CN105954518B - Application of the lectin chip in Urine proteins sugar chain spectrum analysis - Google Patents
Application of the lectin chip in Urine proteins sugar chain spectrum analysis Download PDFInfo
- Publication number
- CN105954518B CN105954518B CN201610342788.3A CN201610342788A CN105954518B CN 105954518 B CN105954518 B CN 105954518B CN 201610342788 A CN201610342788 A CN 201610342788A CN 105954518 B CN105954518 B CN 105954518B
- Authority
- CN
- China
- Prior art keywords
- chip
- agglutinin
- lectin
- incubation
- urine
- 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.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to lectin chips for distinguishing different degrees of diabetic nephropathy.Agglutinin quickly and effectively distinguishes the diabetic nephropathy of CKD early stages and late stage using tetra- kinds of agglutinin combinations of DSA, SBA, GNA and SNA.Kit is lectin chip kit, the immune reagent kit comprising agglutinin.The detection sample of the kit is urine.The present invention has the following advantages：Urinalysis inspection has the advantages that Non-Invasive, simple, quick, and have many advantages, such as safe, not damaged, sample collect it is easy, easily preserve, detection process high sensitivity, test result are accurate.
Description
Technical field
The present invention relates to a kind of for analyzing the method for Urine proteins sugar chain spectrum, and in particular to lectin chip is used to distinguish not
With the diabetic nephropathy of degree.
Background technology
Serious and harmfulness maximum a kind of chronic complicating diseases caused by diabetic nephropathy is diabetes, are diabetes whole bodies
Property one of microangiopathies performance, Clinical symptoms is albuminuria, gradual kidney function damage, hypertension, oedema, and late period occurs tight
Double kidney functional failure.At present, diabetic nephropathy has become the second reason of End-stage renal disease, is only second to various glomerulonephritis
It is scorching.Already as one of major causes of death of diabetic.Therefore the meaning for delaying diabetic nephropathy is prevented in time
It is great.
The detection of diabetic nephropathy at present and course of disease monitor control index are broadly divided into the following aspects：24 hours albuminuria
Amount, endogenous creatinine clearance rate, glomerular filtration rate（GFR）Deng.
Microalbuminuria is to diagnose the mark of diabetic nephropathy.The early nephropathy phase is also named the microalbuminuria phase, that is, urinates
Albumin excretion rate continues in 20~200 micro- gram/minutes.Clinical diabetes nephrosis phase, Urine proteins gradually increase, urinary albumin row
Rate is let out more than 200 micro- gram/minutes, that is, more than 300 milligrams/24 hours, it is small more than 0.5 gram/24 to be equivalent to total urinary protein
When.Uroscopy is carried out in 6 months, there are 2~3 urinary albumin excretion ratios between 20~200 micro- gram/minutes, it is possible to examine
Disconnected early diabetic nephropathy.But microalbuminuria can not specifically as the detection foundation of diabetic nephropathy, it also with
Other multiple complications of diabetes are related, including hypertension, hyperlipidemia, atherosclerosis and angiocardiopathy etc..Cause
There is microalbuminuria and diabetic nephropathy not necessarily has occurred with regard to representing in this, and whether apparent egg is necessarily proceeded to after occurring
Albiduria and then chronic renal, which fail, still has dispute.The sugar for having microalbuminuria is found in the long-term observation of several larger series
The sick patient of urine only has 30%~45% and switchs to clinical albuminuria, separately has the disappearance of 30% microalbuminuria, this is in II in 10 years
It is become apparent from patients with type Ⅰ DM.
Endogenous creatinine clearance rate is reduced when diabetic nephropathy development is to late period, and general endogenous creatinine clearance rate is down to 10~
15ml/min.Endogenous creatinine clearance rate reduces on the one hand reaction detection of glomeruli filtration function decline, on the other hand, with interior myogenic acid anhydride
The reduction of clearance rate also indicates that the exacerbation of renal impairment degree.
Early diabetic nephropathy shows as glomerular filtration rate increase and B ultrasound measures the increase of kidney volume, works as diabetic nephropathy
When developing into uremia, GFR（ml·min-1·1.73 m2）It reduces but Kidney Volume is without being obviously reduced.Change in renal function is sugar
The important behaviour of the sick nephrosis of urine, the leading indicator for reflecting renal function is GFR, can be into according to GFR and other kidney injury evidences
Row chronic kidney disease（CKD）By stages, different therapies is taken.GFR>When 60, patient is in 1 phase of CKD, 2 phases, generally
Think that GFR is normal or slightly reduces, with injury of kidney, the treatment means for delaying progression of disease are generally used in terms for the treatment of, and work as
GFR<When 60, patient is in 3 phases of CKD, 4 phases, and patient GFR moderates or severe decline, general using assessment and complication
And the means such as Rend dialysis.
Serum this acid anhydride of flesh is insufficient there are sensitivity in GFR is estimated, by individual muscle mass, protein intake, internal generation
The limitations such as the factors such as the level of thanking, haemolysis, piarhemia interference.Therefore, there is an urgent need for one kind can quickly, easy, accurate method is used for
The diabetic nephropathy difference GFR stages are distinguished, to be monitored to the diabetic nephropathy course of disease.
Invention content
The present invention provides a kind of method that lectin chip is used to distinguish the diabetic nephropathy of different chronic kidney diseases by stages.
Technical scheme is as follows：
The present invention is had found by lot of experiments
（1）By being found to the research of type 2 diabetes patient and Diabetic Nephropathy patients urine：Agglutinin MAL-II knows
Other Sia2-3Gal β 1-4Glc (NAc) sugar chain structure, the end of agglutinin PTL-I, PTL-II, SJA, DBA and SBA identification
GalNAc, agglutinin AAL identification α-Fucose structures, agglutinin ConA identification branched and end GlcNAc and
Mannose structures and Sia2-6Gal β 1-4Glc (NAc) sugar chain structure of agglutinin SNA identifications are in Diabetic Nephropathy patients
Abundance is higher in urine.And agglutinin PSA, UEA-I and the Fucose α -1,6GlcNAc, Fucose α 1-2Gal β 1- of STL identifications
4Glc (NAc) and GlcNAc oligomer sugar chain structure abundance in type 2 diabetes patient's urine are higher.
（2）By to GFR>The Diabetic Nephropathy patients and GFR of 60 CKD early stages<The sugar of 60 CKD late stages
The research of the sick nephrotic's urine of urine is found：Agglutinin ConA, SNA and RCA120 identification branched and end GlcNAc and
Mannose, Sia2-3Gal β 1-4Glc (NAc) and β-gal sugar chain structures are in the Diabetic Nephropathy patients urine of CKD early stages
In abundance it is higher；The GlcNAc sugar chain structures of agglutinin DSA identifications are in CKD early stages and the Urine of Patients with Diabetes Mellitus of late stage
In abundance it is essentially identical；And the end of the end GalNAc structures and agglutinin GNA identifications of agglutinin SBA, BS-I and DBA identifications
α -1,3 mannose sugar chain structures are held in GFR>In diabetic nephropathy nephrotic's urine of 60 CKD early stages abundance compared with
It is high.
In summary research and the further screening of applicant, applicant proposed use tetra- kinds of DSA, SBA, GNA and SNA
The diabetic nephropathy of CKD early stages and late stage are quickly and effectively distinguished in agglutinin combination.
Application of the agglutinin in the kit for preparing the diabetic nephropathy for distinguishing CKD early stages and late stage：
The agglutinin is selected from the group of DSA, SBA, GNA and SNA composition.
The kit is lectin chip kit, the immune reagent kit comprising agglutinin.
Under the preparation method of the lectin chip kit enters：
1）Take epoxidation chip base spare；
2）Corresponding to urine specimen, choose following 4 kinds of agglutinins SBA, GNA, DSA and SNA and be configured to a concentration of 1mg/ml
Sampling liquid；
3）The sampling liquid that preparation obtains is added in 384 orifice plates；Chip point sample instrument is reused by above-mentioned agglutinin point sample
Liquid point system is prepared into chip in epoxidation chip base；
4）The chip that point makes is protected from light incubation 12 hours in environment of the humidity for 60%-70%；
5）Chip after incubation is vacuumized into drying under the conditions of 37 DEG C, makes a system fully solid in the agglutinin of chip surface
It is scheduled on chip；
6）The lectin chip fixed is positioned in drier and is kept in dark place for 4 DEG C, as the solidifying of urine specimen detection
The plain chip of collection.
The application method of the kit is as follows：
1）Urine specimen stoste is pre-processed, extraction purification Urine proteins, be marked, be used in combination with fluorescence examination marking agent
The fluorescence that the removal of Sephadex G-25 purification columns is not combined with Urine proteins, obtains corresponding Urine proteins sample to be detected；
2）Urine proteins sample to be detected and the lectin chip prepared are incubated to obtain glycoprotein candy chain in sample altogether
Spectrum:
2.1）1% bovine serum albumin(BSA) component V is dissolved in phosphate buffer-polysorbas20 and is configured to confining liquid, will be made
The lectin chip got ready is placed in confining liquid；
2.2）Lectin chip after closing is cleaned successively with phosphate buffer-polysorbas20 and phosphate buffer,
With removal step 2.1）In agglutinin not covalently bound with chip and remaining bovine serum albumin(BSA) component V, then after drying
It is spare；
2.3）To through step 2.2）Lectin chip after drying adds in incubating for 3-5 μ g samples to be detected and 500-600 μ L
Buffer solution is educated, is incubated at room temperature；
2.4）Lectin chip after incubation is clear with 10mM phosphate buffers-polysorbas20 and phosphate buffer successively
It washes, with removal step 2.3）In not with the sample albumen to be detected of lectin chip specific bond, then after drying it is spare；
2.5）To through step 2.4）Lectin chip after drying is scanned with GenePix4000B chip scanners, is coagulated
The fluorescent image that collection element is combined with Urine proteins sugar chain after label；
2.6）Data analysis is carried out to fluorescent image by GenePix6.0 softwares.
The step 2.1）、2.2）With 2.4）In phosphate buffer pH7.4, contain NaCl 8.0g/L, KCl
0.2g/L, KH2PO40.2g/L, Na2HPO4 12H2O 2.9g/L；Phosphate buffer-polysorbas20：10mmol/L phosphate
The polysorbas20s of 0.05-0.5% containing mass fraction in buffer solution.
The step 2.3）In incubation buffer：1% ox blood containing mass fraction is pure in phosphate buffer-polysorbas20
Protein component V and mass fraction 5-10% azanols.
The fluorescent reagent uses Cy3.
The step 1.3）The point sample instrument is using brilliant 48 spotting systems of core smartarrayer.
The step 2.3）It is that 5 μ g samples to be detected and the incubation buffering of 600 μ L are added in the lectin chip after drying
Liquid, and in incubation at room temperature 3 hours.
The detection sample of the kit is urine.
Another aspect of the present invention：The immune reagent kit comprising agglutinin is：In kit in addition to agglutinin, also
It is useful for distinguishing antigen, antibody, the micromolecular compound of the diabetic nephropathy of CKD early stages and late stage, receptor, is fitted at ligand
Gamete.I.e. those skilled in the art anticipated that present invention discover that agglutinin can distinguish simultaneously CKD early stage and late stage
Diabetic nephropathy other antigens, antibody, micromolecular compound, receptor, ligand, aptamer are used cooperatively, further carry
High detection sensitivity and accuracy.
The present invention has the following advantages：Urinalysis inspection has the advantages that Non-Invasive, simple, quick, and with peace
Entirely, not damaged, sample collects easy, easy the advantages that preserving, and detection process high sensitivity, test result are accurate.
Description of the drawings
Fig. 1 GFR>60 early diabetic nephropathy nephrotic's Urine proteins sugar chain spectrum
Fig. 2 GFR<60 middle and advanced stage Diabetic Nephropathy patients Urine proteins sugar chain spectrum
Fig. 3 ROC curve analysis and evaluation agglutinins SNA distinguishes the diagnostic value of CKD early stage/late stage diabetic nephropathies
Fig. 4 ROC curve analysis and evaluation agglutinins SBA distinguishes the diagnostic value of CKD early stage/late stage diabetic nephropathies
Fig. 5 ROC curve analysis and evaluation agglutinins GNA distinguishes the diagnostic value of CKD early stage/late stage diabetic nephropathies.
Specific embodiment
Agglutinin is a kind of protein for having sugar chain specificity, can promoting cell agglutination, it have with sugar chain specificity,
Non-covalent, Reversible binding ability.Lectin chip be by the agglutinin of various separate sources be fixed on aldehyde radical, epoxidation or
On glass chip after modifying by other means, then with the example reaction to be detected such as glycoprotein, thalline, the cell after label, warp
After subsequent processing, to detect the sugar chain structure of detected sample.Lectin chip can easy, quick, high throughput detection and analysis
Protein it is glycosylation modified, provide the sugared structural fingerprint collection of illustrative plates of detected sample in a short time.
Embodiment 1
The preparation method of lectin chip kit is as follows：
1）Take epoxidation chip base spare；
2）Corresponding to urine specimen, choose following 4 kinds of agglutinins DSA, SBA, GNA and SNA and be configured to a concentration of 1mg/ml
Sampling liquid；
3）The sampling liquid that preparation obtains is added in 384 orifice plates；Chip point sample instrument is reused by above-mentioned agglutinin point sample
Liquid point system is prepared into chip in epoxidation chip base, and the point sample instrument is using brilliant 48 spotting systems of core smartarrayer；
4）The chip that point makes is protected from light incubation 12 hours in environment of the humidity for 60%-70%；
5）Chip after incubation is vacuumized into drying under the conditions of 37 DEG C, makes a system fully solid in the agglutinin of chip surface
It is scheduled on chip；
6）The lectin chip fixed is positioned in drier and is kept in dark place for 4 DEG C, as the solidifying of urine specimen detection
The plain chip of collection；
Embodiment 2：
The application method and result judgement of lectin chip kit are as follows：
1）Collect 50 mL of urina sanguinis（Best interlude）, quickly deposit in 4 DEG C of refrigerators.4 DEG C, 14000 g centrifugations 60min is removed
Decontamination collects supernatant.Acetone/trichloroacetic acid method precipitation Urine proteins, -20 DEG C of pre- cold acetone cleaning precipitations, phosphate buffer
Dissolving precipitation.The Urine proteins of extraction are marked after quantitative with fluorescence examination marking agent, then pure using Sephadex G-25
Change the fluorescence that column removal is not combined with Urine proteins, obtain corresponding Urine proteins sample to be detected；
2）Urine proteins sample to be detected is incubated altogether with the lectin chip prepared, to obtain Urine proteins sample to be detected
Middle glycoprotein candy chain spectrum, can distinguish slight severe diabetic nephropathy, specifically according to the difference that different Urinary albumen sugar chains are composed
Operating procedure is as follows：
2.1）1% bovine serum albumin(BSA) component V is dissolved in phosphate buffer-polysorbas20 and is configured to confining liquid, will be made
The lectin chip got ready is placed in confining liquid；
2.2）Lectin chip after closing is cleaned successively with phosphate buffer-polysorbas20 and phosphate buffer,
With removal step 2.1）In agglutinin not covalently bound with chip and remaining bovine serum albumin(BSA) component V, then after drying
It is spare；
2.3）To through step 2.2）Lectin chip after drying adds in 5 μ g samples to be detected and the incubation buffering of 600 μ L
Liquid is incubated at room temperature 3 hours；
2.4）Lectin chip after incubation is cleaned successively with phosphate buffer-polysorbas20 and phosphate buffer,
With removal step 2.3）In not with the sample albumen to be detected of lectin chip specific bond, then after drying it is spare；
2.5）To through step 2.4）Lectin chip after drying is scanned with GenePix4000B chip scanners, is coagulated
The fluorescent image that collection element is combined with glycoprotein candy chain after label；Referring to Fig. 1,2.
2.6）Interim analysis is carried out to fluorescent image by GenePix6.0 softwares, reads 4 kinds of agglutinin fluorescence in image
Signal value is simultaneously analyzed.
The step 2.1）、2.2）With 2.4）In phosphate buffer pH7.4, contain NaCl 8.0g/L, KCl
0.2g/L, KH2PO40.2g/L, Na2HPO4 12H2O 2.9g/L；Phosphate buffer-polysorbas20：10mmol/L phosphate
The polysorbas20s of 0.05-0.5% containing mass fraction in buffer solution.
The step 2.3）In incubation buffer：1% ox blood containing mass fraction is pure in phosphate buffer-polysorbas20
Protein component V and mass fraction 5-10% azanols.
The step 1）Middle fluorescent reagent uses Cy3.
Early-stage study the result shows that, agglutinin element DSA identification glycoprotein candy chain in diabetogenous nephrosis early and late CKD
Abundance is identical in patient's Urine proteins, and therefore, systematic error caused by reduce fluorescence deviation, this experiment is with agglutinin DSA originals
Beginning fluorescence signal value is correction coefficient, when SNA corrected values are less than more than 0.45, SBA corrected values less than 0.06, GNA corrected values
Can determine whether patient when 0.19, when meeting at least two above-mentioned condition is GFR<60 CKD Late-stage diabetic nephrosis.
Embodiment 3：Lectin chip kit is verified
4 kinds of agglutinins in two Urine in Patients samples are detected, original signal and corrected value such as following table one, two institutes
Show, by comparing 3 kinds of agglutinin corrected values, it is GFR as a result to show subject 1>60 CKD early diabetic nephropathies, are examined
Person 2 is GFR<60 CKD Late-stage diabetic nephrosis, with organizing the result of confirmation of biopsy consistent.
Table one：1 Urine proteins sugar chain of subject is composed
| Agglutinin | The sugar chain structure of specific recognition | Original signal value | Corrected value |
| DSA | GlcNAc | 3986 | 1 |
| SBA | Terminal GalNAc(especially GalNAcα1-3Gal) | 730 | 0.183 |
| GNA | Terminalα-1,3 mannos | 838 | 0.210 |
| SNA | Sia2-6Galβ1-4Glc(NAc) | 1087 | 0.273 |
Table two：2 Urine proteins sugar chain of subject is composed
| Agglutinin | The sugar chain structure of specific recognition | Original signal value | Corrected value |
| DSA | GlcNAc | 4258 | 1 |
| SBA | Terminal GalNAc(especially GalNAcα1-3Gal) | 123 | 0.029 |
| GNA | Terminalα-1,3 mannose | 443 | 0.104 |
| SNA | Sia2-6Galβ1-4Glc(NAc) | 3018 | 0.709 |
Embodiment 4：Lectin chip specificity and sensibility ROC analyses
2.7）Extract 10 GFR confirmed through renal biopsy tissue biopsy>60 CKD early diabetic nephropathies nephrosis and 10
GFR<60 CKD Late-stage diabetic nephrotic's Urine proteins, the binding signal of four kinds of agglutinins is detected using the above method, and is counted
Calculation SNA, SBA and GNA binding signal corrected values, specificity and sensitivity using ROC curve Analysis and Screening standard, as a result such as
Shown in Fig. 3-5：
2.8）The Diabetic Nephropathy patients that 23 are made a definite diagnosis（11 GFR>60 CKD early diabetic nephropathy nephrosis, 12
GFR<60 CKD Late-stage diabetic nephrosis）Urine proteins carry out Blind Test experiment according to above-mentioned standard, the results show that solidifying using two kinds
The plain corrected value screening of collection is to GFR>60 CKD early diabetic nephropathy diagnosis of nephropathy accuracy >=81.82%, to GFR<60 CKD
Late-stage diabetic nephrotic's rate of correct diagnosis >=83.33%.
Claims (7)
1. agglutinin answering in the Urine proteins sugar chain spectrum analysis kit for preparing differentiation early stage, Late-stage diabetic nephrotic
With, it is characterised in that：The agglutinin is selected from the group being made of DSA, SBA, GNA and SNA；Prepared by the agglutinin become aggegation
Plain chip and used；It is described to divide into using agglutinin DSA raw fluorescence signals value as correction coefficient, when SNA corrected values are more than
When 0.45, SBA corrected value is less than 0.06, GNA corrected values less than 0.19, patient is can determine whether when meeting at least two above-mentioned condition
For GFR<60 CKD Late-stage diabetic nephrosis；
The preparation method of the lectin chip is as follows：
1）Take epoxidation chip base spare；
2）Corresponding to urine specimen, choose following 4 kinds of agglutinins DSA, SBA, GNA and SNA and be configured to a concentration of 1mg/ml's
Sampling liquid；
3）The sampling liquid that preparation obtains is added in 384 orifice plates；Chip point sample instrument is reused by above-mentioned agglutinin sampling liquid point
System is prepared into chip in epoxidation chip base；
4）The chip that point makes is protected from light incubation 12 hours in environment of the humidity for 60%-70%；
5）Chip after incubation is vacuumized into drying under the conditions of 37 DEG C, a system is made to be sufficiently secured in the agglutinin of chip surface
On chip；
6）The lectin chip fixed is positioned in drier and is kept in dark place for 4 DEG C, the agglutinin as urine specimen detection
Chip.
2. agglutinin as described in claim 1 is in the Urine proteins sugar chain spectrum point for preparing differentiation early stage, Late-stage diabetic nephrotic
Analyse the application in kit, it is characterised in that：The application method of the kit is as follows：
1）Urine specimen stoste is pre-processed, extraction purification Urine proteins are marked with fluorescent labeling reagent, are used in combination
The fluorescence that the removal of Sephadex G-25 purification columns is not combined with Urine proteins, obtains corresponding Urine proteins sample to be detected；
2）Urine proteins sample to be detected and the lectin chip prepared are incubated altogether and composed with obtaining glycoprotein candy chain in sample:
2.1）Mass fraction is dissolved in for 1% bovine serum albumin(BSA) component in phosphate buffer-polysorbas20 and is configured to close
The lectin chip prepared is placed in confining liquid by liquid；
2.2）Lectin chip after closing is cleaned successively with phosphate buffer-polysorbas20 and phosphate buffer, to go
Except step 2.1）In agglutinin not covalently bound with chip and remaining bovine serum albumin(BSA) component, then after drying it is spare；
2.3）To through step 2.2）Lectin chip after drying adds in 3-5 μ g samples to be detected and the incubation of 500-600 μ L is delayed
Fliud flushing, incubation at room temperature；
2.4）Lectin chip after incubation is cleaned successively with phosphate buffer-polysorbas20 and phosphate buffer, to go
Except step 2.3）In not with the sample albumen to be detected of lectin chip specific bond, then after drying it is spare；
2.5）To through step 2.4）Lectin chip after drying is scanned with GenePix4000B chip scanners, obtains agglutinin
The fluorescent image combined with glycoprotein candy chain after label；
2.6）Interim analysis is carried out to fluorescent image by GenePix6.0 softwares.
3. application as claimed in claim 2, which is characterized in that step 2.1）、2.2）With 2.4）In phosphate buffer
PH7.4 contains NaCl 8.0g/L, KCl 0.2g/L, KH2PO40.2g/L, Na2HPO4•12H2O 2.9g/L；Phosphate-buffered
Liquid-polysorbas20：The polysorbas20s of 0.05-0.5% containing mass fraction in 10mmol/L phosphate buffers.
4. application as claimed in claim 2, which is characterized in that step 2.3）In incubation buffer：Phosphate buffer-spit
Contain 1% bovine serum albumin(BSA) component of mass fraction and mass fraction 5-10% azanols in temperature 20；Fluorescent labeling reagent uses Cy3.
5. application as described in claim 1, it is characterised in that：Step 3）The point sample instrument is using brilliant core smartarrayer
48 spotting systems.
6. application as claimed in claim 2, it is characterised in that：Step 2.3）It is that 5 μ g are added in the lectin chip after drying
The incubation buffer of sample to be detected and 600 μ L, and in incubation at room temperature 3 hours.
7. application as described in claim 1, it is characterised in that：In kit in addition to agglutinin, also it is useful for distinguishing GFR>60
In early days/GFR<Antigen, antibody, micromolecular compound, receptor, ligand, the aptamer of 60 Late-stage diabetic nephrosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610342788.3A CN105954518B (en) | 2016-05-23 | 2016-05-23 | Application of the lectin chip in Urine proteins sugar chain spectrum analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610342788.3A CN105954518B (en) | 2016-05-23 | 2016-05-23 | Application of the lectin chip in Urine proteins sugar chain spectrum analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105954518A CN105954518A (en) | 2016-09-21 |
| CN105954518B true CN105954518B (en) | 2018-06-26 |
Family
ID=56909343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610342788.3A Active CN105954518B (en) | 2016-05-23 | 2016-05-23 | Application of the lectin chip in Urine proteins sugar chain spectrum analysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105954518B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239363A (en) * | 2018-10-22 | 2019-01-18 | 西北大学 | A kind of application of the agglutinin probe combination in terms of identifying Activity budget gender based on Urine proteins sugar-type |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111735963A (en) * | 2020-06-15 | 2020-10-02 | 中国人民解放军总医院 | Use of specific lectins for the identification of diabetic/non-diabetic nephropathy based on the carbohydrate chains of sialoglycoproteins |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004067732A (en) * | 2002-08-01 | 2004-03-04 | Tokai Univ | IgG HAVING OLIGOSACCHARIDE AND METHOD FOR EXAMINING DIABETIC NEPHROPATHY AND MEMBRANOUS NEPHROPATHY USING THE SAME |
| EP1710581B1 (en) * | 2003-12-25 | 2014-03-19 | National Institute of Advanced Industrial Science and Technology | Method for analyzing interactions between proteins and sugar chains |
| JP4711190B2 (en) * | 2006-07-28 | 2011-06-29 | 国立大学法人 東京大学 | Test method for glycosylation disorder |
| CN101308141B (en) * | 2007-05-16 | 2012-08-29 | 陕西北美基因股份有限公司 | Method for analyzing glucoprotein |
| JP2010256132A (en) * | 2009-04-23 | 2010-11-11 | Okayama Univ | Method for detecting advance degree of diabetic nephropathy, kit for diagnosing advance degree of diabetic nephropathy, substance becoming index of advance degree of diabetic nephropathy, and method for sorting the substance |
| CN102507920A (en) * | 2011-09-30 | 2012-06-20 | 西安交通大学 | Lectin chip and application thereof |
| CN103336126B (en) * | 2013-05-08 | 2015-12-02 | 西北大学 | A kind of agglutinin test chip for saliva sample and disposal route thereof |
| CN103645325A (en) * | 2013-12-12 | 2014-03-19 | 复旦大学 | Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip |
| CN104502611A (en) * | 2014-12-25 | 2015-04-08 | 西安交通大学医学院第一附属医院 | Kit for detecting protein glycosylation subtype and detection method thereof |
-
2016
- 2016-05-23 CN CN201610342788.3A patent/CN105954518B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239363A (en) * | 2018-10-22 | 2019-01-18 | 西北大学 | A kind of application of the agglutinin probe combination in terms of identifying Activity budget gender based on Urine proteins sugar-type |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105954518A (en) | 2016-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10866240B2 (en) | Method for analyzing PSA and method for distinguishing prostate cancer from prostatic hypertrophy using that method for analyzing PSA | |
| US10196694B2 (en) | Method for analyzing PSA, and a method for distinguishing prostate cancer from prostatic hypertrophy using that method for analyzing PSA | |
| CN103336126B (en) | A kind of agglutinin test chip for saliva sample and disposal route thereof | |
| CN102175879A (en) | Method for detecting alternative biological markers of liver neoplasms in saliva, serum and urine | |
| EP2664922A1 (en) | Method for prediction of prognosis of sepsis | |
| JP2017502307A (en) | Lectin chip for identifying liver diseases based on glycoprotein sugar chains of saliva and use thereof | |
| CN105652002A (en) | Lectin microarray for detecting carbohydrate chain marker based on sialoprotein and detection method of carbohydrate chain marker using same | |
| CN109212227B (en) | Product for screening and evaluating liver disease/cirrhosis related to saliva specific glycoprotein sugar chain structure and application | |
| CN105954518B (en) | Application of the lectin chip in Urine proteins sugar chain spectrum analysis | |
| KR20150061816A (en) | Polypeptide markers for cancer diagnosis derived from blood sample and methods for the diagnosis of cancers using the same | |
| CN105675893A (en) | Lectin chip for detecting carbohydrate chain markers based on blood serum and commonly based on protein in blood serum and saliva as well as kit and application of lectin chip | |
| CN109655608A (en) | A kind of excretion body protein for osteosarcoma diagnosis and its instant detection method | |
| KR20150062915A (en) | Serological markers for cancer diagnosis using blood sample | |
| Zámorová et al. | Analysis of changes in the glycan composition of serum, cytosol and membrane glycoprotein biomarkers of colorectal cancer using a lectin-based protein microarray | |
| CN109239362B (en) | Application of lectin probe combination in identification of Qinling mountain golden monkey pregnancy based on urine protein sugar type | |
| CN105785029A (en) | Lectin microarray for detecting carbohydrate chain marker based on protein in saliva, lectin microarray for detecting carbohydrate chain marker jointly based on protein in saliva and serum, kit and applications of lectin microarrays | |
| KR101143891B1 (en) | A marker for the diagnosis of cancers by using aberrant glycosylation of protein | |
| KR101219516B1 (en) | Polypeptide markers for the diagnosis of cancers and methods for the diagnosis of cancers using the same | |
| KR101100809B1 (en) | Polypeptide markers for the diagnosis of cancers and methods for the diagnosis using the same | |
| JP7457367B2 (en) | Method and kit for determining the possibility of developing IgA nephropathy | |
| CN110988222A (en) | Product for screening and evaluating ultra-early gastric cancer based on saliva specific glycoprotein carbohydrate chain structure and application | |
| CN110702904A (en) | Use of specific lectins for the production of a test tool for the identification of the stage of lung cancer and device | |
| CN111077256A (en) | Method for identifying glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrum | |
| CN109342729A (en) | Application of the specific agglutinin combination in terms of identifying the cancer of the esophagus based on saliva sugar-type | |
| KR102293148B1 (en) | Biomarker for early diagnosing diabetes and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |