CN104764885A - An early-stage screening kit for diabetic nephropathy, a biomarker detecting method and applications - Google Patents
An early-stage screening kit for diabetic nephropathy, a biomarker detecting method and applications Download PDFInfo
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- 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
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- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G—PHYSICS
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- 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
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Abstract
An early-stage screening kit for diabetic nephropathy is disclosed. The kit comprises detection agents. The detection agents comprise a first detection agent, a second detection agent, a third detection agent and a fourth detection agent. The solute of the first detection agent is a first podocalyxin antibody. The solute of the second detection agent is a first IV type collagen antibody. The solute of the third detection agent is a first liver-type fatty acid-binding protein antibody. The solute of the fourth detection agent is a first neutrophils gelatinase-associated lipocalin antibody. The kit adopts podocalyxin, IV type collagen, liver-type fatty acid-binding protein and neutrophils gelatinase-associated lipocalin as biomarkers, and can provide assisted determination for renal injury according to actual detection results, thus providing assistance for differentiating early-stage diabetic nephropathy.
Description
Technical field
The present invention relates to field of biological detection, especially relate to a kind of diabetic nephropathy early screening kit, biological marker object detecting method and application.
Background technology
Diabetic nephropathy is one of chronic microvascular complication that diabetes are the most serious, and finally causes last kidney failure eventually, is diabetic's main causes of death.According to World Health Organization's statistics in 2013, the whole world about has 3.47 hundred million people to suffer from diabetes, and the Diabetes Death case wherein more than 80% occurs in developing country.Simultaneously, the World Health Organization (WHO) predicts the year two thousand thirty, the whole world will be doubled because of the number of Diabetes Death, wherein the diabetes B people of 30% ~ 40% will develop into diabetic nephropathy, the type 1 diabetes patient of 20% ~ 40% also will develop into diabetic nephropathy after 15 ~ 30 years, cause huge and heavy social economical burden.
But, diabetic nephropathy is difficult to find in early days, and clinical diabetes diagnosis of nephropathy golden standard creatinine and albuminuria also can only reflect that nephrolithotomy venereal disease becomes (mesangial cell amplification, basilar memebrane thickening, Podocytes in Renal Tissue, renal cells and interstitial cell damage etc.) indirectly, early diabetic nephropathy (normal protein urine phase, see the following form 1) cannot be diagnosed.
Table 1: diabetic nephropathy clinical stages (based on Mogensen allotment method)
Though renal needle biopsy technology can adjuvant clinical diabetogenous nephrosis disease early diagnosis, this technology traumatic comparatively large, postoperative meeting leads to complications, and technical difficulty is higher, the conventional project that can not check as Diabetic Nephropathy patients.Usually, when clinical definite, Diabetic Nephropathy patients misses best therapic opportunity, causes disease sharply to worsen, irreversible.Therefore, actively find diabetic nephropathy early diagnosis marker and detection kit thereof, and carry out early intervention and have important practical significance.
Protein biomarker (biomarker) is the measurable indicant of one of the existence of reflection disease and state.Disease, stress with in the process of rehabilitation, any physiological change finally can be embodied on protein level, is disease research means the most intuitively.The detection of current protein molecular mark has become the most effective method such as clinical disease early diagnosis, disease somatotype, drug targets screening and medicament research and development.Urine is blood through glomerular filtration, the end product of metabolism that renal tubule and concetrated pipe heavily absorb, drain and secrete and produce, protein classes in urine and the height of content directly reflect urinary system, especially the health status of kidney, may be used for predicting that diabetic nephropathy occurs, development and the situation of prognosis.Meanwhile, large quantifier elimination finds, the clinical and prognosis of multiple protein biomarker cohort and chronic is closely related, shows that synchronization combining measures multiple protein factor and contributes to more fully understanding disease process.
Summary of the invention
Based on this, be necessary to provide a kind of may be used for distinguishing early diabetic nephropathy diabetic nephropathy early screening kit, biological marker object detecting method and application.
A kind of diabetic nephropathy early screening kit, comprises and detects reagent and capture agent;
Described detection reagent comprises the first detection reagent, second and detects reagent, the 3rd detection reagent and the 4th detection reagent, described first solute detecting reagent is the first sufficient glycocalicin antibody, described second solute detecting reagent is the first type Ⅳ collagen protein antibodies, described 3rd solute detecting reagent is the first liver type fatty acid binding protein antibody, and the described 4th solute detecting reagent is the first neutrophil gelatinase-associated lipocalin antibody;
The solute of described capture agent comprises the second sufficient glycocalicin antibody combining the first detectable label composition, the second type Ⅳ collagen protein antibodies combining the second detectable label composition, combines the second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody of the 4th detectable label composition.
In one embodiment, described diabetic nephropathy early screening kit also comprises sufficient glycocalicin standard items, type Ⅳ collagen protein standard substance, liver type fatty acid binding protein standard items and neutrophil gelatinase-associated lipocalin standard items.
In one embodiment, described first sufficient glycocalicin antibody is monoclonal antibody, and described second sufficient glycocalicin antibody is many monoclonal antibodies;
Described first type Ⅳ collagen protein antibodies is monoclonal antibody, and described second type Ⅳ collagen protein antibodies is polyclonal antibody;
Described first liver type fatty acid binding protein antibody is monoclonal antibody, and described second liver type fatty acid binding protein antibody is polyclonal antibody;
Described first neutrophil gelatinase-associated lipocalin antibody is monoclonal antibody, and described second neutrophil gelatinase-associated lipocalin antibody is polyclonal antibody.
In one embodiment, described first detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described second detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described 3rd detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described 4th detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.
A kind of biological marker object detecting method, adopts diabetic nephropathy early screening kit described above, it is characterized in that, comprise the steps:
The substrate being provided with detection site is provided, reagent dropwise will be detected in detection site, by described detection site washes clean after fully reacting;
Described detection site after washes clean is closed, by described detection site washes clean after having closed;
Sample drop to be detected is added in and has closed and in the described detection site of washes clean, fully after reaction by described detection site washes clean;
Capture agent is dripped in described detection site, by described detection site washes clean after fully reacting; And
Whether detect described detection site containing described first detectable label composition, described second detectable label composition, described 3rd detectable label composition and described 4th detectable label composition, and obtain the content of described sample mesopodium glycocalicin to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin according to testing result.
In one embodiment, the content of the sufficient glycocalicin in the sample described to be detected obtained, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin and cutoff value are contrasted, be judged to be the positive when the content of the sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin is all more than cutoff value, be then judged to be feminine gender in addition.
In one embodiment, the content of the sufficient glycocalicin in the sample described to be detected obtained, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin and cutoff value are contrasted, when the content of the sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin have at least one more than cutoff value time be judged to be the positive, be then judged to be feminine gender in addition.
In one embodiment, described sample to be detected comprises the front sample for the treatment of and the rear sample for the treatment of;
The content of sufficient glycocalicin antibody, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin in rear to the content of sample mesopodium glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin before the treatment obtained and treatment sample is contrasted, according to the validity of comparison result assess therapeutic.
In one embodiment, described sample to be detected is urine specimen.
Foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin are preparing the application in injury of kidney diagnostic reagent or injury of kidney diagnostic device as biomarker.
This diabetic nephropathy early screening kit by sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarker, can according to actual testing result, for injury of kidney provides auxiliary judgment, thus provide auxiliary for distinguishing early diabetic nephropathy.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the biological marker object detecting method of an embodiment;
Fig. 2 a is the proof diagram of sufficient glycocalicin in the early stage urine of diabetic nephropathy, and * represents P<0.05;
Fig. 2 b is the proof diagram of type Ⅳ collagen albumen in the early stage urine of diabetic nephropathy, and * represents P<0.05;
Fig. 2 c is the proof diagram of liver type fatty acid binding protein in the early stage urine of diabetic nephropathy, and * represents P<0.05;
Fig. 2 d is the proof diagram of neutrophil gelatinase-associated lipocalin in the early stage urine of diabetic nephropathy, and * represents P<0.05;
Fig. 3 a is the ROC curve map of sufficient glycocalicin;
Fig. 3 b is the ROC curve map of type Ⅳ collagen albumen;
Fig. 3 c is the ROC curve map of liver type fatty acid binding protein;
Fig. 3 d is the ROC curve map of neutrophil gelatinase-associated lipocalin.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments diabetic nephropathy early screening kit, biological marker object detecting method and application are described in further detail below.
Foot glycocalicin (podocalyxin) is the transmembrane glycoprotein that O connects, be positioned at Renal Podocytes podocytic process teleblem district, be the principal ingredient of glomerular basement membrane electrostatic barrier, ensure the integrality of glomerular filtration membrane electrostatic barrier, play an important role in kidney filtering function.
Type Ⅳ collagen albumen (Collagen IV) is that the important set of glomerular basement membrane and extracellular matrix claims composition, filters in integrality play an important role in glomerular basement membrane.Diabetes patient's hyperglycaemia stimulates generation IV collagen, if IV collagen increased deposits on diabetes patient's renal glomerulus extracellular matrix, can produce dispersivity glomerulosclerosis; If deposit on glomerular basement membrane or renal tubule, mesentery amplification, glomerulus interstitial and renal damage can be caused.
Liver type fatty acid binding protein (L-FABP) is expressed in renal proximal tubule cell, participates in renal tubular interstitium cellular energy and produces and metabolic process.In diabetic nephropathy concurrent process, in urine, L-FABP expression becomes positive correlation with urine albumin concentration, imply that in urine, L-FABP is the biomarker of renal tubular interstitium cellular damage.
Neutrophil gelatinase-associated lipocalin (NGAL) is a kind of ferric ion associated proteins, expresses in renal tubule endothelial cell, participates in various biological function, as Apoptosis, the innate immunity, kidney growth growth etc.Early stage at diabetic nephropathy, there is high expressed in NGAL, and the damage of the increase of NGAL expression and renal tubule proximal tubule is proportional in urine, imply that in urine, NGAL is the biomarker of renal tubular interstitium cellular damage.
Therefore, foot glycocalicin (podocalyxin) familial combined hyperlipidemia collagen (Collagen IV) can as the biomarker of glomerular injury, and liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) can as the biomarkers of renal damage.
Selecting above-mentioned 4 kinds of biomarkers for diabetogenous nephrosis disease early diagnosis in the application, detect this 4 kinds of biomarkers, according to the content of these 4 kinds of biomarkers, for injury of kidney provides auxiliary judgment, thus providing auxiliary for distinguishing early diabetic nephropathy.
The diabetic nephropathy early screening kit of one embodiment, comprises substrate, detects reagent and capture agent.
Detect reagent and comprise the first detection reagent, the second detection reagent, the 3rd detection reagent and the 4th detection reagent.
First solute detecting reagent is the first sufficient glycocalicin antibody, second solute detecting reagent is the first type Ⅳ collagen protein antibodies, 3rd solute detecting reagent is the first liver type fatty acid binding protein antibody, and the 4th solute detecting reagent is the first neutrophil gelatinase-associated lipocalin antibody.
The solute of capture agent comprises the second sufficient glycocalicin antibody combining the first detectable label composition, the second type Ⅳ collagen protein antibodies combining the second detectable label composition, combines the second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody of the 4th detectable label composition.
Preferably, detect reagent comprise the second detection reagent, the 3rd detect reagent and the 4th detect reagent, the solute of capture agent comprise combine the second detectable label composition the second type Ⅳ collagen protein antibodies, combine the second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody of the 4th detectable label composition.
Substrate is provided with detection site, and substrate can be nitrocellulose filter, nylon membrane or glass substrate.In present embodiment, nitrocellulose filter selected by substrate.Substrate can omit, when above-mentioned diabetic nephropathy early screening kit uses, and the substrate that other companies of arranging in pairs or groups sell.
In present embodiment, detection site comprises the first detection site, the second detection site, the 3rd detection site and the 4th detection site, first detection site corresponding first detects reagent, second detection site corresponding second detects reagent, 3rd detection site the corresponding 3rd detects reagent, and the 4th detection site the corresponding 4th detects reagent.
In present embodiment, the first detection site, the second detection site, the 3rd detection site and the 4th detection site are 4, avoid error by revision test.
This diabetic nephropathy early screening kit according to actual testing result, for injury of kidney provides auxiliary judgment, thus can provide auxiliary for distinguishing early diabetic nephropathy.
Diabetic nephropathy early screening kit also comprises sufficient glycocalicin standard items, type Ⅳ collagen protein standard substance, liver type fatty acid binding protein standard items and neutrophil gelatinase-associated lipocalin standard items, and these four kinds of standard items are all purchased from Beijing OriGene Biotechnology Co., Ltd..Corresponding, substrate also needs be provided with the first positive control site, the second positive control site, the 3rd positive control site and the 4th positive control site, corresponding first positive control site of foot glycocalicin standard items, corresponding second positive control site of type Ⅳ collagen protein standard substance, corresponding 3rd positive control site of liver type fatty acid binding protein standard items, corresponding 4th positive control site of neutrophil gelatinase-associated lipocalin standard items.
First detectable label composition can be enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.Second detectable label composition can be enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.3rd detectable label composition can be enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.4th detectable label composition can be enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.
In present embodiment, the first detectable label composition, the second detectable label composition, the 3rd detectable label composition and the 4th detectable label composition are horseradish peroxidase (HRP).
In present embodiment, diabetic nephropathy early screening kit also comprises detection liquid.The solute detecting liquid is TMB (3,3', 5,5'-tetramethyl benzidine).
In present embodiment, the first sufficient glycocalicin antibody (available from Sigma, article No.: AMAB90667) is monoclonal antibody, and the second sufficient glycocalicin antibody (available from Sigma, article No.: SAB2500809) is polyclonal antibody.
In present embodiment, the first type Ⅳ collagen protein antibodies (available from Sigma, article No.: C1926) is monoclonal antibody, and the second type Ⅳ collagen protein antibodies (purchased from Abcam company, article No.: ab6586) is polyclonal antibody.
In present embodiment, first liver type fatty acid binding protein antibody (available from Sigma, article No.: WH0002167M1) be monoclonal antibody, the second liver type fatty acid binding protein antibody (Abcam company, article No.: ab101837) is polyclonal antibody.
In present embodiment, first neutrophil gelatinase-associated lipocalin antibody is (purchased from Abcam company, article No.: ab23477) be monoclonal antibody, second neutrophil gelatinase-associated lipocalin antibody (Abcam company, article No.: ab166677) is polyclonal antibody.
This diabetic nephropathy early screening kit by sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarker, can according to actual testing result, for injury of kidney provides auxiliary judgment, thus provide auxiliary for distinguishing early diabetic nephropathy.
The biological marker object detecting method of an embodiment as shown in Figure 1, adopts above-mentioned diabetic nephropathy early screening kit, comprises the steps:
S10, provide the substrate being provided with detection site, reagent dropwise will be detected in detection site, fully after reaction by detection site washes clean.
Detect reagent and comprise the first detection reagent, the second detection reagent, the 3rd detection reagent and the 4th detection reagent.
First solute detecting reagent is the first sufficient glycocalicin antibody, second solute detecting reagent is the first type Ⅳ collagen protein antibodies, 3rd solute detecting reagent is the first liver type fatty acid binding protein antibody, and the 4th solute detecting reagent is the first neutrophil gelatinase-associated lipocalin antibody.
The solute of capture agent comprises the second sufficient glycocalicin antibody combining the first detectable label composition, the second type Ⅳ collagen protein antibodies combining the second detectable label composition, combines the second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody of the 4th detectable label composition.
In present embodiment, detection site comprises the first detection site, the second detection site, the 3rd detection site and the 4th detection site, first detection site corresponding first detects reagent, second detection site corresponding second detects reagent, 3rd detection site the corresponding 3rd detects reagent, and the 4th detection site the corresponding 4th detects reagent.
S10 is specially: detect reagent dropwise by first in the first detection site, reagent dropwise is detected in the second detection site by second, reagent dropwise is detected in the 3rd detection site by the 3rd, reagent dropwise is detected in the 4th detection site, respectively by the first detection site, the second detection site, the 3rd detection site and the 4th detection site washes clean after fully reacting by the 4th.
The operation of washing can adopt conventional P BS dilution, is specifically as follows 0.01mol/L PBS (pH=7.4)+0.05% Tween-20.
S20, the detection site after washes clean to be closed, by detection site washes clean after having closed.
S20 is specially: the first detection site after washes clean, the second detection site, the 3rd detection site and the 4th detection site are closed respectively, has closed rear respectively by the first detection site, the second detection site, the 3rd detection site and the 4th detection site washes clean.
Skimmed milk power or bovine serum albumin(BSA) can be dissolved in PBS (pH=7.4,0.01mol/L) and obtain by confining liquid.
S30, sample drop to be detected is added in and closed and in the detection site of washes clean, fully after reaction by detection site washes clean.
S30 is specially: dripped by sample to be detected respectively in the first detection site having closed also washes clean, the second detection site, the 3rd detection site and the 4th detection site, respectively by the first detection site, the second detection site, the 3rd detection site and the 4th detection site washes clean after fully reacting.
In present embodiment, samples selection urine specimen to be detected.
In general, urine specimen is before detecting, and need to dilute, extension rate can be 100 times.
S40, capture agent to be dripped in detection site, fully after reaction by detection site washes clean.
S40 is specially: dripped by capture agent respectively in the first detection site, the second detection site, the 3rd detection site and the 4th detection site, respectively by the first detection site, the second detection site, the 3rd detection site and the 4th detection site washes clean after fully reacting.
S50, detection site whether containing the first detectable label composition, the second detectable label composition, the 3rd detectable label composition and the 4th detectable label composition, and obtain the content of sample mesopodium glycocalicin to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin according to testing result.
Whether whether S50 is specially: detect respectively containing the first detectable label composition, the second detectable label composition, the 3rd detectable label composition and the 4th detectable label composition in the first detection site, the second detection site, the 3rd detection site and the 4th detection site, and determine in sample to be detected containing sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin according to testing result.
This injury of kidney detection method can provide auxiliary judgment for injury of kidney, thus provides auxiliary for distinguishing early diabetic nephropathy.
Concrete, first cutoff value can be set, then the content of the sufficient glycocalicin in the sample to be detected obtained, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin and cutoff value can be contrasted.
(1) be judged to be the positive when the content of the sufficient glycocalicin in sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin is all more than cutoff value, be then judged to be feminine gender in addition.This decision method is applicable to the further confirmation to preliminary screening results.
(2) when the content of the sufficient glycocalicin in sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin have at least one more than cutoff value time be judged to be the positive, be then judged to be feminine gender in addition.This decision method is applicable to the preliminary examination of large crowd's sample.
Above-mentioned two kinds of decision methods respectively have advantage, can be used alone, also can be combined.
In a preferred embodiment, with serum creatinine (creatinine, Cr) after correcting, the concentration cutoff value of Podocalyxin is 67.15ng/g Cr, the concentration cutoff value of Collagen IV is 2.945 μ g/g Cr, the concentration cutoff value of L-FABP is the concentration cutoff value of 4.75 μ g/g Cr, NGAL is 56.8 μ g/g Cr.
This biological marker object detecting method for assessment of the validity of Renal injury grade method, can also be specially: sample to be detected comprises the front sample for the treatment of and the rear sample for the treatment of; The content of sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin in rear to the content of sample mesopodium glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin before the treatment obtained and treatment sample is contrasted, according to the validity of comparison result assess therapeutic.
The concrete evaluation process of the validity of above-mentioned methods for the treatment of is: if the concentration of the biomarker after treatment in sample finds to decline or remain unchanged compared with sample before treatment, then show that treatment effectively; If find to raise, and have statistically significant meaning, then it is invalid to show.
The biological marker object detecting method of the diabetic nephropathy early screening kit that this employing is above-mentioned, by sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin as biomarker, can according to actual testing result, for injury of kidney provides auxiliary judgment, thus provide auxiliary for distinguishing early diabetic nephropathy.
Present inventor is in conjunction with bibliographical information and development test, find that sufficient glycocalicin (podocalyxin), type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) develop closely related with the generation of the early stage disease of diabetic nephropathy, utilize protein chip to detect to find simultaneously, in Diabetic Nephropathy patients urine, the content of these 4 kinds of marks is significantly higher than normal control (p<0.05), and increases along with advancing of disease.
Detect this 4 kinds of biomarkers, utilize it to combine change and predict diabetic nephropathy, examination early diabetic nephropathy that can be sensitive, special, has comparatively wide market application foreground simultaneously.
Detect these 4 kinds of biomarkers to have the following advantages: 1) from Renal Structure functional perspective simultaneously, diabetic nephropathy earlier damage may betide glomerulus, also renal tubule may be betided, four kinds of biomarkers that this kit is selected both had comprised glomerular injury specific biological mark (sufficient glycocalicin, type Ⅳ collagen albumen), comprise again renal damage specific markers (liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin), therefore utilize these 4 kinds of biomarker combinations to detect simultaneously, help avoid and fail to pinpoint a disease in diagnosis, realize Diabetic Nephropathy early to find, early diagnosis, 2) from diabetic nephropathy molecular pathway, diabetic nephropathy damage mechanisms relates to multiple molecular pathway, as renal fibrosis (type Ⅳ collagen albumen), inflammatory reaction (neutrophil gelatinase-associated lipocalin), oxidative stress (liver type fatty acid binding protein) etc., from multiple molecular pathway predictive disease, contribute to diabetes and early find, therefore, detect these 4 kinds of biomarkers of sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin simultaneously, and by selecting rational decision method, substantially increase sensitivity and the specificity of renal dysfunction detection.
In addition, sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin can also be applied preparing in injury of kidney diagnostic reagent or injury of kidney diagnostic device as biomarker.
Be below specific embodiment, the various instrument occurred in embodiment and reagent if not otherwise specified, all adopt this area conventional instrument or reagent.
Below in conjunction with specific embodiment, the screening of the application's 4 kinds of biomarkers, analysis and application are described in further detail.
Embodiment 1: the screening of diabetogenous nephrosis disease early diagnosis biomarker and checking
One, experimental technique
1, pathology screening
Choose experimenter's totally 205 examples of known health, wherein normal healthy controls 50 example, diabetes B patient 155 example (wherein all patients suffer from the diabetes time limit and are all greater than 2 years); And according to albuminuria situation, 155 routine diabetics are divided, wherein normal protein urine patient 61 example, Microalbuminuria patient 52 example, High-grade Proteinuria patient 42 example.MDRD GRF formula estimated glomerular filtration rate (eGFR).
Note: this research is ratified by ethics examination & verification mechanism, and all experimenters all sign Informed Consent Form.
2, the collection of Urine specimens and process
Collect the urine specimen (30mL) of experimenter's first time in early morning discharge with sterile chamber, 4 DEG C of centrifugal 30min of 8000g, get supernatant, are stored in-80 DEG C and test for protein science.All urine specimens are all collected before not using any drug therapy.When carrying out protein science experiment, mix same, for protein science research from the urine specimen of different patient by stages.
3.iTRAQ quantitative proteomics technology
Utilize ProteoMiner
tMprotein enrichment kit (Bio-Rad, article No.: 163-3006) removes high-abundance proteins, and cold acetone/trichloroacetic acid method extracts urine total protein, and Bradford method measures protein concentration.According to iTRAQ labelling kit (Applied Biosystem Inc. article No.: MS10016) operation steps process, mark different group Urine proteins sample.Operation steps is summarized as follows: get each group of each 100 μ g of Urine proteins sample, often pipe adds the acetone (acetone: sample volume ratio=6:1) of precooling respectively,-20 DEG C precipitate 1 hour, after 12000rpm ultracentrifugation 15min, precipitation are resuspended in 20 μ L and dissolve in damping fluids.Reduction, the halfcystine closed in Urine proteins, trypsinization, then utilize iTRAQ labelled reagent 114,115,116,117 mark respectively Urine proteins that normal protein urinates patient, Microalbuminuria patient, High-grade Proteinuria patient and normal control.Urine proteins peptide after mixed in equal amounts mark, strong cation exchange chromatography, oppositely chromatography is utilized to be separated successively, mass-spectrometric technique carries out analyzing (SCX-RPLC-MS/MS), and the albumen in Mascot software qualification urine, finds differentially expressed protein between disease group and contrast and disease group.
4, ELISA method checking differentially expressed protein
Differential protein (sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and the neutrophil gelatinase-associated lipocalin) expression in normal control and each disease group urine utilizing elisa technique to confirm to search out, is intended to verify the accuracy of mass-spectrometric technique result and these differential proteins accuracy as the early stage biomarker of diabetic nephropathy (Biomarker).Specifically, utilize dilution buffer to be diluted by urine 1:100, then utilize the elisa plate for different protein biomarker to measure the content of biomarker in various disease group urine, each sample duplicate measurements three times.
5, statistical study
Data SPSS 14.0 software (Statistical Product and Service Solutions, " statistical product and service solution " software) processes, and represents with mean+SD.Compare between the group of carrying out normal distribution data with variance analysis (Analysis ofVariance, ANOVA); Accuracy, specificity and the sensitivity of the prediction of various biomarker is analyzed with receiver operator characteristics's curve (ROC).P<0.05 represents to have remarkable significant difference.
6,4 kinds of biomarker comprehensive detection early diabetic nephropathies
Diabetic nephropathy is a kind of Complex Complications, may cause renal damage, or cause glomerular injury at the diabetic nephropathy initial stage.4 kinds of biomarkers in the application, its mesopodium glycocalicin familial combined hyperlipidemia collagen mainly reflects glomerular injury; Liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin mainly reflect renal damage.For diagnosing early diabetic nephropathy accurately, this experiment combinationally uses two or more biomarker, adopts the decision method of (1) and (2) to carry out synthetic determination.
(1) contained in combination all biomarkers are judged to be the positive when being the positive (more than cutoff value), are judged to be feminine gender in addition.This decision method is applicable to the further confirmation to preliminary screening results.
(2) contained in combination biomarker has one to be judged as the positive for time positive (more than cutoff value), is judged as feminine gender in addition.This decision method is applicable to the preliminary examination of large crowd's sample.
This research finds, the kind of biomarker combinations and the difference of quantity, can cause medical diagnosis on disease sensitivity different with specificity.Therefore best biomarker combinations can be selected to diagnose early diabetic nephropathy.
Two, result
1, experimenter's essential information
As shown in table 2 below.
Table 2: Mass spectrometry experiments experimenter health and biochemical indicator
Note: ND:No Difference; * p<0.001vs. normal healthy controls;
#p<0.001vs normal protein urine patient; § p<0.001vs Microalbuminuria patient, HbA
1Cfor glycosylated hemoglobin, Serum Cr refers to serum creatinine; UACR refers to urinary albumin and creatinine ratio.
2, urine protein science result describes
In earlier stage screening stage, protein science is tested and is repeated for 2 times to identify 465 and 478 albumen respectively, in secondary experiment, jointly identify 322 albumen.In secondary experiment, 114/117 (normal protein urine group/normal control), 115/117 (Microalbuminuria group/normal control) and 116/117 (High-grade Proteinuria group/normal control) iTRAQ ratio all demonstrate the albumen of differential expression totally 62 (ratio<0.667 or ratio>1.5).Because the present invention is intended to find diabetic nephropathy early stage (normal protein urine phase or I and II phase) biomarker, 62 albumen that differential expression occurs are further analyzed, found that 15 albumen, in the difference of diabetic nephropathy group, differential expression (ratio<0.667 or ratio>1.5) occur by stages.
By bibliographical information and bioinformatic analysis (Gene Ontology and KEGG Pathway analyzes), the differentially expressed protein that 15 identify is further analyzed, finds that sufficient glycocalicin (podocalyxin), type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) are sent out in process in the renal complications disease that diabetes cause and play very important effect.
3, ELISA method checking differentially expressed protein
ELISA method is utilized to verify the differentially expressed protein that urine protein science testing sieve is selected: sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin, obtain following table 3.
Table 3:ELISA verifies 4 kinds of diabetic nephropathy early sign things
Note: ND:No Difference; * p<0.001vs. normal healthy controls;
★p<0.05vs normal healthy controls;
#p<0.001vs normal protein urine patient; § p<0.001vs Microalbuminuria.
As can be seen from table 3 and Fig. 2 a ~ Fig. 2 d, the expression of diabetes group urine mesopodium glycocalicin (podocalyxin), type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) is significantly higher than normal healthy controls, and increases along with serious (the albuminuria increase) of conditions of patients.
To the correlation analysis of glomerular filtration rate(GFR, albuminuria and 4 kinds of protein markers, obtain following table 4.
Table 4: the correlation analysis of glomerular filtration rate(GFR, albuminuria and 4 kinds of protein markers
As can be seen from Table 4, the expression of above-mentioned 4 kinds of albumen and Urine proteins are remarkable positive correlation, are remarkable negative correlation with eGFR.
Utilize four kinds of biomarkers in receiver operator characteristics's curve (ROC) analysis of diabetes nephrotic and normal healthy controls urine, obtain Fig. 3 a ~ Fig. 3 d.
Following table 5 can be obtained by Fig. 3 a ~ Fig. 3 d.
Table 5: four kinds of biomarker ROC tracing analysis
As can be seen from Table 5, podocalyxin, Collagen IV, L-FABP and NGAL area under curve (AUC) are respectively 0.892 (p<0.01), 0.858 (p<0.01), 0.907 (p<0.01) and 0.901 (p<0.01).Sensitivity and Specificity analysis shows, and when choosing the highest " youden " index, the sensitivity of four kinds of biomarkers in urine, specificity and concentration cutoff value are respectively: podocalyxin (sensitivity 89.8%; Specificity is 76.2%; Concentration cutoff value is 67.15ng/g creatinine), Collagen IV (sensitivity 71.2%; Specificity is 90.5%; Concentration cutoff value is 2.945 μ g/g creatinine), L-FABP (sensitivity 79.6%; Specificity is 95.2%; Concentration cutoff value is 4.75 μ g/g creatinine) and NGAL (sensitivity 84.7%; Specificity is 85.8%; Concentration cutoff value is 49.85ng/mL).
Above result shows that these 4 kinds of protein moleculars may take part in diabetic nephropathy disease and send out process, is believable early diabetic nephropathy biomarker.
4, diabetic nephropathy method of early diagnosis
When carrying out diabetes diagnosis, determine that this 4 kinds of protein marker relative contents are with reference to normal ranges by the measurement result of normal healthy controls and four kinds of protein molecular concentration cutoff values, in conjunction with four kinds of biomarker measurement results, if wherein any one protein concentration exceeds normal range, can tentative diagnosis be early diabetic nephropathy; In conjunction with the index such as GFR, albuminuria, comprehensive assessment is carried out to early diabetic nephropathy simultaneously.
5. many kinds of biomarker combinations carry out diabetogenous nephrosis disease early diagnosis
Carry out four kinds of biomarkers in urine combining the sensitivity and specificity of diagnosing early diabetic nephropathy.Wherein NGAL measured value Cr is corrected, by its cutoff value of ROC tracing analysis.In urine, NGAL concentration cutoff value is 56.8 μ g/g Cr; In urine, L-FABP concentration cutoff value is 4.75 μ g/g Cr; In urine, Collagen IV concentration cutoff value is 2.945 μ g/g Cr; In urine, podocalyxin concentration cutoff value is 67.15ng/g Cr.
Statistical treatment is carried out to various combination, compares sensitivity and the specificity of various combination, obtain following table 6.Wherein, in the combination that symbol " ∩ " represents, the positive is judged as when all biomarkers contained by this combination are the positive (more than cutoff value), meter sensitivity and specificity.In the combination that symbol " ∪ " represents, the biomarker contained by this combination has one to be judged as the positive, meter sensitivity and specificity for time positive (more than cutoff value).
Table 6: combine sensitivity and specificity for diagnosing early diabetic nephropathy protein biomarker
| Combination | Sensitivity | Specificity |
| 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 |
As can be seen from Table 6, according to the change of Integrated biomarker kind and quantity, sensitivity and specificity also can change.This represents that experimentally object selects the combination of biomarker, can be efficient, special for medical diagnosis on disease.
Highly sensitive combination (IV Collagen ∪ NGAL; IV Collagen ∪ Podocalyxin; NGAL ∪ Podocalyxin; L-FABP ∪ Podocalyxin; IV Collagen ∪ NGAL ∪ L-FABP ∪ Podocalyxin) be suitable for first examination; On the contrary, combination (the IV Collagen ∩ NGAL that specificity is high; IV Collagen ∩ Podocalyxin; NGAL ∩ Podocalyxin; L-FABP ∩ Podocalyxin; IV Collagen ∩ NGAL ∩ L-FABP ∩ Podocalyxin) be applicable to the needs such as quadratic search or three inspections carry out the higher judgement inspection of reliability.Such as, if occur when first examination positive, now by the combined method that specificity is higher, secondary judgement is carried out to initial examination result, finally provide effectively and reliable early diabetic nephropathy diagnostic result.
As can be seen from Table 6, " IV Collagen, NGAL, L-FABP and Podocalyxin " this combination, by selecting suitable decision method, can obtain the highest sensitivity and specificity.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a diabetic nephropathy early screening kit, is characterized in that, comprises and detects reagent and capture agent;
Described detection reagent comprises the first detection reagent, second and detects reagent, the 3rd detection reagent and the 4th detection reagent, described first solute detecting reagent is the first sufficient glycocalicin antibody, described second solute detecting reagent is the first type Ⅳ collagen protein antibodies, described 3rd solute detecting reagent is the first liver type fatty acid binding protein antibody, and the described 4th solute detecting reagent is the first neutrophil gelatinase-associated lipocalin antibody;
The solute of described capture agent comprises the second sufficient glycocalicin antibody combining the first detectable label composition, the second type Ⅳ collagen protein antibodies combining the second detectable label composition, combines the second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody of the 4th detectable label composition.
2. diabetic nephropathy early screening kit as claimed in claim 1, it is characterized in that, described diabetic nephropathy early screening kit also comprises sufficient glycocalicin standard items, type Ⅳ collagen protein standard substance, liver type fatty acid binding protein standard items and neutrophil gelatinase-associated lipocalin standard items.
3. diabetic nephropathy early screening kit as claimed in claim 1, it is characterized in that, described first sufficient glycocalicin antibody is monoclonal antibody, and described second sufficient glycocalicin antibody is many monoclonal antibodies;
Described first type Ⅳ collagen protein antibodies is monoclonal antibody, and described second type Ⅳ collagen protein antibodies is polyclonal antibody;
Described first liver type fatty acid binding protein antibody is monoclonal antibody, and described second liver type fatty acid binding protein antibody is polyclonal antibody;
Described first neutrophil gelatinase-associated lipocalin antibody is monoclonal antibody, and described second neutrophil gelatinase-associated lipocalin antibody is polyclonal antibody.
4. diabetic nephropathy early screening kit as claimed in claim 1, it is characterized in that, described first detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described second detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described 3rd detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials;
Described 4th detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactive materials.
5. a biological marker object detecting method, adopts the diabetic nephropathy early screening kit according to any one of Claims 1 to 4, it is characterized in that, comprise the steps:
The substrate being provided with detection site is provided, reagent dropwise will be detected in detection site, by described detection site washes clean after fully reacting;
Described detection site after washes clean is closed, by described detection site washes clean after having closed;
Sample drop to be detected is added in and has closed and in the described detection site of washes clean, fully after reaction by described detection site washes clean;
Capture agent is dripped in described detection site, by described detection site washes clean after fully reacting; And
Whether detect described detection site containing described first detectable label composition, described second detectable label composition, described 3rd detectable label composition and described 4th detectable label composition, and obtain the content of described sample mesopodium glycocalicin to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin according to testing result.
6. biological marker object detecting method as claimed in claim 5, it is characterized in that, the content of the sufficient glycocalicin in the sample described to be detected obtained, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin and cutoff value is also comprised the steps: to contrast, be judged to be the positive when the content of the sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin is all more than cutoff value, be then judged to be feminine gender in addition.
7. biological marker object detecting method as claimed in claim 5, it is characterized in that, also comprise the steps: the sufficient glycocalicin in the sample described to be detected that obtains, type Ⅳ collagen albumen, content and the cutoff value of liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin contrast, sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, the content of liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin have at least one more than cutoff value time be judged to be the positive, in addition then feminine gender is judged to be.
8. biological marker object detecting method as claimed in claim 5, is characterized in that, described sample to be detected comprises the front sample for the treatment of and sample after treating;
The content of sufficient glycocalicin antibody, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin in rear to the content of sample mesopodium glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin before the treatment obtained and treatment sample is contrasted, according to the validity of comparison result assess therapeutic.
9. biological marker object detecting method as claimed in claim 5, it is characterized in that, described sample to be detected is urine specimen.
10. sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin are preparing the application in injury of kidney diagnostic reagent or injury of kidney diagnostic device as biomarker.
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| CN109891241B (en) * | 2016-10-03 | 2022-12-06 | 秀比特生物技术公司 | Examination method capable of specifically diagnosing early stage disease of diabetic nephropathy |
| CN112362878A (en) * | 2020-11-03 | 2021-02-12 | 吉林省富生医疗器械有限公司 | Microalbumin detection freeze-drying reagent and reaction tube pre-stored with same |
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| CN204479593U (en) * | 2015-03-24 | 2015-07-15 | 深圳市易特科信息技术有限公司 | Based on the injury of kidney detection kit of biomarker |
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| WO2010143423A1 (en) * | 2009-06-10 | 2010-12-16 | 国立大学法人 新潟大学 | Method for test on diabetic nephropathy |
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| WO2016149971A1 (en) * | 2015-03-24 | 2016-09-29 | 深圳市贝沃德克生物技术研究院有限公司 | Diabetic nephropathy early detection reagent kit, biomarker testing method and applications |
| CN105092844A (en) * | 2015-07-10 | 2015-11-25 | 深圳市贝沃德克生物技术研究院有限公司 | Pancreatic cancer protein biomarker detection kit and detection system |
| CN105092845A (en) * | 2015-07-10 | 2015-11-25 | 深圳市贝沃德克生物技术研究院有限公司 | Pancreatic cancer protein biomarker and application and detection chip thereof, and detection device |
| WO2017121001A1 (en) * | 2016-01-16 | 2017-07-20 | 深圳市华科安测信息技术有限公司 | Gold-labeled antibody test strip for use in early-stage diabetic nephropathy test |
| WO2017202007A1 (en) * | 2016-05-24 | 2017-11-30 | 深圳市前海安测信息技术有限公司 | Method for preparing colloidal gold test strip for detection of early diabetic nephropathy |
| WO2018076284A1 (en) * | 2016-10-28 | 2018-05-03 | The University Of Hong Kong | Non-polyaminated lcn2 as a biomarker for diagnosis and treatment of cardiometabolic diseases |
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| Publication number | Publication date |
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| CN104764885B (en) | 2016-08-24 |
| WO2016149972A1 (en) | 2016-09-29 |
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