CN107144620A - Diabetic nephropathy detects mark - Google Patents
Diabetic nephropathy detects mark Download PDFInfo
- Publication number
- CN107144620A CN107144620A CN201710205267.8A CN201710205267A CN107144620A CN 107144620 A CN107144620 A CN 107144620A CN 201710205267 A CN201710205267 A CN 201710205267A CN 107144620 A CN107144620 A CN 107144620A
- Authority
- CN
- China
- Prior art keywords
- diabetic nephropathy
- precursor
- protein
- reactive protein
- serum
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44795—Isoelectric focusing
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides diabetic nephropathy detection mark, and the diabetic nephropathy detection mark is made up of 12 kinds of albumen：Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, the antigen of serine/threonine protein kitase WNK4 and CD 5.Compared with existing detection method, detection mark specificity, sensitivity and accuracy height of the invention, the foundation that can be diagnosed as diabetic nephropathy.
Description
Technical field
Mark is detected the present invention relates to diabetic nephropathy.
Background technology
Diabetic nephropathy is the common complication of diabetes, is one of diabetes generalized capillary lesion performance, clinical
Albuminuria is characterized as, serious renal failure occurs in gradual kidney function damage, hypertension, oedema, late period, is diabetic
One of major causes of death, current common detection methods are difficult to early detection diabetic nephropathy.
The content of the invention
Mark is detected the invention provides diabetic nephropathy, uses it for detecting diabetic nephropathy, with good spy
The opposite sex, sensitivity and accuracy rate.
Blood is because sampling is convenient and can preferably reflect body pathology physiology course and preferably coming as disease marker
One of source.But search out specificity and accuracy is high, the protein marker of clinical detection can be used for, acquired a certain degree of difficulty.Glycosuria
Sick nephrosis belongs to multifactor relevant disease, it is difficult to search out single protein marker.Therefore, the present invention provides above-mentioned difference egg
It is used for diabetic nephropathy detection in vain, takes the mode of the composite marker thing to improve the specificity and accuracy of detection, has simultaneously
There is certain objectivity, better than single disease marker.The above-mentioned 12 kinds of serum differential proteins combination of the present invention can be used as diabetes
Nephrosis detects mark, with certain objectivity, specificity and accuracy.It should be noted that, diabetic nephropathy inspection of the present invention
The source of mark is surveyed in addition to serum, diabetic nephropathy detection mark could also be from urine sample.
The present invention provides diabetic nephropathy detection mark, and the diabetic nephropathy detects mark by 12 kinds of protein groups
Into：Before Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin
Body, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1,
The antigen of serine/threonine protein kitase WNK4 and CD 5.
The present invention also provides a kind of method for separating diabetic nephropathy detection mark in serum or urine, the diabetes
Nephrosis detection mark is 12 kinds of albumen described in claim 1, and serum or urine are separated using two-dimensional electrophoresis method for protein
Diabetic nephropathy detection mark in liquid；Dielectrophoresis optimal conditions are：
1）First to isoelectric focusing
The microgram of 24cm applied sample amounts 120
Aquation 50V 12 hours（20℃）Active aquation
The linear 0.5 hour desalination of S1 250V
The linear 2 hours desalinations of S2 1000V
S3 10000V boost for linear 6 hours
The poly- glue of the quick 80000VHr of S4 10000V
S5 500v are kept for quick 3 hours；
Under the conditions of sheet, each sample is repeated 3 times, and first is good to isoelectric focusing result, and protein site is separately good.
2）Second to vertical electrophoresis
Run glue using constant pressure, start to select low-voltage 60V during sample introduction, treat sample point glue bromophenol blue concentration it is into a line it
Afterwards, high voltage, until bromophenol blue indicator goes to bottom position, stops running glue to 200V.
The present invention also provides a kind of diabetic nephropathy detection kit, and the kit includes the serum of Healthy People, in blood
In clear, the expression quantity of diabetic nephropathy detection mark is in normal range (NR)；The diabetic nephropathy detects mark by 12 kinds
Albumen is constituted：Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone combination ball
Amyloid protein precursor, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor montage is different
Structure body 1, the antigen of serine/threonine protein kitase WNK4 and CD 5.
The present invention also provides application of 12 kinds of albumen in mark is detected as diabetic nephropathy；12 kinds of albumen
For：Before Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin
Body, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1,
The antigen of serine/threonine protein kitase WNK4 and CD 5.
The present invention also provides a kind of protein chip for detecting diabetic nephropathy, and the albumen in the protein chip is：Angle egg
White 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor is immunized
Immunoglobulin heavy chain Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, serine/
The antigen of Serineprotein kinase WNK4 and CD 5.
One group of serum differential protein that the present invention is provided is detected available for diabetic nephropathy, for diabetic nephropathy detection
When, more single disease markers improve the specificity and accuracy of detection, while having certain objectivity.The present invention is adopted
With two-dimensional electrophoresis method for protein, deposition condition is optimized, clearly protein electrophoresis result is obtained；By to glycosuria
The electrophoresis result of sick nephrotic's serum and normal healthy controls serum is compared analysis, obtains the two serum differential protein 12
Individual, compared with the control, up-regulated expression protein 10 expresses down-regulation protein 2.Can also further it be adopted on the basis of the present invention
These albumen are carried out with Enzyme-multiplied immune technique to detect one by one or these albumen are carried out using protein chip technology to detect simultaneously,
It is determined that the change in concentration of the serum differential protein detected.Compared with existing detection method, detection mark of the invention is special
The opposite sex, sensitivity and accuracy are high, the foundation that can be diagnosed as diabetic nephropathy.The sample to be checked of the present invention can use serum
And urine, gather and more facilitate；Consumption is 200ug-400ug during detection, much smaller than the blood sampling volume of application other method.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, the reality with the present invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the dielectrophoresis result of normal healthy controls serum；
Fig. 2-5 is the clear dielectrophoresis result of Diabetic Nephropathy Patients；
Fig. 6 is 12 species diversity albumen schematic diagrames of test sera and normal healthy controls serum.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used, is city unless otherwise specified in following embodiments
Sell.
The applicant is compared analysis by serum and urine to Diabetic Nephropathy patients and healthy population, finds：
Compared with the serum and urine of healthy population, there are 12 serum differential proteins in the cleer and peaceful urine of Diabetic Nephropathy Patients：
Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor,
Heavy chain immunoglobulin V iiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, silk
Propylhomoserin/the antigen of Serineprotein kinase WNK4 and CD 5, wherein 10 expressing protein up-regulations, 2 expressing proteins are lowered.The application
This group of differential protein is used in the early detection of diabetic nephropathy by people, finds with good specificity, sensitivity and accurate
Property.
Embodiment 1
The diabetic nephropathy of the present invention detects that mark is：
Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin
Precursor, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer
1, the antigen of serine/threonine protein kitase WNK4 and CD 5.
Embodiment 2
The application separates 12 kinds of albumen in serum or urine using two-dimensional electrophoresis method for protein first（I.e. Keratin 19, is tied
Close globin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor, immunoglobulin
Heavy chain Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, serine/threonine
The antigen of protein kinase WNK4 and CD 5）, button point then is carried out to dielectrophoresis gel discrepancy, mass spectral analysis is done, determines albumen
Title and its size, and the content of protein is compared clearly or in urine to healthy control group and Diabetic Nephropathy Patients
Analysis.Concrete operations are as follows：
First, detectable substance is serum
1st, normal healthy controls serum is prepared：Expression of the normal healthy controls serum collection from above-mentioned 12 kinds of albumen in healthy population, serum
Amount is in the normal range (NR) of specification.
Normal healthy controls serum is dispensed after acquisition, and directly freezed is taken out when in use in -80 DEG C of environment
Defrosting is used.
2nd, the serum of collection Diabetic Nephropathy patients is used as test sera.The minimum 5ml of blood sampling volume.
In use, being removed respectively in test sera and normal healthy controls serum using Affinity2bluegel and ProteinA
Albumin and IgG, the serum not processed and each 400 μ g of serum for removing albumin and IgG are mixed with hydrating fluid.
Aquation formula of liquid is：
Urea 8M 4.805g
CHAPS 4% 0.4g
DTT 65mM 0.098g（Now plus）
Ampholytes 0.2%（W/V） 50ul（40%）（Now plus）
The 10ul of bromophenol blue 0.001%（1% bromophenol blue）
MilliQ water is settled to 10ml.
It is packed as 1ml, -20 DEG C of preservations.
3rd, by two-dimensional electrophoresis method for protein, the albumen in test sera and normal healthy controls serum is separated.This
Condition when applicant is to Two-Dimensional Gel Electrophoresis is optimized：Mainly to first to isoelectric focusing program and second in
Constant pressure run adhesive tape part be optimized, enable to separate above-mentioned 12 kinds of albumen well, electrophoresis result is clear.Specific electrophoresis
Method is as follows：
A. first to isoelectric focusing
1) the aquation sample-loading buffer of -20 DEG C of freezen protectives is taken from refrigerator（I）（Without DTT（Dithiothreitol (DTT), molecular formula is
C4H10O2S2, molecular weight is 154.25）, without Bio-Lyte（Ampholytes））One tubule（1ml/ is managed）, put room-temperature dissolution.
Aquation sample-loading buffer（I）Formula be：
Urea 8M 4.805g
CHAPS 4% 0.4g
DTT 65mM 0.098g（Now plus）
Ampholytes 0.2%（W/V） 50ul（40%）（Now plus）
The 10ul of bromophenol blue 0.001%（1% bromophenol blue）
MilliQ water is settled to 10ml；
It is packed as 1ml, -20 DEG C of preservations.
2) 0.01g DTT, Bio-Lyte each 2.5ml of 4-6,5-7 are added in tubule, are fully mixed.
3) 400ul aquation sample-loading buffers are taken out from tubule（I）, add 200ul samples（Experiment blood after handling
Albumin or normal healthy controls haemocyanin）, fully mix.
4) the prefabricated adhesive tape of IPG of -20 DEG C of freezen protectives is taken from refrigerator（17cm pH 4-7）, 10 points are placed in room temperature
Clock.
5) along edge to the left side for focusing on disk or aquation disk bracket groove, the right side linearly adds sample.Each 1cm or so at groove two ends
It should not be loaded, middle sample liquid must link up.Note：Bubble should not be produced.Otherwise point of protein in adhesive tape is had influence on
Cloth.
6) after all protein examples are all had been added in focusing disk or aquation disk, with the removal of tweezers gently
Protective layer in prefabricated IPG adhesive tape.
7) both positive and negative polarity of adhesive tape is distinguished, it is molten that IPG adhesive tape glue lightly is placed face down on into sample in focusing disk or aquation disk
On liquid so that the positive pole of adhesive tape（Indicate）Corresponding to the positive pole for focusing on disk.Ensure that adhesive tape is contacted with electrode seal.Sample should not be made
Product solution is got in the plastic support film at the adhesive tape back side, because these solution will not be absorbed by adhesive tape.Equally being also noted that does not make
Solution below adhesive tape produces bubble.If having produced bubble, one end of adhesive tape is lightly lifted with tweezers, glue is moved up and down
Bar, until bubble is rushed to beyond adhesive tape.
8) 2-3ml mineral oil is covered in every adhesive tape, the evaporation of liquid in adhesive tape hydration process is prevented.Need slow
Mineral oil is added, along adhesive tape, mineral oil is drop by drop slowly added in plastic support film.
9) to good positive and negative electrode, close the lid.Isoelectric focusing program is set：
First to isoelectric focusing：
The microgram of 24cm applied sample amounts 120
Aquation 50V 12 hours（20℃）Active aquation
The linear 0.5 hour desalination of S1 250V
The linear 2 hours desalinations of S2 1000V
S3 10000V boost for linear 6 hours
The poly- glue of the quick 80000VHr of S4 10000V
S5 500v are kept for quick 3 hours.
Under the conditions of sheet, each sample is repeated 3 times, and first is good to isoelectric focusing result, and protein site is separately good.
10) adhesive tape terminated is focused on.It is balanced immediately, second to SDS-PAGE electrophoresis, adhesive tape is otherwise placed in sample
In aquation disk, -20 DEG C of refrigerators are preserved.
B. second to SDS-PAGE electrophoresis
1) two pieces of the acrylamide gel of preparation 10%.With 80ml gel solutions, per clotting glue 40ml, solution is injected separately into glass
In glass plate interlayer, 1cm space is stayed on top, with MilliQ water, ethanol or water-saturated n-butanol front cover, keeps glue surface smooth.It is poly-
Close 30 minutes.General gel is with after upper liquid layering, showing that gel polymerize substantially.
2) after MilliQ water, ethanol or the water-saturated n-butanol for after gel sets, going to separation gel surface, MilliQ is used
Water is rinsed.
3) adhesive tape taken out from -20 DEG C of refrigerators, places 10 minutes prior to room temperature, dissolves it.
4) adhesive tape level pad I is prepared.
Adhesive tape equalizing and buffering mother liquor：
Urea 6M 36g
SDS 2% 2g
This-HCL 0.375M pH8.8 25ml
The 20ml of glycerine 20%
MilliQ water constant volume is to 100ml；
10 pipes are distributed into, often pipe 10ml, -20 DEG C of refrigerators are preserved.
Adhesive tape level pad I：
Adhesive tape equalizing and buffering mother liquor 10ml
DTT 0.2g
Fully mix, it is now with the current.
5) dry thick filter paper is first placed on the table, and the adhesive tape glue surface focused on is placed on dry thick filter paper upward.Will be another
The thick filter paper MilliQ water-soakeds of part, squeeze and remove excessive moisture, be then placed directly within adhesive tape, gently blot the mineral oil in adhesive tape
And redundant sample.This vertical stripe occurred when can reduce gel-colored.
6) adhesive tape is transferred in swelling disk, each piece adhesive tape of groove, 5ml adhesive tape balance is added in the groove for have adhesive tape
Buffer solution I.Sample hydration disk is placed on horizontal shaker and slowly rocked 15 minutes.
7) adhesive tape level pad II is prepared：
Adhesive tape equalizing and buffering mother liquor 10ml
Iodoacetamide 0.24g.
Fully mix, matching while using.
8) after balance terminates for the first time, thoroughly outwell or sop up the adhesive tape level pad I in sample hydration disk.It is used in combination
Filter paper draws unnecessary equilibrium liquid（Adhesive tape is erected on filter paper, in order to avoid loss albumen or damage gel surface）.Add adhesive tape
Level pad II, continuation is slowly rocked 15 minutes on horizontal shaker.
9) liquid unnecessary between glass plate above SDS-PAGE polyacrylamide gels is sucked with filter paper.By what is handled well
Second puts on the table to gel, and long glass plate is under, and short glass plate is upward.
10) agarose sealing liquid is dissolved by heating.
11) by 10 × electrophoretic buffer, 10 times are diluted with graduated cylinder, into 1 × electrophoretic buffer.Rush buffer solution surface
Bubble.
10 × electrophoretic buffer：
Tris alkali 30g
Glycine 144g
SDS 10g
MilliQ water 1L；
After mixing, room temperature preservation, used time dilution.
12) after second of balance terminates, thoroughly outwell or sop up the adhesive tape level pad II in sample hydration disk.And
Unnecessary equilibrium liquid is drawn with filter paper（Adhesive tape is erected on filter paper, in order to avoid loss albumen or damage gel surface）.
13) IPG adhesive tape is removed from sample hydration disk, one end of adhesive tape is clamped with tweezers makes glue surface soak end completely 1
In × electrophoretic buffer.Then adhesive tape glue surface is placed on the long glass plate of gel upward.Remaining adhesive tape is equally operated.
14) PAGE gel for being placed with adhesive tape is transferred on encapsulating frame, short glass plate one is facing to oneself.Solidifying
The top of glue adds low melting-point agarose sealing liquid.
15) with the syringe needle of tweezers, spatula or tack, lightly adhesive tape is pushed down on, is allowed to solidifying with polyacrylamide
Glue glue surface is completely attached to.It is careful not to produce any bubble below adhesive tape.Adhesive tape is being pushed away with tweezers, spatula or tack syringe needle
When, it should be noted that it is the support membrane for promoting the gel back side, glue surface should not be encountered.
16) place 5 minutes, low melting-point agarose sealing liquid is thoroughly solidified.
17) after low melting-point agarose sealing liquid completely solidification.Gel is transferred in electrophoresis tank.
18) after electrophoresis tank adds 1 × electrophoretic buffer, switch on power, glue is run using constant pressure, is during beginning sample introduction
Low-voltage 60V, after treating that sample point glue bromophenol blue concentration is into a line, high voltage 200V, until bromophenol blue indicator is run
To bottom position, stop running glue.The whole race glue time is probably 6 hours.When bromophenol blue indicator reaches bottom margin i.e.
Electrophoresis can be stopped.
19) after electrophoresis terminates, layer glass is gently pried open, gel is taken out, and corner cut is with marking（Gloves are worn, dirt is prevented
Contaminate glue surface）.
20) dyed：Use green skies silver staining kit.Dyed according to the method for kit, during operation
Hand is avoided to touch glue surface as far as possible, in order to avoid the pollution of albumen.
4th, mass spectral analysis：
Button point is carried out to dielectrophoresis gel discrepancy, mass spectral analysis is done, protein name and its size is determined, and it is right to health
Content according to protein in group and test sera is compared.
5th, electrophoresis and mass spectral results are analyzed：
Find there are 12 differential proteins in test sera and normal healthy controls serum by electrophoresis result and mass spectrometry results：Angle
Protein 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor is exempted from
Epidemic disease immunoglobulin heavy chain V iiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, silk ammonia
Acid/the antigen of Serineprotein kinase WNK4 and CD 5.The expression quantity of above-mentioned 12 species diversity albumen is changed：Relative to health
For control serum, in test sera：Before Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4
Body, sex hormone binding globulin precursor, heavy chain immunoglobulin V iiiregionhil, complement component C4, k eratin 6 l, C is anti-
Answer amyloid protein precursor splicing isomer 1 and the up-regulation of the antigens of CD 5 this 10 species diversity expressing quantities, and hoptoglobin and serine/
Serineprotein kinase WNK4 expression quantity is lowered.Specifically it is shown in Table 1 and Fig. 1-6.
Fig. 1 is the dielectrophoresis result of normal healthy controls serum；
Fig. 2-5 is the clear dielectrophoresis result of Diabetic Nephropathy Patients；
Fig. 6 is 12 species diversity albumen schematic diagrames of test sera and normal healthy controls serum；Wherein, 12 represent：Before platelet factor 4
Body；16 represent：Sex hormone binding globulin precursor；19 represent：The antigens of CD 5；22 represent：K eratin 6 l；26 represent：C reacts
Albumen；37 represent：Serine/threonine protein kitase WNK4；49 represent：Heavy chain immunoglobulin V iiiregionhil；54
Represent：Complement component C4；73 represent：Apo E；86 represent：Hoptoglobin；99 represent：C reactive protein precursor montage is different
Structure body 1；102 represent：Keratin 19.
The serum differential protein of the Diabetic Nephropathy patients of table 1 and normal healthy controls
2nd, detectable substance is urine
1st, normal healthy controls urine is prepared：Expression of the normal healthy controls urine capture from above-mentioned 12 kinds of albumen in healthy population, urine
Amount is in the normal range (NR) of specification.
2nd, collection experiment urine；
Urine sample collecting need to be finished in 2 hours, if can not if refrigerate and preserved in 4 DEG C of refrigerators, it is small to preserve 6 altogether
When；
The precipitation of Urine proteins：Take 5ml urine respectively, 20%TCA/ acetone precipitations precipitation urine albumen, vacuum drying rear overhang in
200 microlitres of IEF buffer solutions（Shanghai Yu Bo bio tech ltd, production code member：Amresco M243）In.
3rd, the linear IPG immobilized ph gradient strips by above-mentioned treated urine specimen by 4-7 of pH value carry out Two-Dimensional Gel Electrophoresis
Experiment, carries out button point to dielectrophoresis gel discrepancy, does mass spectral analysis, electrophoresis and mass spectral results are analyzed.Specific behaviour
Make step identical with the operating procedure of serum.
Existing diabetic nephropathy is only to detect that one or several albumen indexs lack specificity, so acatalepsia
Really, the misdiagnosis rate of especially early diabetes is higher.The present invention is used to detect that the protein marker of diabetic nephropathy has 12 kinds,
Specificity increases than original detection unique identification thing, can reach 89.8%；Sensitivity is also up to more than 90%.
165 Diabetic Nephropathy patients are detected using the detection method of the present invention, work as Keratin 19, with reference to pearl egg
In vain, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor, heavy chain immunoglobulin V
Iiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1 and the antigens of CD 5 this 10 species diversity
Expressing quantity is raised, and timing under hoptoglobin and serine/threonine protein kitase WNK4 expression quantity, is judged with sugar
The sick nephrosis of urine, as a result detects 147, clinical coincidence rate（Accuracy rate）Reach more than 89%.
The one kind of serum to be checked in following methods with diabetic nephropathy that the method for the application present invention is detected
Further detected, can further improve specificity, sensitivity and the accuracy of detection：
（1）With enzyme linked immunological kit (Shanghai Yao Chao bioengineering Co., Ltd) Enzyme-multiplied immune technique to 12 serum difference eggs
Detected one by one in vain, the change in concentration size of the serum differential protein detected can be further determined that.
（2）12 species diversity albumen are prepared into protein chip according to the known method of this area, using protein chip technology
12 serum differential proteins are carried out to detect simultaneously, it is determined that the change in concentration of the serum differential protein detected.
Embodiment 3
The diabetic nephropathy detection kit of the present invention：
1st, the expression quantity of above-mentioned 12 kinds of albumen in the serum of 2-3 healthy population of collection, serum is in the normal range (NR) of specification.
It is frozen in after collection in -80 DEG C of environment.It is used as standard female serum；
2nd, solution used during electrophoresis is prepared（If there is improved place, write exactly herein, it is corresponding with embodiment 2）；
3rd, on-gauge plate is prepared：Fig. 1 and Fig. 6 is printed, on-gauge plate is used as.After detection, according to on-gauge plate or standard female serum
Electrophoresis result is judged.
The detection method of the detection kit of the present invention is carried out according to embodiment 2（Omit and mass spectral analysis is carried out to discrepancy
The step of）.
Testing result basis for estimation：Work as Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor
4 precursors, sex hormone binding globulin precursor, heavy chain immunoglobulin V iiiregionhil, complement component C4, k eratin 6 l, C
Reactive protein precursor splicing isomer 1 and this 10 species diversity expressing quantity up-regulation of the antigens of CD 5, and hoptoglobin and silk ammonia
Timing under acid/Serineprotein kinase WNK4 expression quantity, judges to suffer from diabetic nephropathy.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (5)
1. diabetic nephropathy detects mark, it is characterised in that：The diabetic nephropathy detection mark is made up of 12 kinds of albumen：
Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor,
Heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, silk ammonia
Acid/the antigen of Serineprotein kinase WNK4 and CD 5.
2. a kind of separate the method that diabetic nephropathy detects mark in serum or urine, the diabetic nephropathy detects mark
For 12 kinds of albumen described in claim 1, it is characterised in that：Separated using two-dimensional electrophoresis method for protein in serum or urine
Diabetic nephropathy detection mark；Dielectrophoresis optimal conditions are：
1）First to isoelectric focusing：
Aquation 50V 12 hours（20℃）Active aquation
The linear 0.5 hour desalination of S1 250V
The linear 2 hours desalinations of S2 1000V
S3 10000V boost for linear 6 hours
The poly- glue of the quick 80000VHr of S4 10000V
S5 500v are kept for quick 3 hours；
2）Second to vertical electrophoresis
Run glue using constant pressure, start to use low-voltage 60V during sample introduction, treat sample point glue bromophenol blue concentration it is into a line it
Afterwards, high voltage, until bromophenol blue indicator goes to bottom position, stops running glue to 200V.
3. a kind of diabetic nephropathy detection kit, it is characterised in that：The kit includes the serum of Healthy People, in serum
In, the expression quantity of diabetic nephropathy detection mark is in normal range (NR)；The diabetic nephropathy detects mark by 12 hatching eggs
White composition：Keratin 19, hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone combination ball egg
Cynanchum glaucescens body, heavy chain immunoglobulin Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor montage isomery
Body 1, the antigen of serine/threonine protein kitase WNK4 and CD 5.
Application of 4.12 kinds of albumen in mark is detected as diabetic nephropathy；12 kinds of albumen is：Keratin 19, with reference to
Globin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor, immunoglobulin weight
Chain Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, serine/threonine egg
The white antigen of kinases WNK4 and CD 5.
5. a kind of protein chip for detecting diabetic nephropathy, it is characterised in that：Albumen in the protein chip is：Keratin
19, ball is immunized in hoptoglobin, apo E, c reactive protein, platelet factor 4 precursor, sex hormone binding globulin precursor
Ferritin heavy chain Viiiregionhil, complement component C4, k eratin 6 l, c reactive protein precursor splicing isomer 1, serine/Soviet Union
The antigen of propylhomoserin protein kinase WNK4 and CD 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710205267.8A CN107144620A (en) | 2017-03-31 | 2017-03-31 | Diabetic nephropathy detects mark |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710205267.8A CN107144620A (en) | 2017-03-31 | 2017-03-31 | Diabetic nephropathy detects mark |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107144620A true CN107144620A (en) | 2017-09-08 |
Family
ID=59783869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710205267.8A Pending CN107144620A (en) | 2017-03-31 | 2017-03-31 | Diabetic nephropathy detects mark |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107144620A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129949A (en) * | 2020-08-18 | 2020-12-25 | 兰州百源基因技术有限公司 | Retinol binding protein detection kit, preparation method and use method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101661032A (en) * | 2008-08-29 | 2010-03-03 | 生物远景技术有限公司 | Biomarker of Parkinson disease |
| WO2010040571A2 (en) * | 2008-10-10 | 2010-04-15 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Method for a genome wide identification of expression regulatory sequences and use of genes and molecules derived thereof for the diagnosis and therapy of metabolic and/or tumorous diseases |
| CN101759798A (en) * | 2009-10-30 | 2010-06-30 | 上海交通大学医学院附属第九人民医院 | Marker membrane protein for purifying and enriching intradermal multipotentiality cells |
| CN101978248A (en) * | 2008-01-07 | 2011-02-16 | 动态通量生命科学仪器有限公司 | Discovery tool with integrated microfluidic biomarker optical detection array device and methods for use |
| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
| CN103299192A (en) * | 2010-09-21 | 2013-09-11 | 普罗蒂阿米克斯国际有限公司 | Biomarkers associated with pre-diabetes, diabetes and diabetes related conditions |
-
2017
- 2017-03-31 CN CN201710205267.8A patent/CN107144620A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101978248A (en) * | 2008-01-07 | 2011-02-16 | 动态通量生命科学仪器有限公司 | Discovery tool with integrated microfluidic biomarker optical detection array device and methods for use |
| CN101661032A (en) * | 2008-08-29 | 2010-03-03 | 生物远景技术有限公司 | Biomarker of Parkinson disease |
| WO2010040571A2 (en) * | 2008-10-10 | 2010-04-15 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Method for a genome wide identification of expression regulatory sequences and use of genes and molecules derived thereof for the diagnosis and therapy of metabolic and/or tumorous diseases |
| CN101759798A (en) * | 2009-10-30 | 2010-06-30 | 上海交通大学医学院附属第九人民医院 | Marker membrane protein for purifying and enriching intradermal multipotentiality cells |
| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
| CN103299192A (en) * | 2010-09-21 | 2013-09-11 | 普罗蒂阿米克斯国际有限公司 | Biomarkers associated with pre-diabetes, diabetes and diabetes related conditions |
Non-Patent Citations (6)
| Title |
|---|
| 刘文斌 等: "2型糖尿病患者并发肾病状况及相关因素分析", 《新疆医学》 * |
| 姚俊玲 等: "结合珠蛋白在糖尿病肾病早期诊断中的意义", 《中华临床医师杂志》 * |
| 李春梅 等: "同型半胱氨酸与性激素结合球蛋白联合检测在糖尿病肾病早期诊断中的意义", 《浙江医学》 * |
| 王衍慧 等: "糖尿病肾病患者血浆动态蛋白质组学研究", 《山东医药》 * |
| 郑吉平 等: "2型糖尿病人D-二聚体、CRP监测对糖尿病视网膜病变和肾病的临床意义", 《中西医结合心血管病杂志》 * |
| 黄潇苇 等: "慢性肾功能不全患者CYFRA21-1水平与肾功能指标的相关性分析", 《标记免疫分析与临床》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129949A (en) * | 2020-08-18 | 2020-12-25 | 兰州百源基因技术有限公司 | Retinol binding protein detection kit, preparation method and use method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gachon et al. | Immunological and electrophoretic studies of human tear proteins | |
| Javois | Immunocytochemical methods and protocols | |
| Fahey et al. | Preparation of antisera specific for 6.6 S γ-globulins, β2A-globulins, γ1-macroglobulins, and for type I and II common γ-globulin determinants | |
| Wang et al. | A simple affinity spin tube filter method for removing high‐abundant common proteins or enriching low‐abundant biomarkers for serum proteomic analysis | |
| Axelsen | Quantitative immunoelectrophoretic methods as tools for a polyvalent approach to standardization in the immunochemistry of Candida albicans | |
| JPH05264553A (en) | Antigen prepared for detecting helicobacter pyroly | |
| Hanson | The serological relationship between human milk and blood plasma | |
| KR20170118858A (en) | The immunological measurement method of L-FABP and the measurement reagent used in the method | |
| Abate et al. | Canine serum protein patterns using high-resolution electrophoresis (HRE) | |
| Miller et al. | Proteins of rat serum: II. Influence of some biological parameters of the two‐dimensional electrophoresis pattern | |
| Lehmann et al. | Capillary electrophoresis of human serum proteins and apolipoproteins | |
| Wiederkehr et al. | Two-dimensional gel electrophoresis of cerebrospinal fluid proteins from patients with various neurological diseases. | |
| CN107144620A (en) | Diabetic nephropathy detects mark | |
| Freeman et al. | Human serum protein fractionation by gel filtration | |
| Wallace et al. | Visualization of protein—SDS complexes in polyacrylamide gels by chilling | |
| Tissot et al. | Two‐dimensional electrophoretic analysis of cryoproteins: A report of 335 samples | |
| Kshirsagar et al. | A map of urine proteins based on one-dimensional SDS-polyacrylamide gel electrophoresis and Western blotting using one microliter of unconcentrated urine | |
| CN106496328A (en) | The preparation method of hyaluronic acid binding protein polyclonal antibody | |
| CN103376323A (en) | Application of apolipoprotein C-III as marker of obesity-diabetes | |
| Carey et al. | A critical evaluation of the proposal that serum apolipoproteins are the major constituents of the human erythrocyte membrane | |
| Betgovargez et al. | Characterization of proteins in the human serum proteome | |
| Keir et al. | Nitrocellulose immunofixation following agarose electrophoresis in the study of immunoglobulin G subgroups in unconcentrated cerebrospinal fluid | |
| Ek | Identification of the Pr prealbumin proteins in horse serum | |
| Lapin et al. | Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate‐electrophoresis method and rapid two‐dimensional electrophoresis | |
| Larsen et al. | Serum proteins in the hedgehog |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170908 |
|
| RJ01 | Rejection of invention patent application after publication |