.
  • the S20 is specifically: the first detection site, the second detection site, the third detection site, and the fourth detection site after the washing are respectively closed, and the first detection site and the second detection bit are respectively respectively after the closure is completed.
  • the spot, the third detection site, and the fourth detection site are washed clean.
  • S30 is specifically: the sample to be detected is respectively dropped on the first detection site, the second detection site, the third detection site, and the fourth detection site which are closed and washed, and fully reacted and then respectively The detection site, the second detection site, the third detection site, and the fourth detection site are washed clean.
  • 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 S40 is specifically: the capture reagent is separately added to the first detection site, the second detection site, the third detection site, and the fourth detection site, and the first detection site and the second detection are respectively fully reacted.
  • the site, the third detection site, and the fourth detection site are washed clean.
  • 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.
  • S50 is specifically: detecting whether the first detection site, the second detection site, the third detection site, and the fourth detection site respectively include the first detectable marker component, the second detectable marker component, and the third detectable
  • the component and the fourth detectable label component are labeled, and based on the detection result, it is determined whether the sample to be detected contains podophyllotoxin, type IV collagen, hepatic 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 diabetic nephropathy early screening kit, as a biomarker by podoside protein, type IV collagen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin 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
  • Simultaneous detection of these four biomarkers has the following advantages: 1) From the perspective of renal structural function, early damage of diabetic nephropathy may occur in glomeruli, and may also occur in renal tubules.
  • the four biomarkers selected in this kit are Contains glomerular damage-specific biomarkers (foot glycoprotein, type IV collagen), and also contains tubular specific markers of liver damage (hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin), Simultaneous detection of these four biomarkers can help to avoid missed diagnosis and early detection and early diagnosis of diabetic kidney injury. 2) From the molecular pathway of diabetic nephropathy, the mechanism of diabetic nephropathy involves multiple molecular pathways, such as the kidney.
  • Fibrosis type IV collagen
  • inflammatory response neutral gelatinase-associated lipocalin
  • oxidative stress hepatic fatty acid binding protein
  • predicting disease from multiple molecular pathways contributing to early diabetes Found; therefore, simultaneous detection of podoside protein, type IV collagen, hepatic fatty acid binding protein and neutrophil
  • the four biomarkers of lipase-associated lipocalin and the sensitivity and specificity of renal function damage detection are greatly improved by selecting a reasonable judgment method.
  • podoside protein, type IV collagen, hepatic fatty acid binding protein and neutrophil gelatinase-associated lipocalin can also be used as biomarkers in the preparation of a kidney injury diagnostic reagent or a kidney injury diagnostic device.
  • Example 1 Screening and verification of biomarkers for early diagnosis of diabetic nephropathy
  • 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 were classified according to proteinuria. There were 61 patients with normal proteinuria, 52 patients with microalbuminuria, and 42 patients with massive proteinuria. MDRD The GRF formula estimates glomerular filtration rate (eGFR).
  • 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.
  • 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.
  • 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 normal proteinuria
  • HbA1C is glycated hemoglobin
  • Serum Cr refers to serum creatinine
  • UACR refers to the ratio of urinary albumin to creatinine.
  • 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).
  • 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 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 ⁇ Podocalyxin
  • a combination with high specificity IV Collagen ⁇ NGAL; IV Collagen ⁇ Podocalyxin; NGAL ⁇ Podocalyxin; L-FABP ⁇ Podocalyxin; IV Collagen ⁇ NGAL ⁇ L-FABP ⁇ Podocalyxin
  • IV Collagen ⁇ NGAL ⁇ L-FABP ⁇ Podocalyxin 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.
  • Table 6 "IV The combination of Collagen, NGAL, L-FABP and Podocalyxin can achieve the highest sensitivity and specificity by selecting the appropriate determination method.

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Abstract

一种糖尿病肾病早期筛查试剂盒，包括检测试剂；所述检测试剂包括第一检测试剂、第二检测试剂、第三检测试剂和第四检测试剂；所述第一检测试剂的溶质为第一足糖萼蛋白抗体，所述第二检测试剂的溶质为第一IV型胶原蛋白抗体，所述第三检测试剂的溶质为第一肝型脂肪酸结合蛋白抗体，所述第四检测试剂的溶质为第一中性粒细胞明胶酶相关脂质运载蛋白抗体。这种糖尿病肾病早期筛查试剂盒通过足糖萼蛋白、IV型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物，可以根据实际检测结果，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。

Description

糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用
技术领域
本发明涉及生物检测领域，尤其是涉及一种糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用。
背景技术
糖尿病肾病是糖尿病最严重的慢性微血管并发症之一，并最终引起终末肾衰竭，是糖尿病患者死亡的主要原因。据世界卫生组织2013年统计，全世界约有3.47亿人患有糖尿病，其中超过80%的糖尿病死亡病例发生在发展中国家。同时，世界卫生组织预测到2030年，全球因糖尿病死亡的人数将翻一番，其中30%~40%的2型糖尿病人将发展为糖尿病肾病，20%~40%的1型糖尿病患者在15~30年后也将发展为糖尿病肾病，造成了巨大且沉重的社会经济负担。
然而，糖尿病肾病早期很难发现，且临床糖尿病肾病诊断黄金标准肌酐和蛋白尿也只能间接反映肾脏实质性病变（肾小球系膜细胞扩增、基底膜加厚、足细胞损伤、肾小管上皮细胞及间质细胞损伤等），无法诊断早期糖尿病肾病。
肾穿刺活检技术虽可以辅助临床糖尿病肾病早期诊断，但该技术的创伤性较大，术后会引起并发症，且技术难度较高，不能作为糖尿病肾病患者检查的常规项目。通常当临床确诊时，糖尿病肾病患者错过了最佳的治疗时机，致使疾病急剧恶化，不可逆转。因此，积极寻找糖尿病肾病早期诊断标志物及其检测试剂盒，并进行早期干预具有重要的现实意义。
蛋白生物标志物(biomarker)是反映疾病存在和状态的一种可测量的指示物。在疾病、应激和康复的过程中，任何的生理变化最终都能在蛋白水平上得到体现，是疾病研究最直观的手段。当前蛋白分子标志物的检测已成为临床疾病早期诊断、疾病分型、药物靶标筛选及药物研发等最有效的方法。尿液是血液在经过肾小球过滤，肾小管及集合管重吸收、排泄及分泌而产生的代谢终产物，尿液中的蛋白种类及含量的高低直接反映了泌尿系统，尤其是肾脏的健康状态，可以用于预测糖尿病肾病发生、发展及预后的情况。同时，大量的研究发现，多个蛋白生物标志物组群与慢性病患者的临床及预后密切相关，表明同步联合测定多个蛋白因子有助于更全面的了解疾病进程。
发明内容
基于此，有必要提供一种可以用于区分早期糖尿病肾病的糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用。
一种糖尿病肾病早期筛查试剂盒，包括检测试剂和捕获试剂；
所述检测试剂包括第一检测试剂、第二检测试剂、第三检测试剂和第四检测试剂，所述第一检测试剂的溶质为第一足糖萼蛋白抗体，所述第二检测试剂的溶质为第一Ⅳ型胶原蛋白抗体，所述第三检测试剂的溶质为第一肝型脂肪酸结合蛋白抗体，所述第四检测试剂的溶质为第一中性粒细胞明胶酶相关脂质运载蛋白抗体；
所述捕获试剂的溶质包括结合了第一可检测标记成分的第二足糖萼蛋白抗体、结合了第二可检测标记成分的第二Ⅳ型胶原蛋白抗体、结合了第三可检测标记成分的第二肝型脂肪酸结合蛋白抗体和结合了第四可检测标记成分的第二中性粒细胞明胶酶相关脂质运载蛋白抗体。
足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物在制备肾损伤诊断试剂或肾损伤诊断装置中的应用。
这种糖尿病肾病早期筛查试剂盒通过足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物，可以根据实际检测结果，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
附图说明
图1为一实施方式的生物标志物检测方法的流程图；
图2a为足糖萼蛋白在糖尿病肾病早期尿液中的验证图，*表示P<0.05；
图2b为Ⅳ型胶原蛋白在糖尿病肾病早期尿液中的验证图，*表示P<0.05；
图2c为肝型脂肪酸结合蛋白在糖尿病肾病早期尿液中的验证图，*表示P<0.05；
图2d为中性粒细胞明胶酶相关脂质运载蛋白在糖尿病肾病早期尿液中的验证图，*表示P<0.05；
图3a为足糖萼蛋白的ROC曲线图；
图3b为Ⅳ型胶原蛋白的ROC曲线图；
图3c为肝型脂肪酸结合蛋白的ROC曲线图；
图3d为中性粒细胞明胶酶相关脂质运载蛋白的ROC曲线图。
具体实施方式
下面主要结合附图及具体实施例对糖尿病肾病早期筛查试剂盒、生物标志物检测方法及应用作进一步详细的说明。
足糖萼蛋白（podocalyxin）是O连接的跨膜糖蛋白，位于肾脏足细胞足突顶膜区，是肾小球基底膜电荷屏障的主要成分，保证肾小球滤过膜电荷屏障的完整性，在肾脏滤过功能中发挥着重要作用。 Ⅳ型胶原蛋白（Collagen Ⅳ）是肾小球基底膜及系膜基质的重要组称成分，在肾小球基底膜滤过完整性中发挥着重要作用。糖尿病人高血糖刺激产生Ⅳcollagen，若增加的Ⅳcollagen沉积到糖尿病人肾脏肾小球系膜基质上，会产生弥散性肾小球硬化症；若沉积到肾小球基底膜或肾小管上，会导致系膜扩增、肾小球间质化和肾小管损伤。 肝型脂肪酸结合蛋白(L-FABP)在肾近端小管细胞内表达，参与肾小管间质细胞能量产生及代谢过程。在糖尿病肾病并发过程中，尿液中L-FABP表达水平与尿白蛋白浓度成正相关，预示着尿液中L-FABP是肾小管间质细胞损伤的生物标志物。 中性粒细胞明胶酶相关脂质运载蛋白(NGAL)是一种铁离子结合蛋白，在肾小管内皮细胞中表达，参与多种生物学功能，如细胞凋亡、天然免疫、肾脏生长发育等。在糖尿病肾病早期，NGAL在尿液中出现高表达，且NGAL表达量的增加与肾小管近端小管的损伤呈正比，预示着尿液中NGAL是肾小管间质细胞损伤的生物标志物。 因此，足糖萼蛋白（podocalyxin）和Ⅳ型胶原蛋白（Collagen Ⅳ）可以作为肾小球损伤的生物标志物，肝型脂肪酸结合蛋白(L-FABP)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)可以作为肾小管损伤的生物标志物。
本申请中选择上述4种生物标志物用于糖尿病肾病早期诊断，检测这4种生物标志物，根据这4种生物标志物的含量，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
一实施方式的糖尿病肾病早期筛查试剂盒，包括基板、检测试剂和捕获试剂。 检测试剂包括第一检测试剂、第二检测试剂、第三检测试剂和第四检测试剂。
第一检测试剂的溶质为第一足糖萼蛋白抗体，第二检测试剂的溶质为第一Ⅳ型胶原蛋白抗体，第三检测试剂的溶质为第一肝型脂肪酸结合蛋白抗体，第四检测试剂的溶质为第一中性粒细胞明胶酶相关脂质运载蛋白抗体。
捕获试剂的溶质包括结合了第一可检测标记成分的第二足糖萼蛋白抗体、结合了第二可检测标记成分的第二Ⅳ型胶原蛋白抗体、结合了第三可检测标记成分的第二肝型脂肪酸结合蛋白抗体和结合了第四可检测标记成分的第二中性粒细胞明胶酶相关脂质运载蛋白抗体。
优选的，检测试剂包括第二检测试剂、第三检测试剂和第四检测试剂，捕获试剂的溶质包括结合了第二可检测标记成分的第二Ⅳ型胶原蛋白抗体、结合了第三可检测标记成分的第二肝型脂肪酸结合蛋白抗体和结合了第四可检测标记成分的第二中性粒细胞明胶酶相关脂质运载蛋白抗体。
基板上设有检测位点，基板可以为硝酸纤维素膜、尼龙膜或玻璃基片。本实施方式中，基板选择硝酸纤维素膜。基板可以省略，在上述糖尿病肾病早期筛查试剂盒使用时，搭配其他公司售卖的基板即可。
本实施方式中，检测位点包括第一检测位点、第二检测位点、第三检测位点和第四检测位点，第一检测位点对应第一检测试剂，第二检测位点对应第二检测试剂，第三检测位点对应第三检测试剂，第四检测位点对应第四检测试剂。
本实施方式中，第一检测位点、第二检测位点、第三检测位点和第四检测位点均为4个，通过重复试验避免误差。
这种糖尿病肾病早期筛查试剂盒可以根据实际检测结果，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
糖尿病肾病早期筛查试剂盒还包括足糖萼蛋白标准品、Ⅳ型胶原蛋白标准品、肝型脂肪酸结合蛋白标准品和中性粒细胞明胶酶相关脂质运载蛋白标准品，这四种标准品均购自北京傲锐东源生物科技有限公司。对应的，基板上还需要设有第一阳性对照位点、第二阳性对照位点、第三阳性对照位点和第四阳性对照位点，足糖萼蛋白标准品对应第一阳性对照位点，Ⅳ型胶原蛋白标准品对应第二阳性对照位点，肝型脂肪酸结合蛋白标准品对应第三阳性对照位点，中性粒细胞明胶酶相关脂质运载蛋白标准品对应第四阳性对照位点。
第一可检测标记成分可以为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质。第二可检测标记成分可以为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质。第三可检测标记成分可以为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质。第四可检测标记成分可以为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质。
本实施方式中，第一可检测标记成分、第二可检测标记成分、第三可检测标记成分和第四可检测标记成分均为辣根过氧化物酶（HRP）。
本实施方式中，糖尿病肾病早期筛查试剂盒还包括检测液。检测液的溶质为TMB（3,3',5,5'-四甲基联苯胺）。
本实施方式中，第一足糖萼蛋白抗体（购自Sigma公司，货号：AMAB90667）为单克隆抗体，第二足糖萼蛋白抗体（购自Sigma公司，货号：SAB2500809）为多克隆抗体。
本实施方式中，第一Ⅳ型胶原蛋白抗体（购自Sigma公司，货号：C1926）为单克隆抗体，第二Ⅳ型胶原蛋白抗体(购自Abcam公司，货号：ab6586)为多克隆抗体。
本实施方式中，第一肝型脂肪酸结合蛋白抗体（购自Sigma公司，货号：WH0002167M1）为单克隆抗体，第二肝型脂肪酸结合蛋白抗体(Abcam公司，货号：ab101837)为多克隆抗体。
本实施方式中，第一中性粒细胞明胶酶相关脂质运载蛋白抗体（购自Abcam公司，货号：ab23477）为单克隆抗体，第二中性粒细胞明胶酶相关脂质运载蛋白抗体（Abcam公司，货号：ab166677）为多克隆抗体。
这种糖尿病肾病早期筛查试剂盒通过足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物，可以根据实际检测结果，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
如图1所示的一实施方式的生物标志物检测方法，采用上述的糖尿病肾病早期筛查试剂盒，包括如下步骤：
S10、提供设有检测位点的基板，将检测试剂滴加在检测位点上，充分反应后将检测位点洗涤干净。
检测试剂包括第一检测试剂、第二检测试剂、第三检测试剂和第四检测试剂。
第一检测试剂的溶质为第一足糖萼蛋白抗体，第二检测试剂的溶质为第一Ⅳ型胶原蛋白抗体，第三检测试剂的溶质为第一肝型脂肪酸结合蛋白抗体，第四检测试剂的溶质为第一中性粒细胞明胶酶相关脂质运载蛋白抗体。
捕获试剂的溶质包括结合了第一可检测标记成分的第二足糖萼蛋白抗体、结合了第二可检测标记成分的第二Ⅳ型胶原蛋白抗体、结合了第三可检测标记成分的第二肝型脂肪酸结合蛋白抗体和结合了第四可检测标记成分的第二中性粒细胞明胶酶相关脂质运载蛋白抗体。
本实施方式中，检测位点包括第一检测位点、第二检测位点、第三检测位点和第四检测位点，第一检测位点对应第一检测试剂，第二检测位点对应第二检测试剂，第三检测位点对应第三检测试剂，第四检测位点对应第四检测试剂。
S10具体为：将第一检测试剂滴加在第一检测位点，将第二检测试剂滴加在第二检测位点，将第三检测试剂滴加在第三检测位点，将第四检测试剂滴加在第四检测位点，充分反应后分别将第一检测位点、第二检测位点、第三检测位点和第四检测位点洗涤干净。
洗涤的操作可以采用传统PBS稀释液，具体可以为0.01mol/L PBS（pH=7.4）+0.05%吐温-20。
S20、将洗涤干净后的检测位点封闭，封闭完成后将检测位点洗涤干净。
S20具体为：分别将洗涤干净后的第一检测位点、第二检测位点、第三检测位点和第四检测位点封闭，封闭完成后分别将第一检测位点、第二检测位点、第三检测位点和第四检测位点洗涤干净。
封闭液可以将脱脂奶粉或牛血清白蛋白溶于PBS（pH=7.4，0.01mol/L）得到。
S30、将待检测样本滴加在封闭完成并洗涤干净的检测位点上，充分反应后将检测位点洗涤干净。
S30具体为：将待检测样本分别滴加在封闭完成并洗涤干净的第一检测位点、第二检测位点、第三检测位点和第四检测位点上，充分反应后分别将第一检测位点、第二检测位点、第三检测位点和第四检测位点洗涤干净。
本实施方式中，待检测样本选择尿液样本。
一般来说，尿液样本在进行检测前，需要进行稀释，稀释倍数可以为100倍。
S40、将捕获试剂滴加在检测位点上，充分反应后将检测位点洗涤干净。
S40具体为：将捕获试剂分别滴加在第一检测位点、第二检测位点、第三检测位点和第四检测位点上，充分反应后分别将第一检测位点、第二检测位点、第三检测位点和第四检测位点洗涤干净。
S50、检测位点是否含有第一可检测标记成分、第二可检测标记成分、第三可检测标记成分和第四可检测标记成分，并且根据检测结果得到待检测样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量。
S50具体为：分别检测第一检测位点、第二检测位点、第三检测位点和第四检测位点上是否含有第一可检测标记成分、第二可检测标记成分、第三可检测标记成分和第四可检测标记成分，并且根据检测结果确定待检测样本中是否含有足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白。
这种肾损伤检测方法可以为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
具体的，可以先设置截断值，接着可以将得到的待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量与截断值进行对比。
（1）当待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量均在截断值以上时判定为阳性，此外则判定为阴性。这种判定方法适用于对初步筛查结果的进一步确认。
（2）当待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量至少有一个在截断值以上时判定为阳性，此外则判定为阴性。这种判定方法适用于大人群样本的初步筛查。
上述二种判定方法各有优点，可以单独使用，也可以结合使用。
在一个优选的实施例中，用血肌酐（creatinine，Cr）校正后，Podocalyxin的浓度截断值为67.15ng/g Cr，Collagen Ⅳ的浓度截断值为2.945μg/g Cr，L-FABP的浓度截断值为4.75μg/g Cr，NGAL的浓度截断值为56.8μg/g Cr。
这种生物标志物检测方法还可以用于评估肾损伤治疗方法的有效性，具体为：待检测样本包括治疗前样本和治疗后样本；将得到的治疗前样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量与治疗后样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量进行对比，根据比对结果评估治疗方法的有效性。
上述治疗方法的有效性的具体评估过程为：如果治疗后样本中的生物标志物的浓度与治疗前样本相比发现下降或者维持不变，则表明治疗有效；如果发现升高，且具有统计学显著意义，则表明无效。
这种采用上述的糖尿病肾病早期筛查试剂盒的生物标志物检测方法，通过足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物，可以根据实际检测结果，为肾损伤提供辅助判断，从而为区分早期糖尿病肾病提供辅助。
本申请发明人结合文献报道及研究试验，发现足糖萼蛋白（podocalyxin）、Ⅳ型胶原蛋白（Collagen Ⅳ）、肝型脂肪酸结合蛋白(L-FABP)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)与糖尿病肾病早期疾病的发生发展密切相关，同时利用蛋白芯片检测发现，在糖尿病肾病患者尿液中这4种标志物的含量显著高于正常对照（p<0.05），且随着疾病的发展而增加。
同时检测这4种生物标志物，利用其联合变化对糖尿病肾病进行预测，可灵敏、特异的筛查早期糖尿病肾病，拥有较为广阔的市场应用前景。
同时检测这4种生物标志物具有以下优点：1）从肾脏结构功能角度看，糖尿病肾病早期损伤可能发生于肾小球，亦可能发生于肾小管，本试剂盒选用的四种生物标志物既包含肾小球损伤特异生物标志物（足糖萼蛋白、Ⅳ型胶原蛋白），又包含肾小管损伤特异标志物（肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白），因此利用这4种生物标志物组合同时检测，有助于避免漏诊，实现糖尿病肾损伤早发现、早诊断；2）从糖尿病肾病分子通路上看，糖尿病肾病损伤机制涉及到多个分子通路，如肾脏纤维化（Ⅳ型胶原蛋白）、炎症反应（中性粒细胞明胶酶相关脂质运载蛋白）、氧化应激（肝型脂肪酸结合蛋白）等，从多个分子通路预测疾病，有助于糖尿病早发现；因此，同时检测足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白这4种生物标志物，并且通过选择合理的判定方法，大大提高了肾功能损伤检测的灵敏度和特异度。
此外，足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白还可以作为生物标志物在制备肾损伤诊断试剂或肾损伤诊断装置中得到应用。
以下为具体实施例，实施例中出现的各种仪器和试剂如果没有特别说明，均采用本领域常规仪器或试剂。
下面结合具体实施例对本申请的4种生物标志物的筛选、分析和应用做进一步的详细说明。
实施例1：糖尿病肾病早期诊断生物标志物的筛选及验证
一、实验方法
1、病理筛选
选取已知身体状况的受试者共205例，其中健康对照50例，2型糖尿病患者155例（其中所有患者患糖尿病年限均大于2年）；并按照蛋白尿情况对155例糖尿病患者进行划分，其中正常蛋白尿患者61例，微量蛋白尿患者52例，大量蛋白尿患者42例。MDRD GRF公式估算肾小球滤过率（eGFR）。
注：本研究已被伦理审核机构批准，所有受试者均签署知情同意书。
2、尿标本的收集和处理
用无菌容器收集受试者清晨第一次排出的尿液样本（30mL），4℃ 8000g离心30min，取上清，储存于-80℃用于蛋白组学实验。所有尿液标本均在未使用任何药物治疗前收集。在进行蛋白组学实验时，将同一分期来自不同患者的尿液样本混合，用于蛋白组学研究。
3. iTRAQ定量蛋白组学技术
利用ProteoMinerTM蛋白富集试剂盒（Bio-Rad, 货号: 163-3006）去除高丰度蛋白，冷丙酮/三氯乙酸法提取尿液总蛋白，Bradford法测定蛋白浓度。按照iTRAQ标记试剂盒（Applied Biosystem Inc. 货号: MS10016）操作步骤处理、标记不同组尿蛋白样本。操作步骤简述如下：取各组尿蛋白样本各100μg，每管分别加入预冷的丙酮（丙酮：样本体积比=6：1），-20℃沉淀1小时，12000rpm超速离心15min后将沉淀重悬于20μL溶解缓冲液中。还原、封闭尿蛋白中的半胱氨酸，胰酶消化，然后利用iTRAQ标记试剂114、115、116、117分别标记正常蛋白尿患者、微量蛋白尿患者、大量蛋白尿患者和正常对照的尿蛋白。等量混合标记后的尿蛋白肽，依次利用强离子交换层析、反向层析进行分离，质谱技术进行分析（SCX-RPLC-MS/MS），Mascot软件鉴定尿液中的蛋白，寻找疾病组和对照以及疾病组之间差异表达蛋白。
4、ELISA法验证差异表达蛋白
利用ELISA技术确认寻找到的差异蛋白（足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白）在正常对照和各疾病组尿液中的表达量，旨在验证质谱技术结果的准确性及这些差异蛋白作为糖尿病肾病早期生物标志物（Biomarker）的准确性。具体地说，利用稀释缓冲液将尿液1：100稀释，然后利用针对不同蛋白生物标志物的ELISA板测量不同疾病组尿液中生物标志物的含量，每个样本重复测量三次。
5、统计分析
数据用SPSS 14.0软件（Statistical Product and Service Solutions，“统计产品与服务解决方案”软件）处理，以平均值±标准差表示。用方差分析(Analysis of Variance，ANOVA)进行正态分布数据的组间比较；用受试者工作特性曲线（ROC）分析各种生物标志物预测的准确性、特异性及灵敏性。P<0.05表示具有显著统计学差异。
6、4种生物标志物综合检测早期糖尿病肾病
糖尿病肾病是一种复杂并发症，在糖尿病肾病初期可能会导致肾小管损伤，或者导致肾小球损伤。本申请中的4种生物标志物，其中足糖萼蛋白和Ⅳ型胶原蛋白主要反映肾小球损伤；肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白主要反映肾小管损伤。为准确的诊断早期糖尿病肾病，本实验组合使用二种或二种以上生物标志物，采用（1）和（2）的判定方法进行综合判定。
（1）组合中所含的所有生物标志物均为阳性（截断值以上）时判定为阳性，除此以外判定为阴性。这种判定方法适用于对初步筛查结果的进一步确认。
（2）组合中所含的生物标志物有一个为阳性（截断值以上）时即判断为阳性，除此以外判断为阴性。这种判定方法适用于大人群样本的初步筛查。
本研究发现，生物标志物组合的种类和数量的差异，会导致疾病诊断灵敏度和特异度不同。因此可以选择最佳的生物标志物组合来诊断早期糖尿病肾病。
二、结果
1、受试者基本信息如下表2所示。
表2：质谱实验受试者身体状况及生化指标
　 健康对照(n=50) 正常蛋白尿患者(n=61) 微量蛋白尿患者(n=52) 大量蛋白尿患者(n=42) P 值
性别（男 / 女） 30/20 27/34 23/29 22/20 ND
年龄 58±10.2 63±12 62±6.9 60±14 ND
患病时间 - 4.5±3.6 7.5±6.2 10±7.7 0.012
血糖 4.3±2.12 9.41±3.21 8.81±2.56 7.87±2.84 <0.001
HbA1C (%) 4.2±0.44 7.6±1.6 7.2±1.4 7.4±2.5 <0.001
收缩压（ mmHg ） 118±12 128±10 125±15 131±22 0.017
舒张压（ mmHg ） 72±10 76±8 80±12 81±9 0.101
Serum Cr (mg/dL) 0.9±0.3 0.9±0.4 1.1±0.9 2.1±1.8* <0.001
MDRD_eGFR 86±9.5 84.2±22.3 76±25.8*# 45.9±21.6*#§ <0.001
UACR (mg/g) 7.2 ±2.1 13.1±7.5* 93.1 ±22.5*# 650.3±125*#§ <0.001
注：ND: No Difference; *p<0.001 vs. 健康对照；#p<0.001 vs 正常蛋白尿患者；§p<0.001 vs 微量蛋白尿患者，HbA1C为糖化血红蛋白，Serum Cr指血肌酐; UACR指尿白蛋白与肌酐比值。
2、尿液蛋白组学结果描述
前期筛选阶段，蛋白组学实验2次重复分别鉴定到465和478个蛋白，在二次实验中共同鉴定到322个蛋白。在二次实验中114/117（正常蛋白尿组/正常对照）、115/117(微量蛋白尿组/正常对照)及116/117（大量蛋白尿组/正常对照）iTRAQ比值均显示出差异表达的蛋白共62个（ratio<0.667或ratio>1.5）。因本发明旨在寻找糖尿病肾病早期（正常蛋白尿期或Ⅰ和Ⅱ期）生物标志物，对发生差异表达的62个蛋白进行进一步分析，结果发现有15个蛋白在糖尿病肾病组的不同分期发生差异表达（ratio<0.667或ratio>1.5）。
通过文献报道和生物信息学分析（Gene Ontology和KEGG Pathway分析）对15个鉴定到的差异表达蛋白进行进一步分析，发现足糖萼蛋白（podocalyxin）、Ⅳ型胶原蛋白（Collagen Ⅳ）、肝型脂肪酸结合蛋白(L-FABP)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)在糖尿病引起的肾病并发症病发过程中发挥着非常重要的作用。
3、ELISA法验证差异表达蛋白
利用ELISA法验证尿液蛋白组学实验筛选出的差异表达蛋白：足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白，得到下表3。
表3：ELISA验证4种糖尿病肾病早期标志物
　 健康对照 (n=55) 正常蛋白尿患者 (n=40) 微量蛋白尿患者 (n=53) 大量蛋白尿患者 (n=35) P 值
性别（男 / 女） 35/20 18/22 29/24 17/18 ND
年龄 58±11 60±12 62±5 63±11 ND
HbA1C (%) 4.1±0.6 7.3±1.1 7.5±1.2 7.0±2.2 <0.001
MDRD_eGFR 86±9 82.2±20.3 72±25.8★# 42±22.5*#§ 0.021
UACR (mg/g) 6.1 ±2.5 23.1±7.2* 212.1 ±25.5*# 850.3±125*#§ <0.001
Podocalyxin (μg/g creatinine) 34.3±10.8 100±32.8* 129.4±52.2* 251.7±103.5*#§ <0.001
Collagen Ⅳ (μg/g creatinine) 1.63±0.8 2.23±1.22★ 6.18±2.75*# 15.4±6.2*#§ <0.001
L-FABP ( μ g/g creatinine) 1.6±0.9 4.8±2.3* 8.6±3.5*# 64±24.8*#§ <0.001
NGAL (ng/mL) 6.6±2.1 55.6±22.1* 105.8±30.5*# 258.6±85.9*#§ <0.001
注：ND: No Difference; *p<0.001 vs. 健康对照；★p<0.05 vs 健康对照；#p<0.001 vs 正常蛋白尿患者；§p<0.001 vs 微量蛋白尿。
由表3和图2a~图2d可以看出，糖尿病组尿液中足糖萼蛋白（podocalyxin）、Ⅳ型胶原蛋白（Collagen Ⅳ）、肝型脂肪酸结合蛋白(L-FABP)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的表达量显著高于健康对照，且随着患者病情的严重（蛋白尿增加）而增加。
对肾小球滤过率、蛋白尿与4种蛋白标志物的相关性分析，得到下表4。
表4：肾小球滤过率、蛋白尿与4种蛋白标志物的相关性分析
　 肾小球滤过率（ eGFR ） 尿白蛋白肌酐比率（ UACR ）
Podocalyxin R=-0.984* R= 0.958*
Collagen R= -0.996# R= 0.963*
L-FABP R= -0.989* R= 0.998#
NGAL R= -0.984* R= 0.976*
由表4可以看出，上述4种蛋白的表达量和尿蛋白呈显著正相关，与eGFR呈显著负相关。 利用受试者工作特性曲线（ROC）分析糖尿病肾病患者和健康对照尿液中的四种生物标志物，得到图3a~图3d。 由图3a~图3d可以得到下表5。
表5：四种生物标志物ROC曲线分析
测试结果变量 曲线下面积 渐近显著性水平 渐近 95% 置信区间 下限 渐近 95% 置信区间 上限
Collagen 0.858 0.005 0.777 0.938
NGAL 0.901 0.001 0.825 0.976
LFABP 0.907 0.003 0.837 0.976
PDOX 0.892 0.003 0.816 0.968
由表5可以看出，podocalyxin、Collagen Ⅳ、L-FABP和NGAL曲线下面积（AUC）分别为0.892（p<0.01）、0.858（p<0.01）、0.907（p<0.01）和0.901（p<0.01）。敏感性和特异性分析显示，当选取最高“youden”指数时，尿液中四种生物标志物的灵敏度、特异度和浓度截断值分别为：podocalyxin（灵敏度89.8%；特异度为76.2%；浓度截断值为67.15ng/g creatinine）、Collagen Ⅳ（灵敏度71.2%；特异度为90.5%；浓度截断值为2.945 μg/g creatinine）、L-FABP（灵敏度79.6%；特异度为95.2%；浓度截断值为4.75 μg/g creatinine）和NGAL（灵敏度84.7%；特异度为85.8%；浓度截断值为49.85ng/mL）。
以上结果表明这4种蛋白分子可能参与了糖尿病肾病病发过程，为可信的早期糖尿病肾病生物标志物。
4、糖尿病肾病早期诊断方法
在进行糖尿病诊断时，可通过健康对照的测定结果及四种蛋白分子浓度截断值来确定这4种蛋白标志物相对含量参考正常范围，结合四种生物标志物测量结果，若其中任何一种蛋白浓度超出正常范围则可初步诊断为早期糖尿病肾病；同时结合GFR、蛋白尿等指标对早期糖尿病肾病进行综合评估。
5．多种生物标志物组合进行糖尿病肾病早期诊断
将尿中四种生物标志物进行组合来诊断早期糖尿病肾病的灵敏度和特异度。其中将NGAL测量值用Cr校正，通过ROC曲线分析其截断值。尿中NGAL浓度截断值为56.8μg/g Cr；尿中L-FABP浓度截断值为4.75 μg/g Cr；尿中Collagen Ⅳ浓度截断值为2.945 μg/g Cr；尿中podocalyxin浓度截断值为67.15ng/g Cr。
对各种组合进行统计处理，比较不同组合的灵敏度和特异度，得到下表6。其中，在符号“∩”表示的组合中，该组合所含的所有生物标志物均为阳性（截断值以上）时即判断为阳性，计算灵敏度和特异度。在符号“∪”表示的组合中，该组合所含的生物标志物有一个为阳性（截断值以上）时即判断为阳性，计算灵敏度和特异度。
表6：用于诊断早期糖尿病肾病蛋白生物标志物组合灵敏度和特异性
组合 灵敏度 特异度
IV Collagen ∪ NGAL 0.933 0.665
IV Collagen ∩ NGAL 0.55 0.942
IV Collagen ∪ Podocalyxin 0.92 0.594
IV Collagen ∩ Podocalyxin 0.547 0.983
NGAL ∪ Podocalyxin 0.968 0.615
NGAL ∩ Podocalyxin 0.527 0.941
L-FABP ∪ Podocalyxin 0.923 0.505
L-FABP ∩ Podocalyxin 0.541 0.969
IV Collagen ∪ NGAL∪ L-FABP ∪Podocalyxin 0.989 0.475
IV Collagen ∩ NGAL∩ L-FABP ∩Podocalyxin 0.536 0.983
由表6可以看出，根据组合生物标志物种类和数量的变化，灵敏度和特异度也会发生变化。这表示根据实验目的选择生物标志物的组合，能高效、特异的用于疾病诊断。
灵敏度高的组合（IV Collagen ∪ NGAL；IV Collagen ∪ Podocalyxin；NGAL ∪ Podocalyxin； L-FABP ∪ Podocalyxin；IV Collagen ∪ NGAL∪ L-FABP ∪Podocalyxin）适合于初次筛查；相反，特异度高的组合（IV Collagen ∩ NGAL；IV Collagen ∩ Podocalyxin；NGAL ∩ Podocalyxin；L-FABP ∩ Podocalyxin；IV Collagen ∩ NGAL∩ L-FABP ∩Podocalyxin）适合二次检查或三次检查等需要进行可靠性更高的判断检查。例如，若在初次筛查时出现阳性，此时通过特异度较高的组合方法对初次检查结果进行二次判定，最终给出有效且可靠的早期糖尿病肾病诊断结果。 由表6可以看出，“IV Collagen、NGAL、L-FABP和Podocalyxin”这个组合，可以通过选择合适的判定方法，获得最高的灵敏度和特异度。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (13)

  1. 一种糖尿病肾病早期筛查试剂盒，其特征在于，包括检测试剂和捕获试剂；
    所述检测试剂包括第一检测试剂、第二检测试剂、第三检测试剂和第四检测试剂，所述第一检测试剂的溶质为第一足糖萼蛋白抗体，所述第二检测试剂的溶质为第一Ⅳ型胶原蛋白抗体，所述第三检测试剂的溶质为第一肝型脂肪酸结合蛋白抗体，所述第四检测试剂的溶质为第一中性粒细胞明胶酶相关脂质运载蛋白抗体；
    所述捕获试剂的溶质包括结合了第一可检测标记成分的第二足糖萼蛋白抗体、结合了第二可检测标记成分的第二Ⅳ型胶原蛋白抗体、结合了第三可检测标记成分的第二肝型脂肪酸结合蛋白抗体和结合了第四可检测标记成分的第二中性粒细胞明胶酶相关脂质运载蛋白抗体。
  2. 如权利要求1所述的糖尿病肾病早期筛查试剂盒，其特征在于，所述糖尿病肾病早期筛查试剂盒还包括足糖萼蛋白标准品、Ⅳ型胶原蛋白标准品、肝型脂肪酸结合蛋白标准品和中性粒细胞明胶酶相关脂质运载蛋白标准品。
  3. 如权利要求1所述的糖尿病肾病早期筛查试剂盒，其特征在于，所述第一足糖萼蛋白抗体为单克隆抗体，所述第二足糖萼蛋白抗体为多单克隆抗体； 所述第一Ⅳ型胶原蛋白抗体为单克隆抗体，所述第二Ⅳ型胶原蛋白抗体为多克隆抗体； 所述第一肝型脂肪酸结合蛋白抗体为单克隆抗体，所述第二肝型脂肪酸结合蛋白抗体为多克隆抗体； 所述第一中性粒细胞明胶酶相关脂质运载蛋白抗体为单克隆抗体，所述第二中性粒细胞明胶酶相关脂质运载蛋白抗体为多克隆抗体。
  4. 如权利要求1所述的糖尿病肾病早期筛查试剂盒，其特征在于，所述第一可检测标记成分为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质； 所述第二可检测标记成分为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质； 所述第三可检测标记成分为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质； 所述第四可检测标记成分为酶、辅基、荧光物质、发光物质、生物发光物质或放射活性物质。
  5. 一种生物标志物检测方法，采用如权利要求1所述的糖尿病肾病早期筛查试剂盒，其特征在于，包括如下步骤：
    提供设有检测位点的基板，将检测试剂滴加在检测位点上，充分反应后将所述检测位点洗涤干净； 将洗涤干净后的所述检测位点封闭，封闭完成后将所述检测位点洗涤干净； 将待检测样本滴加在封闭完成并洗涤干净的所述检测位点上，充分反应后将所述检测位点洗涤干净； 将捕获试剂滴加在所述检测位点上，充分反应后将所述检测位点洗涤干净；以及 检测所述检测位点是否含有所述第一可检测标记成分、所述第二可检测标记成分、所述第三可检测标记成分和所述第四可检测标记成分，并且根据检测结果得到所述待检测样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量。
  6. 一种生物标志物检测方法，采用如权利要求2所述的糖尿病肾病早期筛查试剂盒，其特征在于，包括如下步骤：
    提供设有检测位点的基板，将检测试剂滴加在检测位点上，充分反应后将所述检测位点洗涤干净； 将洗涤干净后的所述检测位点封闭，封闭完成后将所述检测位点洗涤干净； 将待检测样本滴加在封闭完成并洗涤干净的所述检测位点上，充分反应后将所述检测位点洗涤干净； 将捕获试剂滴加在所述检测位点上，充分反应后将所述检测位点洗涤干净；以及 检测所述检测位点是否含有所述第一可检测标记成分、所述第二可检测标记成分、所述第三可检测标记成分和所述第四可检测标记成分，并且根据检测结果得到所述待检测样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量。
  7. 一种生物标志物检测方法，采用如权利要求3所述的糖尿病肾病早期筛查试剂盒，其特征在于，包括如下步骤：
    提供设有检测位点的基板，将检测试剂滴加在检测位点上，充分反应后将所述检测位点洗涤干净； 将洗涤干净后的所述检测位点封闭，封闭完成后将所述检测位点洗涤干净； 将待检测样本滴加在封闭完成并洗涤干净的所述检测位点上，充分反应后将所述检测位点洗涤干净； 将捕获试剂滴加在所述检测位点上，充分反应后将所述检测位点洗涤干净；以及 检测所述检测位点是否含有所述第一可检测标记成分、所述第二可检测标记成分、所述第三可检测标记成分和所述第四可检测标记成分，并且根据检测结果得到所述待检测样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量。
  8. 一种生物标志物检测方法，采用如权利要求4所述的糖尿病肾病早期筛查试剂盒，其特征在于，包括如下步骤：
    提供设有检测位点的基板，将检测试剂滴加在检测位点上，充分反应后将所述检测位点洗涤干净； 将洗涤干净后的所述检测位点封闭，封闭完成后将所述检测位点洗涤干净； 将待检测样本滴加在封闭完成并洗涤干净的所述检测位点上，充分反应后将所述检测位点洗涤干净； 将捕获试剂滴加在所述检测位点上，充分反应后将所述检测位点洗涤干净；以及 检测所述检测位点是否含有所述第一可检测标记成分、所述第二可检测标记成分、所述第三可检测标记成分和所述第四可检测标记成分，并且根据检测结果得到所述待检测样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量。
  9. 如权利要求5所述的生物标志物检测方法，其特征在于，还包括如下步骤：将得到的所述待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量与截断值进行对比，当所述待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量均在截断值以上时判定为阳性，此外则判定为阴性。
  10. 如权利要求5所述的生物标志物检测方法，其特征在于，还包括如下步骤：将得到的所述待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量与截断值进行对比，当所述待检测样本中的足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量至少有一个在截断值以上时判定为阳性，此外则判定为阴性。
  11. 如权利要求5所述的生物标志物检测方法，其特征在于，所述待检测样本包括治疗前样本和治疗后样本；
    将得到的治疗前样本中足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量与治疗后样本中的足糖萼蛋白抗体、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量进行对比，根据比对结果评估治疗方法的有效性。
  12. 如权利要求5所述的生物标志物检测方法，其特征在于，所述待检测样本为尿液样本。
  13. 足糖萼蛋白、Ⅳ型胶原蛋白、肝型脂肪酸结合蛋白和中性粒细胞明胶酶相关脂质运载蛋白作为生物标志物在制备肾损伤诊断试剂或肾损伤诊断装置中的应用。
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