CN204479594U - Based on the diabetic nephropathy early detection kit of biomarker - Google Patents

Based on the diabetic nephropathy early detection kit of biomarker Download PDF

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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
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reagent tube
detection
antibody
biomarker
group
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张贯京
张俊然
谷东风
克里斯基捏·普拉纽克
艾琳娜·古列莎
波达别特·伊万
刘遥峰
卢卡·穆桑特
杨轶
石江波
朱孔林
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Shenzhen Beiwo Deke Biotechnology Research Institute Co Ltd
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Shenzhen Qianhai AnyCheck Information Technology Co Ltd
Shenzhen E Techco Information Technology Co Ltd
Shenzhen Beiwo Deke Biotechnology Research Institute Co Ltd
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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

Based on the diabetic nephropathy early detection kit of biomarker
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.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
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

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
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

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