CN204479594U - Based on the diabetic nephropathy early detection kit of biomarker - Google Patents
Based on the diabetic nephropathy early detection kit of biomarker Download PDFInfo
- Publication number
- CN204479594U CN204479594U CN201520169728.7U CN201520169728U CN204479594U CN 204479594 U CN204479594 U CN 204479594U CN 201520169728 U CN201520169728 U CN 201520169728U CN 204479594 U CN204479594 U CN 204479594U
- Authority
- CN
- China
- Prior art keywords
- reagent tube
- detection
- antibody
- biomarker
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 210
- 239000000090 biomarker Substances 0.000 title claims abstract description 82
- 208000007342 Diabetic Nephropathies Diseases 0.000 title claims abstract description 81
- 208000033679 diabetic kidney disease Diseases 0.000 title claims abstract description 80
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 112
- 102000013519 Lipocalin-2 Human genes 0.000 claims abstract description 94
- 108010051335 Lipocalin-2 Proteins 0.000 claims abstract description 94
- 102100026745 Fatty acid-binding protein, liver Human genes 0.000 claims abstract description 84
- 101710188974 Fatty acid-binding protein, liver Proteins 0.000 claims abstract description 84
- 101710189565 Fatty acid-binding protein, liver-type Proteins 0.000 claims abstract description 84
- 102000004266 Collagen Type IV Human genes 0.000 claims abstract description 60
- 108010042086 Collagen Type IV Proteins 0.000 claims abstract description 60
- 101800001015 Glycocalicin Proteins 0.000 claims abstract description 42
- 102400000446 Glycocalicin Human genes 0.000 claims abstract description 42
- 239000000203 mixture Substances 0.000 claims description 70
- 239000000243 solution Substances 0.000 claims description 60
- 239000000463 material Substances 0.000 claims description 24
- 239000003795 chemical substances by application Substances 0.000 claims description 22
- 239000000758 substrate Substances 0.000 claims description 17
- 239000000126 substance Substances 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 230000029918 bioluminescence Effects 0.000 claims description 12
- 238000005415 bioluminescence Methods 0.000 claims description 12
- 239000012857 radioactive material Substances 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 12
- 238000007689 inspection Methods 0.000 claims description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 4
- 239000000020 Nitrocellulose Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 229920001220 nitrocellulos Polymers 0.000 claims description 4
- CMZYGFLOKOQMKF-UHFFFAOYSA-N 1-(3,5-dimethylphenyl)-3,5-dimethylbenzene Chemical group CC1=CC(C)=CC(C=2C=C(C)C=C(C)C=2)=C1 CMZYGFLOKOQMKF-UHFFFAOYSA-N 0.000 claims description 3
- 239000004677 Nylon Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 229920001778 nylon Polymers 0.000 claims description 3
- 230000006378 damage Effects 0.000 abstract description 19
- 210000003734 kidney Anatomy 0.000 abstract description 14
- 208000027418 Wounds and injury Diseases 0.000 abstract description 11
- 208000014674 injury Diseases 0.000 abstract description 11
- 238000012360 testing method Methods 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 description 45
- 210000002700 urine Anatomy 0.000 description 45
- 102000004169 proteins and genes Human genes 0.000 description 44
- 238000000034 method Methods 0.000 description 28
- 102000008186 Collagen Human genes 0.000 description 26
- 229920001436 collagen Polymers 0.000 description 26
- 108010035532 Collagen Proteins 0.000 description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 24
- 102000004401 podocalyxin Human genes 0.000 description 21
- 108090000917 podocalyxin Proteins 0.000 description 21
- 201000010099 disease Diseases 0.000 description 19
- 230000035945 sensitivity Effects 0.000 description 17
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 16
- 206010012601 diabetes mellitus Diseases 0.000 description 16
- 239000013641 positive control Substances 0.000 description 12
- 239000013642 negative control Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 9
- 229940109239 creatinine Drugs 0.000 description 8
- 238000013399 early diagnosis Methods 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 206010001580 Albuminuria Diseases 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 206010027525 Microalbuminuria Diseases 0.000 description 5
- 230000024924 glomerular filtration Effects 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 208000013901 Nephropathies and tubular disease Diseases 0.000 description 4
- 206010029164 Nephrotic syndrome Diseases 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000000585 glomerular basement membrane Anatomy 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 208000009928 nephrosis Diseases 0.000 description 4
- 231100001027 nephrosis Toxicity 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000005239 tubule Anatomy 0.000 description 4
- 230000005779 cell damage Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000003990 molecular pathway Effects 0.000 description 3
- 201000001474 proteinuria Diseases 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 201000001376 Familial Combined Hyperlipidemia Diseases 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 210000000557 podocyte Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 1
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 108050008290 Serpin H1 Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011981 development test Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000012854 evaluation process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 210000003584 mesangial cell Anatomy 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 210000005234 proximal tubule cell Anatomy 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008085 renal dysfunction Effects 0.000 description 1
- 201000002793 renal fibrosis Diseases 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000012437 strong cation exchange chromatography Methods 0.000 description 1
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The utility model discloses a kind of diabetic nephropathy early detection kit based on biomarker, comprise and detect Reagent Tube group; Described detection Reagent Tube group comprises two or three in the first detection Reagent Tube, the second detection Reagent Tube, the 3rd detection Reagent Tube and the 4th detection Reagent Tube; Described first detects Reagent Tube comprises the first sufficient glycocalicin antibody-solutions, described second detects Reagent Tube comprises the first type Ⅳ collagen protein antibodies solution, described 3rd detects Reagent Tube comprises the first liver type fatty acid binding protein antibody-solutions, and the described 4th detects Reagent Tube comprises the first neutrophil gelatinase-associated lipocalin antibody-solutions.This diabetic nephropathy early detection kit based on biomarker by two kinds in sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin or three kinds as biomarker, can according to actual testing result, for injury of kidney provides auxiliary judgment, thus provide auxiliary for distinguishing early diabetic nephropathy.
Description
Technical field
The utility model relates to field of biological detection, especially relates to a kind of diabetic nephropathy early detection kit based on biomarker.
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 detect Reagent Tube group box, and carrying 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.
Utility model content
Based on this, be necessary to provide a kind of diabetic nephropathy early detection kit based on biomarker that may be used for distinguishing early diabetic nephropathy.
Based on a diabetic nephropathy early detection kit for biomarker, comprise and detect Reagent Tube group, substrate and capture agent pipe group;
Described detection Reagent Tube group comprises two or three in the first detection Reagent Tube, the second detection Reagent Tube, the 3rd detection Reagent Tube and the 4th detection Reagent Tube, described first detects Reagent Tube comprises the first sufficient glycocalicin antibody-solutions, described second detects Reagent Tube comprises the first type Ⅳ collagen protein antibodies solution, described 3rd detects Reagent Tube comprises the first liver type fatty acid binding protein antibody-solutions, and the described 4th detects Reagent Tube comprises the first neutrophil gelatinase-associated lipocalin antibody-solutions;
Described substrate is provided with the detection site of arrayed, described detection site comprises two or three in the first detection site, the second detection site, the 3rd detection site and the 4th detection site, described first detection site corresponding described first detects Reagent Tube, described second detection site corresponding described second detects Reagent Tube, described 3rd detection site the corresponding described 3rd detects Reagent Tube, and described 4th detection site the corresponding described 4th detects Reagent Tube;
Described capture agent pipe group comprises the second sufficient glycocalicin antibody-solutions combining the first detectable label composition, the second type Ⅳ collagen protein antibodies solution combining the second detectable label composition, the second liver type fatty acid binding protein antibody-solutions combining the 3rd detectable label composition and two kinds or three kinds of combining in the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition;
Described detection Reagent Tube group is mated mutually with described detection site, and described detection Reagent Tube group is mated mutually with described capture agent pipe group.
In one embodiment, described detection Reagent Tube group comprises described first detection Reagent Tube and described 4th detection Reagent Tube;
Described detection site comprises described first detection site and described 4th detection site;
The second sufficient glycocalicin antibody-solutions of the first detectable label composition and described the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition is combined described in described capture agent pipe group comprises.
In one embodiment, described detection Reagent Tube group comprises described second detection Reagent Tube, described 3rd detection Reagent Tube and described 4th detection Reagent Tube;
Described detection site comprises described second detection site, described 3rd detection site and described 4th detection site;
Combine described in described capture agent pipe group comprises the second type Ⅳ collagen protein antibodies solution of the second detectable label composition, described in combine the second liver type fatty acid binding protein antibody-solutions of the 3rd detectable label composition and described the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition.
In one embodiment, described first detection site, described second detection site, described 3rd detection site and described 4th detection site are 4.
In one embodiment, described substrate is nitrocellulose filter, nylon membrane or glass substrate.
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.
In one embodiment, described first detectable label composition, described second detectable label composition, described 3rd detectable label composition and described 4th detectable label composition are horseradish peroxidase.
In one embodiment, the described diabetic nephropathy early detection kit based on biomarker also comprises two or three in sufficient glycocalicin standard QC group, type Ⅳ collagen protein standard QC group, liver type fatty acid binding protein standard QC group and neutrophil gelatinase-associated lipocalin standard QC group;
Described sufficient glycocalicin standard QC group comprises sufficient glycocalicin standard solution, described type Ⅳ collagen protein standard QC group comprises type Ⅳ collagen protein standard substance solution, described liver type fatty acid binding protein standard QC group comprises liver type fatty acid binding protein standard solution, and described neutrophil gelatinase-associated lipocalin standard QC group comprises neutrophil gelatinase-associated lipocalin standard solution.
In one embodiment, the described diabetic nephropathy early detection kit based on biomarker also comprises inspection liquid-measuring tube group, and described inspection liquid-measuring tube group comprises 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution.
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.
This diabetic nephropathy early detection kit based on biomarker by two kinds in sufficient glycocalicin, type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin or three kinds 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 structural representation of the diabetic nephropathy early detection kit based on biomarker 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 the diabetic nephropathy early detection kit based on biomarker is 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 detection kit based on biomarker of one embodiment, comprises and detects Reagent Tube group, substrate and capture agent pipe group.
Detection Reagent Tube group comprises two or three in the first detection Reagent Tube, the second detection Reagent Tube, the 3rd detection Reagent Tube and the 4th detection Reagent Tube.
First detects Reagent Tube comprises the first sufficient glycocalicin antibody-solutions, second detects Reagent Tube comprises the first type Ⅳ collagen protein antibodies solution, 3rd detects Reagent Tube comprises the first liver type fatty acid binding protein antibody-solutions, and the 4th detects Reagent Tube comprises the first neutrophil gelatinase-associated lipocalin antibody-solutions.
Substrate can be nitrocellulose filter, nylon membrane or glass substrate.Substrate is provided with the detection site of arrayed, detection site comprises two or three in the first detection site, the second detection site, the 3rd detection site and the 4th detection site, first detection site corresponding first detects Reagent Tube, second detection site corresponding second detects Reagent Tube, 3rd detection site the corresponding 3rd detects Reagent Tube, and the 4th detection site the corresponding described 4th detects Reagent Tube.
Capture agent pipe group comprises the second sufficient glycocalicin antibody-solutions combining the first detectable label composition, the second type Ⅳ collagen protein antibodies solution combining the second detectable label composition, the second liver type fatty acid binding protein antibody-solutions combining the 3rd detectable label composition and two kinds or three kinds of combining in the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition.
Detect Reagent Tube group mutually to mate with detection site.Here, detect Reagent Tube group to be specially with the meaning that detection site is mated mutually: detect Reagent Tube group and comprise the first detection Reagent Tube (or second detects Reagent Tube, or the 3rd detects Reagent Tube, or the 4th detects Reagent Tube) time, detection site comprises the first detection site (or the second detection site, or the 3rd detection site, or the 4th detection site).
Detect Reagent Tube group mutually to mate with capture agent pipe group.Here, detect Reagent Tube group to be specially with the meaning that capture agent pipe group is mated mutually: detect Reagent Tube group and comprise the first detection Reagent Tube (or second detects Reagent Tube, or the 3rd detects Reagent Tube, or the 4th detects Reagent Tube) time, capture agent pipe group comprises the second sufficient glycocalicin antibody-solutions combining the first detectable label composition and (or combines the second type Ⅳ collagen protein antibodies solution of the second detectable label composition, or combine the second liver type fatty acid binding protein antibody-solutions of the 3rd detectable label composition, or combine the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition).
Preferably, detect Reagent Tube group and comprise the first detection Reagent Tube and the 4th detection Reagent Tube, detection site comprises the first detection site the 4th detection site, and capture agent pipe group comprises the second sufficient glycocalicin antibody-solutions combining the first detectable label composition and the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition.
Preferably, detect Reagent Tube group and comprise the second detection Reagent Tube, the 3rd detection Reagent Tube and the 4th detection Reagent Tube, detection site comprises the second detection site, the 3rd detection site and the 4th detection site, and the solute of capture agent pipe group combines the second type Ⅳ collagen protein antibodies solution of the second detectable label composition, combine the second liver type fatty acid binding protein antibody-solutions of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition.
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).
This injury of kidney detection kit can also comprise sufficient glycocalicin standard QC group, type Ⅳ collagen protein standard QC group, liver type fatty acid binding protein standard QC group and neutrophil gelatinase-associated lipocalin standard QC group.
Foot glycocalicin standard QC group comprises sufficient glycocalicin standard solution, type Ⅳ collagen protein standard QC group comprises type Ⅳ collagen protein standard substance solution, liver type fatty acid binding protein standard QC group comprises liver type fatty acid binding protein standard solution and neutrophil gelatinase-associated lipocalin standard QC group comprises in neutrophil gelatinase-associated lipocalin standard solution two kinds or three kinds.These four kinds of standard items are all purchased from Beijing OriGene Biotechnology Co., Ltd..
Corresponding, detection site also comprises positive control site and negative control site.
This diabetic nephropathy early detection kit based on biomarker can also comprise inspection liquid-measuring tube group 80.Inspection liquid-measuring tube group 80 comprises 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution (TMB).
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 detection kit based on biomarker according to actual testing result, for injury of kidney provides auxiliary judgment, thus can provide auxiliary for distinguishing early diabetic nephropathy.
Composition graphs 1, the second detection Reagent Tube is comprised to detect Reagent Tube group, 3rd detects Reagent Tube and the 4th detects Reagent Tube, detection site comprises the second detection site, 3rd detection site and the 4th detection site, the solute of capture agent pipe group combines the second type Ⅳ collagen protein antibodies solution of the second detectable label composition, the the second liver type fatty acid binding protein antibody-solutions combining the 3rd detectable label composition and the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition are example, the diabetic nephropathy early detection kit based on biomarker of one embodiment comprises detection Reagent Tube group 10, substrate 20 and capture agent pipe group 30.
Detect Reagent Tube group 10 and comprise the second detection Reagent Tube 14, the 3rd detection Reagent Tube 16 and the 4th detection Reagent Tube 18.
Substrate 20 is provided with the detection site of arrayed.In present embodiment, nitrocellulose filter selected by substrate.
Detection site comprises the second detection site 24, the 3rd detection site 26 and the 4th detection site 28.
Second detection site 24 corresponding second detects corresponding 3rd detection Reagent Tube the 16, four detection site 28 the corresponding 4th of Reagent Tube the 14, three detection site 26 and detects Reagent Tube 18.
In present embodiment, the second detection site 24, the 3rd detection site 26 and the 4th detection site 28 are 4, avoid error by revision test.
In present embodiment, the diabetic nephropathy early detection kit based on biomarker also comprises type Ⅳ collagen protein standard QC group 50, liver type fatty acid binding protein standard QC group 60 and neutrophil gelatinase-associated lipocalin standard QC group 70.
Type Ⅳ collagen protein standard QC group 50 comprises type Ⅳ collagen protein standard substance solution, liver type fatty acid binding protein standard QC group 60 comprises liver type fatty acid binding protein standard solution, and neutrophil gelatinase-associated lipocalin standard QC group 70 comprises neutrophil gelatinase-associated lipocalin standard solution.These three kinds of standard items are all purchased from Beijing OriGene Biotechnology Co., Ltd..
Corresponding, detection site also comprises positive control site 202 and negative control site 204.Positive control site 202 and negative control site 204 are 3.Wherein, 3 positive control site 202 are corresponding with type Ⅳ collagen protein standard QC group 50, liver type fatty acid binding protein standard QC group 60 and neutrophil gelatinase-associated lipocalin standard QC group 70 respectively.
Capture agent pipe group 30 comprise combine the second detectable label composition the second type Ⅳ collagen protein antibodies solution, combine the second liver type fatty acid binding protein antibody-solutions of the 3rd detectable label composition and combine the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition.
This diabetic nephropathy early detection kit based on biomarker 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.
Below the detection method of the biomarker adopting the above-mentioned diabetic nephropathy early detection kit based on biomarker is introduced, comprises the steps:
S10, the solution detected in Reagent Tube group 10, type Ⅳ collagen protein standard QC group 50, liver type fatty acid binding protein standard QC group 60 and neutrophil gelatinase-associated lipocalin standard QC group 70 is dripped respectively in the detection site of substrate 20.
S10 is specially: detect Reagent Tube 14 by second, 3rd detects the first type Ⅳ collagen protein antibodies solution in Reagent Tube 16 and the 4th detection Reagent Tube 18, first liver type fatty acid binding protein antibody-solutions and the first neutrophil gelatinase-associated lipocalin antibody-solutions drip the second detection site 24 at substrate 20 respectively, in 3rd detection site 26 and the 4th detection site 28, simultaneously by type Ⅳ collagen protein standard QC group 50, three kinds of standard solutions in liver type fatty acid binding protein standard QC group 60 and neutrophil gelatinase-associated lipocalin standard QC group 70 drip respectively on three positive control site, by detection site washes clean after abundant reaction.
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: closed in the second detection site 24 after washes clean, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204 respectively, has closed rear respectively by the second detection site 24, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204 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 on the second detection site 24 having closed also washes clean, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204, respectively by the second detection site 24, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204 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 pipe group to be dripped in detection site, fully after reaction by detection site washes clean.
S40 is specially: dripped by the solution in capture agent pipe group 30 respectively on the second detection site 24, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204, respectively by the second detection site 24, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204 washes clean after fully reacting.
S50, detection detection site whether containing 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 second detectable label composition, the 3rd detectable label composition and the 4th detectable label composition on the second detection site 24, the 3rd detection site 26, the 4th detection site 28, positive control site 202 and negative control site 204, and determine in sample to be detected containing 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 type Ⅳ collagen albumen in sample to be detected, 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 type Ⅳ collagen albumen in sample to be detected, 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, after correcting with serum creatinine (creatinine, 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.
The detection method of this biomarker 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 type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin in rear to the content of type Ⅳ collagen albumen, liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin in sample 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, then it is invalid to show.
The detection method of the biomarker of the diabetic nephropathy early detection kit based on biomarker that this employing is above-mentioned, by 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.
Foot 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.
Detect type Ⅳ collagen albumen (Collagen IV), liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) three kinds of biomarkers simultaneously, utilize it to combine change to predict diabetic nephropathy, examination early diabetic nephropathy that can be sensitive, special, has comparatively wide market application foreground.
Detect type Ⅳ collagen albumen (Collagen IV) simultaneously, liver type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) have the following advantages: 1) from Renal Structure functional perspective, diabetic nephropathy earlier damage may betide glomerulus, also renal tubule may be betided, three kinds of biomarkers that this kit is selected both had comprised glomerular injury specific biological mark (type Ⅳ collagen albumen), comprise again renal damage specific markers (liver type fatty acid binding protein and neutrophil gelatinase-associated lipocalin), therefore utilize these three 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 type Ⅳ collagen albumen, liver type fatty acid binding protein and these three kinds of biomarkers of neutrophil gelatinase-associated lipocalin simultaneously, and by selecting rational decision method, substantially increase sensitivity and the specificity of renal dysfunction detection.
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.
(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 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, jointly identifies 322 albumen in twice experiment.In twice 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 utility model 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 | 0.989 | 0.4707 |
| IV Collagen∩NGAL∩L-FABP | 0.532 | 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) be suitable for first examination; On the contrary, combination (the IV Collagen ∩ NGAL that specificity is high; IVCollagen ∩ Podocalyxin; NGAL ∩ Podocalyxin; L-FABP ∩ Podocalyxin; IVCollagen ∩ NGAL ∩ L-FABP) 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, " NGAL and Podocalyxin ", " IV Collagen, NGAL and L-FABP ", these two combinations, by selecting suitable decision method, can obtain higher sensitivity and specificity.
The above embodiment only have expressed several embodiment of the present utility model, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the utility model the scope of the claims.It should be pointed out that for the person of ordinary skill of the art, without departing from the concept of the premise utility, can also make some distortion and improvement, these all belong to protection domain of the present utility model.Therefore, the protection domain of the utility model patent should be as the criterion with claims.
Claims (10)
1. based on a diabetic nephropathy early detection kit for biomarker, it is characterized in that, comprise and detect Reagent Tube group, substrate and capture agent pipe group;
Described detection Reagent Tube group comprises two or three in the first detection Reagent Tube, the second detection Reagent Tube, the 3rd detection Reagent Tube and the 4th detection Reagent Tube, described first detects Reagent Tube comprises the first sufficient glycocalicin antibody-solutions, described second detects Reagent Tube comprises the first type Ⅳ collagen protein antibodies solution, described 3rd detects Reagent Tube comprises the first liver type fatty acid binding protein antibody-solutions, and the described 4th detects Reagent Tube comprises the first neutrophil gelatinase-associated lipocalin antibody-solutions;
Described substrate is provided with the detection site of arrayed, described detection site comprises two or three in the first detection site, the second detection site, the 3rd detection site and the 4th detection site, described first detection site corresponding described first detects Reagent Tube, described second detection site corresponding described second detects Reagent Tube, described 3rd detection site the corresponding described 3rd detects Reagent Tube, and described 4th detection site the corresponding described 4th detects Reagent Tube;
Described capture agent pipe group comprises the second sufficient glycocalicin antibody-solutions combining the first detectable label composition, the second type Ⅳ collagen protein antibodies solution combining the second detectable label composition, the second liver type fatty acid binding protein antibody-solutions combining the 3rd detectable label composition and two kinds or three kinds of combining in the second neutrophil gelatinase-associated lipocalin antibody-solutions of the 4th detectable label composition;
Described detection Reagent Tube group is mated mutually with described detection site, and described detection Reagent Tube group is mated mutually with described capture agent pipe group.
2. as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, described detection Reagent Tube group comprises described first and detects Reagent Tube and described 4th detection Reagent Tube;
Described detection site comprises described first detection site and described 4th detection site;
The second sufficient glycocalicin antibody-solutions of the first detectable label composition and described the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition is combined described in described capture agent pipe group comprises.
3. as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, described detection Reagent Tube group comprises described second and detects Reagent Tube, described 3rd detection Reagent Tube and described 4th detection Reagent Tube;
Described detection site comprises described second detection site, described 3rd detection site and described 4th detection site;
Combine described in described capture agent pipe group comprises the second type Ⅳ collagen protein antibodies solution of the second detectable label composition, described in combine the second liver type fatty acid binding protein antibody-solutions of the 3rd detectable label composition and described the second neutrophil gelatinase-associated lipocalin antibody-solutions combining the 4th detectable label composition.
4., as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, described first detection site, described second detection site, described 3rd detection site and described 4th detection site are 4.
5., as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, described substrate is nitrocellulose filter, nylon membrane or glass substrate.
6., as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, 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.
7. as claimed in claim 6 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, described first detectable label composition, described second detectable label composition, described 3rd detectable label composition and described 4th detectable label composition are horseradish peroxidase.
8. as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, the described diabetic nephropathy early detection kit based on biomarker also comprises two or three in sufficient glycocalicin standard QC group, type Ⅳ collagen protein standard QC group, liver type fatty acid binding protein standard QC group and neutrophil gelatinase-associated lipocalin standard QC group;
Described sufficient glycocalicin standard QC group comprises sufficient glycocalicin standard solution, described type Ⅳ collagen protein standard QC group comprises type Ⅳ collagen protein standard substance solution, described liver type fatty acid binding protein standard QC group comprises liver type fatty acid binding protein standard solution, and described neutrophil gelatinase-associated lipocalin standard QC group comprises neutrophil gelatinase-associated lipocalin standard solution.
9. as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, it is characterized in that, the described diabetic nephropathy early detection kit based on biomarker also comprises inspection liquid-measuring tube group, described inspection liquid-measuring tube group comprises 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution.
10., as claimed in claim 1 based on the diabetic nephropathy early detection kit of biomarker, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520169728.7U CN204479594U (en) | 2015-03-24 | 2015-03-24 | Based on the diabetic nephropathy early detection kit of biomarker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520169728.7U CN204479594U (en) | 2015-03-24 | 2015-03-24 | Based on the diabetic nephropathy early detection kit of biomarker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN204479594U true CN204479594U (en) | 2015-07-15 |
Family
ID=53635381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201520169728.7U Expired - Fee Related CN204479594U (en) | 2015-03-24 | 2015-03-24 | Based on the diabetic nephropathy early detection kit of biomarker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN204479594U (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2016149972A1 (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 screening reagent kit, biomarker testing method and applications |
| 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 |
| CN113917153A (en) * | 2021-09-22 | 2022-01-11 | 北京松果天目健康管理有限公司 | Application of urine protein marker in preparation of kit for detecting clinical stages of diabetic nephropathy |
-
2015
- 2015-03-24 CN CN201520169728.7U patent/CN204479594U/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2016149972A1 (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 screening reagent kit, biomarker testing method and applications |
| 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 |
| CN113917153A (en) * | 2021-09-22 | 2022-01-11 | 北京松果天目健康管理有限公司 | Application of urine protein marker in preparation of kit for detecting clinical stages of diabetic nephropathy |
| CN113917153B (en) * | 2021-09-22 | 2023-11-24 | 北京松果天目健康管理有限公司 | Application of urine protein marker in preparation of kit for detecting clinical stage of diabetic nephropathy |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104764885A (en) | An early-stage screening kit for diabetic nephropathy, a biomarker detecting method and applications | |
| Puthumana et al. | Urine interleukin 18 and lipocalin 2 are biomarkers of acute tubular necrosis in patients with cirrhosis: a systematic review and meta-analysis | |
| CN204479594U (en) | Based on the diabetic nephropathy early detection kit of biomarker | |
| CN104764886A (en) | An early-stage detection kit for diabetic nephropathy, a biomarker detecting method and applications | |
| EP2442106B1 (en) | Method for test on diabetic nephropathy | |
| CN204479593U (en) | Based on the injury of kidney detection kit of biomarker | |
| Huang et al. | A novel time-resolved fluoroimmunoassay for the quantitative detection of antibodies against the phospholipase A2 receptor | |
| Omoruyi et al. | Evaluation of the performance of urine albumin, creatinine and albumin–creatinine ratio assay on two POCT analyzers relative to a central laboratory method | |
| Wang et al. | Development of a novel chemiluminescence immunoassay for the detection of procalcitonin | |
| Ghasemi et al. | Predictive accuracy of urinary neutrophil gelatinase associated lipocalin (NGAL) for renal parenchymal involvement in children with acute pyelonephritis | |
| EP2442105B1 (en) | Test method on renal diseases | |
| Kamel et al. | The potential use of urinary transferrin, urinary adiponectin, urinary Retinol Binding Protein, and serum zinc alpha 2 glycoprotein levels as novel biomarkers for early diagnosis of diabetic nephropathy: a case-control study | |
| CN103808944B (en) | Biomarker VWF and ADAMTS13 and the purposes in liver cirrhosis diagnosis reagent thereof | |
| CN105699665A (en) | Detection kit of lipocalin related to neutrophil gelatinase | |
| Liu et al. | Methodological evaluation and comparison of five urinary albumin measurements | |
| CN103776914A (en) | Biomarker of male infertility in unknown aetiology and application method of biomarker | |
| Raila et al. | Influence of kidney function on urinary excretion of albumin and retinol-binding protein in dogs with naturally occurring renal disease | |
| Gao et al. | Comparison of free plasma metanephrines enzyme immunoassay with 131 I-MIBG scan in diagnosis of pheochromocytoma | |
| Wu et al. | Diagnostic value of progranulin in patients with lupus nephritis and its correlation with disease activity | |
| Chen et al. | A new criteria for screening macroprolactinemia using polyethylene glycol treatment combined with different assays for prolactin. | |
| Trasolini et al. | Fecal leukocyte esterase, an alternative biomarker to fecal calprotectin in inflammatory bowel disease: a Pilot series | |
| CN105372431A (en) | Serum specific marker proteins for sarcoidosis and kit thereof | |
| Yu et al. | Establishment of Galectin-3 time-resolved fluoroimmunoassay and its application in idiopathic membranous nephropathy | |
| US20180275135A1 (en) | Biomarker panel to identify steroid resistance in childhood idiopathic nephrotic syndrome | |
| JP2020106382A (en) | Method of detecting non-alcoholic steatohepatitis (nash) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHENZHEN BEIWODEKE BIO-TECHNOLOGY RESEARCH INSTITU Free format text: FORMER OWNER: SHENZHEN YITEKE INFORMATION TECHNOLOGY CO., LTD. Effective date: 20150901 Free format text: FORMER OWNER: SHENZHEN QIANHAIAN INFORMATION TECHNOLOGY CO., LTD. SHENZHEN BEIWODEKE BIO-TECHNOLOGY RESEARCH INSTITUTE CO., LTD. Effective date: 20150901 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20150901 Address after: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A Patentee after: SHENZHEN BEIWO DEKE BIOTECHNOLOGY RESEARCH INSTITUTE CO., LTD. Address before: 518000 Guangdong city of Shenzhen province Nanshan District science and Technology Park high-tech South seven Digital Technology Park building B1 3B Patentee before: Shenzhen Yi Teke Information Technology Co., Ltd Patentee before: SHENZHEN QIANHAI ANCE INFORMATION TECHNOLOGY CO., LTD. Patentee before: SHENZHEN BEIWO DEKE BIOTECHNOLOGY RESEARCH INSTITUTE CO., LTD. |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150715 Termination date: 20200324 |