CN104764885B - Diabetic nephropathy early screening kit, biological marker object detecting method and application - Google Patents
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- 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
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Abstract
The invention discloses a kind of diabetic nephropathy early screening kit, including detectable；Described detectable includes the first detectable, the second detectable, the 3rd detectable and the 4th detectable；The solute of described first detectable is the first foot glycocalicin antibody, the solute of described second detectable is the first type Ⅳ collagen protein antibodies, the solute of described 3rd detectable is the first liver type fatty acid binding protein antibody, and the solute of described 4th detectable is the first neutrophil gelatinase-associated lipocalin NGAL antibody.This diabetic nephropathy early screening kit passes through foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin NGAL as biomarker, can be according to actually detected result, there is provided auxiliary judgment for injury of kidney, thus provide auxiliary for distinguishing early 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 end kidney eventually
Exhaustion, is diabetics main causes of death.According to World Health Organization's statistics in 2013, the whole world is about
Having 3.47 hundred million people to suffer from diabetes, wherein the Diabetes Death case more than 80% occurs in developing country.
Meanwhile, World Health Organization (WHO) predicts the year two thousand thirty, and the whole world will be doubled, wherein because of the number of Diabetes Death
The type 2 diabetes mellitus people of 30%～40% would develop into diabetic nephropathy, and the type 1 diabetes patient of 20%～40% exists
Also would develop into diabetic nephropathy after 15～30 years, cause huge and heavy social economical burden.
But, diabetic nephropathy is difficult in early days find, and clinical diabetes diagnosis of nephropathy golden standard creatinine and
Albuminuria also can only indirectly reflect nephrolithotomy sexually transmitted disease (STD) become (mesangial cell amplification, basement membrane thicken,
Podocytes in Renal Tissue, renal cells and Interstitial cell damage etc.), it is impossible to diagnosis early diabetic nephropathy is (just
Often the albuminuria phase, see table 1).
Table 1: diabetic nephropathy clinical stages (based on Mogensen allotment method)
Though renal needle biopsy technology can be with adjuvant clinical diabetogenous nephrosis disease early diagnosis, but this technology is traumatic
Relatively big, postoperative meeting causes complication, and technical difficulty is higher, it is impossible to check as diabetic nephropathy patient
Conventional project.Generally when clinical definite, diabetic nephropathy patient misses optimal therapic opportunity, causes
Disease drastically deteriorates, irreversible.Therefore, diabetic nephropathy early diagnosis marker and inspection thereof are actively found
Test agent box, and carry out early intervention and have important practical significance.
Protein biomarker (biomarker) is that reflection disease exists and the measurable indicant of one of state.
Disease, stress with rehabilitation during, any physiological change finally can obtain body on protein level
Existing, it is disease research means the most intuitively.The detection of current protein molecular mark has become clinical disease early
The most efficient methods such as phase diagnosis, the screening of disease typing, drug targets and medicament research and development.Urine is that blood exists
The metabolic end product heavily absorbing through glomerular filtration, renal tubules and collecting tubule, drain and secreting and produce,
Protein classes in urine and the height of content directly reflect the health status of urinary system, especially kidney,
May be used for predicting that diabetic nephropathy occurs, develops and the situation of prognosis.Meanwhile, substantial amounts of research finds,
Multiple protein biomarker groups are closely related with the clinic of chronic and prognosis, show synchronization combining
Measure multiple protein factor and contribute to more fully understanding disease process.
Summary of the invention
Based on this, it is necessary to provide a kind of and may be used for distinguishing the diabetic nephropathy of early diabetic nephropathy in early days
Kit for screening, biological marker object detecting method and application.
A kind of diabetic nephropathy early screening kit, including detectable and capture agent；
Described detectable includes the first detectable, the second detectable, the 3rd detectable and the 4th inspection
Test agent, the solute of described first detectable is the first foot glycocalicin antibody, described second detectable
Solute be the first type Ⅳ collagen protein antibodies, the solute of described 3rd detectable is the first liver type fatty acid
Associated proteins antibody, the solute of described 4th detectable is the first neutrophilic granulocyte gelatinase related lipid fortune
Carry protein antibodies；
The solute of described capture agent includes that the second foot glycocalicin combining the first detectable label composition resists
Body, combine the second type Ⅳ collagen protein antibodies of the second detectable label composition, combine the 3rd and can detect
Second liver type fatty acid binding protein antibody of marked member and combine the second of the 4th detectable label composition
Neutrophil gelatinase-associated lipocalin NGAL antibody.
In one embodiment, described diabetic nephropathy early screening kit also includes foot glycocalicin standard
Product, type Ⅳ collagen protein standard substance, liver type fatty acid binding protein standard substance and neutrophilic granulocyte gelatinase phase
Close lipocalin protein standard substance.
In one embodiment, described first foot glycocalicin antibody is monoclonal antibody, described second foot sugar
Calyx protein antibodies is polyclonal antibody；
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, described second liver type fatty acid knot
Hop protein antibody is polyclonal antibody；
Described first neutrophil gelatinase-associated lipocalin NGAL antibody is monoclonal antibody, described
Two neutrophil gelatinase-associated lipocalin NGAL antibody are polyclonal antibody.
In one embodiment, described first detectable label composition is enzyme, prothetic group, fluorescent material, luminescence
Material, bioluminescence material or radioactive materials；
Described second detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence thing
Matter or radioactive materials；
Described 3rd detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence thing
Matter or radioactive materials；
Described 4th detectable label composition is enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence thing
Matter or radioactive materials.
A kind of biological marker object detecting method, uses diabetic nephropathy early screening kit described above, its
It is characterised by, comprises the steps:
The substrate being provided with detection site is provided, detectable is dripped in detection site, fully will after reaction
Described detection site washes clean；
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, fully by described detection site washes clean after reaction；
And
Detect whether described detection site contains described first detectable label composition, described second can detect mark
Note composition, described 3rd detectable label composition and described 4th detectable label composition, and according to detection
Result obtains described sample mesopodium glycocalicin to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein
Content with neutrophil gelatinase-associated lipocalin NGAL.
In one embodiment, the sufficient glycocalicin in the sample described to be detected that will obtain, type Ⅳ collagen egg
In vain, the content of liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin NGAL with block
Value contrasts, when the sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, liver type fatty acid
Judge when the content of associated proteins and neutrophil gelatinase-associated lipocalin NGAL is all more than cutoff value
For the positive, the most then it is judged to feminine gender.
In one embodiment, the sufficient glycocalicin in the sample described to be detected that will obtain, type Ⅳ collagen egg
In vain, the content of liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin NGAL with block
Value contrasts, when the sufficient glycocalicin in described sample to be detected, type Ⅳ collagen albumen, liver type fatty acid
The content of associated proteins and neutrophil gelatinase-associated lipocalin NGAL at least one cutoff value with
It is judged to the positive time upper, is the most then judged to feminine gender.
In one embodiment, described sample to be detected includes treating sample after front sample and treatment；
Sample mesopodium glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein before the treatment that will obtain
Resist with the sufficient glycocalicin in sample after the content of neutrophil gelatinase-associated lipocalin NGAL and treatment
Body, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophilic granulocyte gelatinase related lipid delivery egg
White content contrasts, according to the effectiveness of comparison result assessment Therapeutic Method.
In one embodiment, described sample to be detected is urine specimen.
Foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophilic granulocyte gelatinase phase
Close lipocalin protein as biomarker preparation injury of kidney diagnostic reagent or the injury of kidney diagnostic equipment in
Application.
This diabetic nephropathy early screening kit is by foot glycocalicin, type Ⅳ collagen albumen, liver type fat
Fat acid binding protein and neutrophil gelatinase-associated lipocalin NGAL are as biomarker, Ke Yigen
Border testing result factually, provides auxiliary judgment for injury of kidney, thus provides auxiliary for distinguishing early diabetic nephropathy
Help.
Accompanying drawing explanation
Fig. 1 is the flow chart of the biological marker object detecting method of an embodiment；
Fig. 2 a is foot glycocalicin proof diagram in diabetic nephropathy in early days urine, and * represents P < 0.05；
Fig. 2 b is type Ⅳ collagen albumen proof diagram in diabetic nephropathy in early days urine, and * represents P < 0.05；
Fig. 2 c is liver type fatty acid binding protein proof diagram in diabetic nephropathy in early days urine, and * represents
P<0.05；
Fig. 2 d is neutrophil gelatinase-associated lipocalin NGAL testing in diabetic nephropathy in early days urine
Card figure, * represents P < 0.05；
Fig. 3 a is the ROC curve figure of foot glycocalicin；
Fig. 3 b is the ROC curve figure of type Ⅳ collagen albumen；
Fig. 3 c is the ROC curve figure of liver type fatty acid binding protein；
Fig. 3 d is the ROC curve figure of neutrophil gelatinase-associated lipocalin NGAL.
Detailed description of the invention
Below mainly in combination with drawings and the specific embodiments to diabetic nephropathy early screening kit, biological marker
Object detecting method and application are described in further detail.
Foot glycocalicin (podocalyxin) is the transmembrane glycoprotein that O connects, and is positioned at Renal Podocytes podocytic process
Teleblem district, is the main component of glomerular basement membrane electrostatic barrier, it is ensured that glomerular filtration membrane electrostatic barrier
Integrity, plays 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,
Play an important role in glomerular basement membrane filters integrity.Diabetes patient's hyperglycemia stimulates generation IV
Collagen, if IV collagen increased deposits to, on diabetes patient's renal glomerulus extracellular matrix, can produce
Dispersivity glomerulosclerosis；If depositing on glomerular basement membrane or renal tubules, mesentery can be caused to expand,
Glomerule interstitial and injury of renal tubular.
Liver type fatty acid binding protein (L-FABP) is expressed in renal proximal tubule cell, participates in renal tubular interstitium
Cellular energy produces and metabolic process.In diabetic nephropathy concurrent process, L-FABP expression in urine
Become positive correlation with urine albumin concentration, imply that in urine, L-FABP is the biology of renal tubular interstitium cell injury
Mark.
Neutrophil gelatinase-associated lipocalin NGAL (NGAL) is a kind of iron ion associated proteins, at kidney
Tubule epithelium cell is expressed, participates in various biological function, such as apoptosis, the natural immunity, kidney life
Long growth etc..At diabetic nephropathy in early days, NGAL occurs high expressed, and NGAL expression in urine
Increase proportional with the damage of renal tubules proximal tubule, imply that in urine, NGAL is that renal tubular interstitium is thin
The biomarker of cellular damage.
Therefore, foot glycocalicin (podocalyxin) familial combined hyperlipidemia collagen protein (Collagen IV) can be made
For the biomarker of glomerular injury, liver type fatty acid binding protein (L-FABP) and neutrophilic granulocyte gelatin
Enzyme associated lipocalin (NGAL) can be as the biomarker of injury of renal tubular.
The application selects above-mentioned 4 kinds of biomarkers for diabetogenous nephrosis disease early diagnosis, detect these 4 kinds
Biomarker, according to the content of these 4 kinds of biomarkers, provides auxiliary judgment for injury of kidney, thus is
Distinguish early diabetic nephropathy and auxiliary is provided.
The diabetic nephropathy early screening kit of one embodiment, tries including substrate, detectable and capture
Agent.
Detectable includes the first detectable, the second detectable, the 3rd detectable and the 4th detection examination
Agent.
The solute of the first detectable is the first foot glycocalicin antibody, and the solute of the second detectable is first
Type Ⅳ collagen protein antibodies, the solute of the 3rd detectable is the first liver type fatty acid binding protein antibody, the
The solute of four detectable is the first neutrophil gelatinase-associated lipocalin NGAL antibody.
The solute of capture agent include combining the first detectable label composition the second foot glycocalicin antibody,
Combine the second type Ⅳ collagen protein antibodies of the second detectable label composition, combine the 3rd detectable label
Second liver type fatty acid binding protein antibody of composition is neutral with combine the 4th detectable label composition second
Granulocyte gelatinase associated lipocalin antibody.
Preferably, detectable includes the second detectable, the 3rd detectable and the 4th detectable, catches
The solute obtaining reagent includes combining the second type Ⅳ collagen protein antibodies of the second detectable label composition, combination
The second liver type fatty acid binding protein antibody of the 3rd detectable label composition and combine the 4th and can detect mark
Second neutrophil gelatinase-associated lipocalin NGAL antibody of note composition.
Substrate is provided with detection site, and substrate can be nitrocellulose filter, nylon membrane or glass substrate.This
In embodiment, substrate selects nitrocellulose filter.Substrate can omit, at above-mentioned diabetic nephropathy in early days
When kit for screening uses, the substrate that other companies of arranging in pairs or groups sell.
In present embodiment, detection site includes the first detection site, the second detection site, the 3rd detecting position
Point and the 4th detection site, corresponding first detectable of the first detection site, the second detection site correspondence second
Detectable, corresponding 3rd detectable of the 3rd detection site, corresponding 4th detectable of the 4th detection site.
In present embodiment, the first detection site, the second detection site, the 3rd detection site and the 4th detection
Site is 4, avoids error by repeating test.
This diabetic nephropathy early screening kit can provide auxiliary according to actually detected result for injury of kidney
Help judgement, thus provide auxiliary for distinguishing early diabetic nephropathy.
Diabetic nephropathy early screening kit also includes foot glycocalicin standard substance, type Ⅳ collagen protein standard
Product, liver type fatty acid binding protein standard substance and neutrophil gelatinase-associated lipocalin NGAL standard substance,
These four standard substance are purchased from Beijing OriGene Biotechnology Co., Ltd..Corresponding, substrate also needs to
It is provided with the first positive control site, the second positive control site, the 3rd positive control site and the 4th positive right
According to site, corresponding first positive control site of foot glycocalicin standard substance, type Ⅳ collagen protein standard substance is corresponding
Second positive control site, corresponding 3rd positive control site of liver type fatty acid binding protein standard substance, neutral
Corresponding 4th positive control site of granulocyte gelatinase associated lipocalin standard substance.
First detectable label composition can be enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence thing
Matter or radioactive materials.Second detectable label composition can be enzyme, prothetic group, fluorescent material, shiner
Matter, bioluminescence material or radioactive materials.3rd detectable label composition can be enzyme, prothetic group, glimmering
Stimulative substance, luminescent substance, bioluminescence material or radioactive materials.4th detectable label composition is permissible
For 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 can detect
Marked member and the 4th detectable label composition are horseradish peroxidase (HRP).
In present embodiment, diabetic nephropathy early screening kit also includes detecting liquid.The solute of detection liquid
For TMB (3,3', 5,5'-tetramethyl benzidine).
In present embodiment, the first foot glycocalicin antibody (purchased from Sigma company, article No.: AMAB90667)
For monoclonal antibody, the second foot glycocalicin antibody (purchased from Sigma company, article No.: SAB2500809)
For polyclonal antibody.
In present embodiment, the first type Ⅳ collagen protein antibodies (purchased from Sigma company, article No.: C1926)
For monoclonal antibody, the second type Ⅳ collagen protein antibodies (purchased from Abcam company, article No.: ab6586) is many
Clonal antibody.
In present embodiment, the first liver type fatty acid binding protein antibody (purchased from Sigma company, article No.:
WH0002167M1) it is monoclonal antibody, the second liver type fatty acid binding protein antibody (Abcam company,
Article No.: ab101837) it is polyclonal antibody.
In present embodiment, the first neutrophil gelatinase-associated lipocalin NGAL antibody is (purchased from Abcam
Company, article No.: ab23477) it is monoclonal antibody, the second neutrophilic granulocyte gelatinase related lipid delivery egg
Bai Kangti (Abcam company, article No.: ab166677) is polyclonal antibody.
This diabetic nephropathy early screening kit is by foot glycocalicin, type Ⅳ collagen albumen, liver type fat
Fat acid binding protein and neutrophil gelatinase-associated lipocalin NGAL are as biomarker, Ke Yigen
Border testing result factually, provides auxiliary judgment for injury of kidney, thus provides auxiliary for distinguishing early diabetic nephropathy
Help.
The biological marker object detecting method of an embodiment as shown in Figure 1, uses above-mentioned diabetic nephropathy
Early screening kit, comprises the steps:
S10, provide and be provided with the substrate of detection site, detectable is dripped in detection site, fully reacts
After by detection site washes clean.
Detectable includes the first detectable, the second detectable, the 3rd detectable and the 4th detection examination
Agent.
The solute of the first detectable is the first foot glycocalicin antibody, and the solute of the second detectable is first
Type Ⅳ collagen protein antibodies, the solute of the 3rd detectable is the first liver type fatty acid binding protein antibody, the
The solute of four detectable is the first neutrophil gelatinase-associated lipocalin NGAL antibody.
The solute of capture agent include combining the first detectable label composition the second foot glycocalicin antibody,
Combine the second type Ⅳ collagen protein antibodies of the second detectable label composition, combine the 3rd detectable label
Second liver type fatty acid binding protein antibody of composition is neutral with combine the 4th detectable label composition second
Granulocyte gelatinase associated lipocalin antibody.
In present embodiment, detection site includes the first detection site, the second detection site, the 3rd detecting position
Point and the 4th detection site, corresponding first detectable of the first detection site, the second detection site correspondence second
Detectable, corresponding 3rd detectable of the 3rd detection site, corresponding 4th detectable of the 4th detection site.
S10 is particularly as follows: by the first detectable dropping in the first detection site, exist the second detectable dropping
Second detection site, by the 3rd detectable dropping in the 3rd detection site, exists the 4th detectable dropping
Respectively by the first detection site, the second detection site, the 3rd detecting position after 4th detection site, fully reaction
Point and the 4th detection site washes clean.
The operation of washing can use tradition PBS diluent, is specifically as follows 0.01mol/L PBS (pH=7.4)
+ 0.05% tween 20.
S20, by after washes clean detection site close, by detection site washes clean after having closed.
S20 particularly as follows: respectively by the first detection site after washes clean, the second detection site, the 3rd detection
Site and the 4th detection site are closed, after having closed respectively by the first detection site, the second detection site,
3rd detection site and the 4th detection site washes clean.
Defatted milk powder or bovine serum albumin can be dissolved in PBS (pH=7.4,0.01mol/L) and obtain by confining liquid
Arrive.
S30, sample drop to be detected is added in and has closed and in the detection site of washes clean, fully after reaction
By detection site washes clean.
S30 is particularly as follows: drip sample to be detected respectively and closing and the first detecting position of washes clean
In point, the second detection site, the 3rd detection site and the 4th detection site, fully respectively by first after reaction
Detection site, the second detection site, the 3rd detection site and the 4th detection site washes clean.
In present embodiment, samples selection urine specimen to be detected.
In general, urine specimen, before detecting, needs to be diluted, and extension rate can be 100
Times.
S40, capture agent is dripped in detection site, fully by detection site washes clean after reaction.
S40 is particularly as follows: drip capture agent respectively in the first detection site, the second detection site, the 3rd inspection
On location point and the 4th detection site, fully after reaction respectively by the first detection site, the second detection site,
3rd detection site and the 4th detection site washes clean.
S50, detection site whether contain the first detectable label composition, the second detectable label composition, the 3rd
Detectable label composition and the 4th detectable label composition, and obtain in sample to be detected according to testing result
Foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein fat relevant with neutrophilic granulocyte gelatinase
The content of matter transporter.
S50 is particularly as follows: detect the first detection site, the second detection site, the 3rd detection site and the 4th respectively
Whether contain the first detectable label composition, the second detectable label composition in detection site, the 3rd can detect
Marked member and the 4th detectable label composition, and determine in sample to be detected whether contain according to testing result
There are sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein relevant with neutrophilic granulocyte gelatinase
Lipocalin protein.
This injury of kidney detection method can be that injury of kidney provides auxiliary judgment, thus for distinguishing early diabetes
Nephropathy provides auxiliary.
Concrete, cutoff value, the sufficient glycocalyx egg in the sample to be detected that then can will obtain can be first set
In vain, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophilic granulocyte gelatinase related lipid delivery egg
White content contrasts with cutoff value.
(1) when the sufficient glycocalicin in sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein
It is judged to the positive during with the content of neutrophil gelatinase-associated lipocalin NGAL all more than cutoff value,
The most then it is judged to feminine gender.This decision method is applicable to further confirm that preliminary screening results.
(2) when the sufficient glycocalicin in sample to be detected, type Ⅳ collagen albumen, liver type fatty acid binding protein
With the content of neutrophil gelatinase-associated lipocalin NGAL at least one more than cutoff value time judge
For the positive, the most then it is judged to feminine gender.This decision method is applicable to the preliminary examination of big crowd's sample.
Above-mentioned two kinds of decision methods are respectively arranged with advantage, can be used alone, it is also possible to be used in combination.
In a preferred embodiment, after correcting with serum creatinine (creatinine, Cr), Podocalyxin
Concentration cutoff value be 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 4.75 μ g/g Cr, and the concentration cutoff value of NGAL is 56.8 μ g/g Cr.
This biological marker object detecting method can be also used for assessing the effectiveness of Renal injury grade method, specifically
Include treating sample after front sample and treatment for: sample to be detected；Sample mesopodium glycocalyx before the treatment that will obtain
Albumen, type Ⅳ collagen albumen, liver type fatty acid binding protein and the delivery of neutrophilic granulocyte gelatinase related lipid
The content of the albumen sufficient glycocalicin in sample, type Ⅳ collagen albumen, liver type fatty acid after treatment are combined egg
White and neutrophil gelatinase-associated lipocalin NGAL content contrasts, and assesses according to comparison result
The effectiveness of Therapeutic Method.
The concrete evaluation process of the effectiveness of above-mentioned Therapeutic Method is: if the biological marker after Zhi Liao in sample
The concentration of thing finds to decline or remain unchanged compared with sample before treatment, then show that treatment is effectively；If sent out
Now 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, passes through
Foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein fat relevant with neutrophilic granulocyte gelatinase
Matter transporter, as biomarker, can provide auxiliary judgment according to actually detected result for injury of kidney,
Thus provide auxiliary for distinguishing early diabetic nephropathy.
Present inventor combines document report and development test, the sufficient glycocalicin (podocalyxin) of discovery,
Type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophilic granulocyte gelatin
The generation development of enzyme associated lipocalin (NGAL) and diabetic nephropathy disease in early days is closely related, simultaneously
Utilizing protein chip detection to find, in diabetic nephropathy patient urine, the content of these 4 kinds of marks is significantly high
In normal control (p < 0.05), and increase along with advancing of disease.
Detect this 4 kinds of biomarkers simultaneously, utilize it to combine change and diabetic nephropathy is predicted, can
Sensitive, special examination early diabetic nephropathy, has the most wide market application foreground.
Detect these 4 kinds of biomarkers simultaneously and have the advantage that 1) in terms of Renal Structure functional perspective, sugar
The sick nephropathy earlier damage of urine is it may happen that in glomerule, also it may happen that in renal tubules, this test kit is selected
Four kinds of biomarkers had both comprised glomerular injury specific biological mark (foot glycocalicin, type Ⅳ collagen egg
In vain), injury of renal tubular specific markers (liver type fatty acid binding protein and neutrophilic granulocyte gelatinase are comprised again
Associated lipocalin), detect hence with these 4 kinds of biomarker combinations simultaneously, help avoid leakage
Examine, it is achieved Diabetic Nephropathy early finds, early diagnosis；2) from diabetic nephropathy molecular pathway, glycosuria
Sick nephropathy damage mechanisms relates to multiple molecular pathway, as anti-in renal fibrosis (type Ⅳ collagen albumen), inflammation
Should (neutrophil gelatinase-associated lipocalin NGAL), oxidative stress (liver type fatty acid binding protein)
Deng, from multiple molecular pathway predictive diseases, contribute to diabetes and early find；Therefore, the sufficient glycocalyx of detection simultaneously
Albumen, type Ⅳ collagen albumen, liver type fatty acid binding protein and the delivery of neutrophilic granulocyte gelatinase related lipid
These 4 kinds of biomarkers of albumen, and by selecting rational decision method, substantially increase renal function and damage
The sensitivity of triage survey and specificity.
Additionally, foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophilic granulocyte are bright
Glue enzyme associated lipocalin is also used as biomarker in preparation injury of kidney diagnostic reagent or injury of kidney
The diagnostic equipment is applied.
Be below specific embodiment, in embodiment occur various instruments and reagent if not otherwise specified,
All use this area conventional instrument or reagent.
Below in conjunction with specific embodiment to the screening of 4 kinds of biomarkers, the analysis of the application and should be used as into
The detailed description of one step.
Embodiment 1: the screening of diabetogenous nephrosis disease early diagnosis biomarker and checking
One, experimental technique
1, pathology screening
Choosing experimenter's totally 205 example of known health, wherein normal healthy controls 50 example, type 2 diabetes mellitus is suffered from
Person 155 example (the most all patients suffer from the diabetes time limit and are all higher than 2 years)；And according to albuminuria situation to 155
Example diabetics divides, wherein normal albuminuria 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 audited mechanism by ethics and ratified, and all experimenters all sign Informed Consent Form.
2, the collection of Urine specimens and process
Collect the urine specimen (30mL) discharged for the first time in experimenter early morning with sterile chamber, 4 DEG C of 8000g from
Heart 30min, takes supernatant, is stored in-80 DEG C and tests for protein science.All urine specimens are not all using
Collect before any Drug therapy.When carrying out protein science experiment, by same by stages from the urine of different patients
Liquid sample mixes, for protein science research.
3.iTRAQ quantitative proteomics technology
Utilize ProteoMinerTMProtein enrichment test kit (Bio-Rad, article No.: 163-3006) removes Gao Feng
Degree albumen, 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) operating procedure processes, mark
Note difference group urine protein sample.Operating procedure is summarized as follows: take each group of each 100 μ g of urine protein sample, often pipe point
Not adding the acetone (acetone: sample volume ratio=6:1) of pre-cooling ,-20 DEG C precipitate 1 hour, and 12000rpm surpasses
After the centrifugal 15min of speed, precipitation is resuspended in 20 μ L to dissolve in buffer.Reduction, half Guang closed in urine protein
Propylhomoserin, trypsinization, then utilize iTRAQ labelled reagent 114,115,116,117 labelling respectively normal
Albuminuria patient, Microalbuminuria patient, High-grade Proteinuria patient and the urine protein of normal control.Equivalent is mixed
Close the urine protein peptide after labelling, utilize strong cation exchange chromatography, reversely chromatography to separate successively, mass spectrum skill
Art is analyzed (SCX-RPLC-MS/MS), and the albumen in urine identified by Mascot software, finds disease
Differentially expressed protein between group and comparison and disease group.
4, ELISA method checking differentially expressed protein
Utilize differential protein (foot glycocalicin, type Ⅳ collagen albumen, liver that elisa technique confirms to search out
Type fatty acid binding protein and neutrophil gelatinase-associated lipocalin NGAL) in normal control and each disease
Expression in sick group urine, it is intended to the accuracy of checking mass-spectrometric technique result and these differential proteins are as sugar
The accuracy of urine sick nephropathy biomarker (Biomarker) in early days.Specifically, dilution buffer is utilized
Urine 1:100 is diluted, then utilizes the elisa plate for different protein biomarker to measure different disease
The content of biomarker, each sample repeated measure three times in sick group urine.
5, statistical analysis
Data SPSS 14.0 software (Statistical Product and Service Solutions, " statistical product
With service solution " software) process, represent with mean+SD.With variance analysis (Analysis of
Variance, ANOVA) carry out comparing between the group of normal distribution data；With receiver operator characteristics's curve (ROC)
Analyze accuracy, specificity and the susceptiveness of the prediction of various biomarker.P < 0.05 represents have notable system
Difference learned by meter.
6,4 kinds of biomarker comprehensive detection early diabetic nephropathies
Diabetic nephropathy is a kind of Complex Complications, may result in injury of renal tubular at the diabetic nephropathy initial stage,
Or cause glomerular injury.4 kinds of biomarkers in the application, its mesopodium glycocalicin familial combined hyperlipidemia glue
Former albumen mainly reflects glomerular injury；Liver type fatty acid binding protein fat relevant with neutrophilic granulocyte gelatinase
Matter transporter mainly reflects injury of renal tubular.For diagnosing early diabetic nephropathy accurately, this experiment is combined
Use two or more biomarker, use the decision method of (1) and (2) to carry out synthetic determination.
(1) all biomarkers contained in combination are judged to the positive when being the positive (more than cutoff value),
It is judged to feminine gender in addition.This decision method is applicable to further confirm that preliminary screening results.
(2) biomarker contained in combination has one to be to be judged as sun time positive (more than cutoff value)
Property, it is judged as feminine gender in addition.This decision method is applicable to the preliminary examination of big crowd's sample.
The study find that the kind of biomarker combinations and the difference of quantity can cause medical diagnosis on disease sensitive
Spend different with specificity.Therefore optimal 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's health and biochemical indicator
Note: ND:No Difference；* p < 0.001vs. normal healthy controls；#The normal albuminuria of p < 0.001vs is suffered from
Person；§ p < 0.001vs Microalbuminuria patient, HbA1CFor glycolated hemoglobin, Serum Cr refers to serum creatinine；
UACR refers to urinaryalbumin and creatinine ratio.
2, urine protein science result describes
Early stage screening stage, protein science experiment repeats for 2 times to identify 465 and 478 albumen respectively,
Secondary experiment identifies 322 albumen jointly.114/117 (normal albuminuria group/normal in secondary is tested
Comparison), 115/117 (Microalbuminuria group/normal control) and 116/117 (High-grade Proteinuria group/normal control)
ITRAQ ratio all demonstrates totally 62, the albumen (ratio<0.667 or ratio>1.5) of differential expression.Yin Ben
Invention is intended to find diabetic nephropathy (normal albuminuria phase or I and II phase) biomarker in early days, to sending out
62 albumen of raw differential expression are further analyzed, found that have 15 albumen at diabetic nephropathy
There is differential expression (ratio<0.667 or ratio>1.5) in the difference of group by stages.
By document report and bioinformatic analysis (Gene Ontology and KEGG Pathway analyzes)
15 differentially expressed proteins identified are further analyzed, discovery foot glycocalicin (podocalyxin),
Type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophilic granulocyte gelatin
Enzyme associated lipocalin (NGAL) plays non-during the renal complications disease that diabetes cause is sent out
The most important effect.
3, ELISA method checking differentially expressed protein
Utilize the ELISA method checking differentially expressed protein selected of urine protein science testing sieve: foot glycocalicin,
Type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin NGAL,
Obtain table 3 below.
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 albuminuria patient；§ p < 0.001vs Microalbuminuria.
By table 3 and Fig. 2 a～Fig. 2 d it can be seen that diabetic groups urine mesopodium glycocalicin (podocalyxin),
Type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophilic granulocyte gelatin
The expression of enzyme associated lipocalin (NGAL) is significantly higher than normal healthy controls, and tight along with conditions of patients
Weigh (albuminuria increase) and increase.
To glomerular filtration rate, albuminuria and the correlation analysis of 4 kinds of protein markers, obtain table 4 below.
Table 4: glomerular filtration rate, albuminuria and the correlation analysis of 4 kinds of protein markers
As can be seen from Table 4, the expression of above-mentioned 4 kinds of albumen and urine protein are notable positive correlation, with eGFR
In notable negative correlation.
Utilize in receiver operator characteristics's curve (ROC) analysis of diabetes nephrotic and normal healthy controls urine
Four kinds of biomarkers, obtain Fig. 3 a～Fig. 3 d.
Table 5 below can be obtained by Fig. 3 a～Fig. 3 d.
5: four kinds of biomarker ROC curve of table are analyzed
As can be seen from Table 5, below podocalyxin, Collagen IV, L-FABP and NGAL curve
Long-pending (AUC) is 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, when choosing the highest " youden " index, in urine four
Plant the sensitivity of biomarker, specificity and concentration cutoff value and be respectively as follows: podocalyxin (sensitivity
89.8%；Specificity is 76.2%；Concentration cutoff value is 67.15ng/g creatinine), Collagen IV (spirit
Sensitivity 71.2%；Specificity is 90.5%；Concentration cutoff value is 2.945 μ g/g creatinine), L-FABP (spirit
Sensitivity 79.6%；Specificity is 95.2%；Concentration cutoff value is 4.75 μ g/g creatinine) and NGAL (spirit
Sensitivity 84.7%；Specificity is 85.8%；Concentration cutoff value is 49.85ng/mL).
Result above shows that these 4 kinds of protein moleculars may take part in diabetic nephropathy disease and send out process, for believable
Early diabetic nephropathy biomarker.
4, diabetic nephropathy method of early diagnosis
When carrying out diabetes diagnosis, can be cut by the measurement result of normal healthy controls and four kinds of albumen molecular concentrations
Disconnected value determines that these 4 kinds of protein marker relative amounts, with reference to normal range, are surveyed in conjunction with four kinds of biomarkers
Amount result, if any of which protein concentration is beyond normal range, can tentative diagnosis be early diabetes kidney
Sick；In combination with the index such as GFR, albuminuria, early diabetic nephropathy is carried out comprehensive assessment.
The most kinds of biomarker combinations carry out diabetogenous nephrosis disease early diagnosis
Urine will be combined to diagnose the sensitivity of early diabetic nephropathy and special at four kinds of biomarkers
Degree.Wherein NGAL measured value Cr is corrected, analyze its cutoff value by ROC curve.NGAL in urine
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。
Various combinations are carried out statistical disposition, compares sensitivity and the specificity of various combination, obtain table 6 below.
Wherein, in the combination that symbol " ∩ " represents, all biomarkers contained by this combination are the positive and (block
More than value) time be judged as the positive, meter sensitivity and specificity.In the combination that symbol " ∪ " represents, should
Biomarker contained by combination has one to be to be judged as the positive time positive (more than cutoff value), calculates spirit
Sensitivity and specificity.
Table 6: be used for diagnosing the combination sensitivity of early diabetic nephropathy protein biomarker and specificity
As can be seen from Table 6, according to Integrated biomarker kind and the change of quantity, sensitivity and special
Degree also can change.This represents the combination selecting biomarker according to experiment purpose, 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) it is suitable for first examination；On the contrary, combination (the IV Collagen that specificity is high
∩NGAL；IV Collagen∩Podocalyxin；NGAL∩Podocalyxin；L-FABP∩
Podocalyxin；IV Collagen ∩ NGAL ∩ L-FABP ∩ Podocalyxin) it is suitable for quadratic search or three
Secondary inspections etc. need to carry out the higher judgement of reliability and check.Such as, if the positive occurs when first examination,
The combined method higher now by specificity carries out secondary judgement to initial examination result, is finally given effectively
And reliable early diabetic nephropathy diagnostic result.
As can be seen from Table 6, " IV Collagen, NGAL, L-FABP and Podocalyxin " this group
Close, can be by selecting suitable decision method, it is thus achieved that the highest sensitivity and specificity.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more 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 this area
Those of ordinary skill for, without departing from the inventive concept of the premise, it is also possible to make some deformation and
Improving, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended
Claim is as the criterion.
Claims (8)
1. a diabetic nephropathy early screening kit, it is characterised in that include detectable and capture examination
Agent；
Described detectable is by the first detectable, the second detectable, the 3rd detectable and the 4th detection
Reagent forms, and the solute of described first detectable is the first foot glycocalicin antibody, described second detection examination
The solute of agent is the first type Ⅳ collagen protein antibodies, and the solute of described 3rd detectable is the first liver type fat
Acid binding protein antibody, the solute of described 4th detectable is the first neutrophilic granulocyte gelatinase related lipid
Transporter antibody；
The solute of described capture agent includes that the second foot glycocalicin combining the first detectable label composition resists
Body, combine the second type Ⅳ collagen protein antibodies of the second detectable label composition, combine the 3rd and can detect
Second liver type fatty acid binding protein antibody of marked member and combine the second of the 4th detectable label composition
Neutrophil gelatinase-associated lipocalin NGAL antibody；
Described first foot glycocalicin antibody is monoclonal antibody, and described second foot glycocalicin antibody is many grams
Grand antibody；
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, described second liver type fatty acid knot
Hop protein antibody is polyclonal antibody；
Described first neutrophil gelatinase-associated lipocalin NGAL antibody is monoclonal antibody, described
Two neutrophil gelatinase-associated lipocalin NGAL antibody are polyclonal antibody.
2. diabetic nephropathy early screening kit as claimed in claim 1, it is characterised in that described sugar
The sick nephropathy early screening kit of urine also includes foot glycocalicin standard substance, type Ⅳ collagen protein standard substance, liver
Type fatty acid binding protein standard substance and neutrophil gelatinase-associated lipocalin NGAL standard substance.
3. diabetic nephropathy early screening kit as claimed in claim 1, it is characterised in that described the
One detectable label composition is enzyme, prothetic group, luminescent substance or radioactive materials；
Described second detectable label composition is enzyme, prothetic group, luminescent substance or radioactive materials；
Described 3rd detectable label composition is enzyme, prothetic group, luminescent substance or radioactive materials；
Described 4th detectable label composition is enzyme, prothetic group, luminescent substance or radioactive materials.
4. diabetic nephropathy early screening kit as claimed in claim 3, it is characterised in that described the
One detectable label composition is fluorescent material or bioluminescence material.
5. diabetic nephropathy early screening kit as claimed in claim 3, it is characterised in that described the
Two detectable label compositions are fluorescent material or bioluminescence material.
6. diabetic nephropathy early screening kit as claimed in claim 3, it is characterised in that described the
Three detectable label compositions are fluorescent material or bioluminescence material.
7. diabetic nephropathy early screening kit as claimed in claim 3, it is characterised in that described the
Four detectable label compositions are fluorescent material or bioluminescence material.
8. foot glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophilic granulocyte gelatinase
Associated lipocalin is preparing diabetogenous nephrosis disease early diagnosis reagent or diabetogenous nephrosis as biomarker
Application in disease early diagnosis device.
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| PCT/CN2015/076924 WO2016149972A1 (en) | 2015-03-24 | 2015-04-18 | Diabetic nephropathy early screening reagent kit, biomarker testing method and applications |
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| CN104764886B (en) * | 2015-03-24 | 2016-08-24 | 深圳市贝沃德克生物技术研究院有限公司 | Diabetic nephropathy Test paper box, biological marker object detecting method and application in early days |
| 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 |
| 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 |
| CN205317786U (en) * | 2016-01-16 | 2016-06-15 | 深圳市易特科信息技术有限公司 | A gold -labeled antibody test paper strip for early diabetic nephropathy detects |
| CN106066399A (en) * | 2016-05-24 | 2016-11-02 | 深圳市前海安测信息技术有限公司 | The preparation method of the colloidal gold strip of detection early diabetic nephropathy |
| KR102355835B1 (en) * | 2016-10-03 | 2022-01-25 | 휴비트 제노믹스 가부시키가이샤 | Test method enabling specific diagnosis of early conditions of 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 |
| 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 |
| CN112946303B (en) * | 2021-02-23 | 2023-10-20 | 江苏省中医院 | TAG54:2-FA18:1 and application of composition thereof in diagnosis of diabetes and diabetic nephropathy |
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| EP2442106B1 (en) * | 2009-06-10 | 2015-08-19 | Masanori Hara | Method for test on diabetic nephropathy |
| JPWO2013183596A1 (en) * | 2012-06-06 | 2016-01-28 | 国立大学法人名古屋大学 | Biomarkers for kidney disease and uses thereof |
| US20130338021A1 (en) * | 2012-06-14 | 2013-12-19 | Wisconsin Alumni Research Foundation | Methods and compositions for the detection of complications of diabetes |
| CN204479594U (en) * | 2015-03-24 | 2015-07-15 | 深圳市易特科信息技术有限公司 | Based on the diabetic nephropathy early detection kit of biomarker |
| CN104764886B (en) * | 2015-03-24 | 2016-08-24 | 深圳市贝沃德克生物技术研究院有限公司 | Diabetic nephropathy Test paper box, biological marker object detecting method and application in early days |
| 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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