CN109521107A - A kind of method and its detection kit for identifying kidney biomarker - Google Patents
A kind of method and its detection kit for identifying kidney biomarker Download PDFInfo
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Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying kidney.The biomarker is the ratio of the free mannose and glucose and mannose concentration that obtain in serum by high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.Detection method is high performance liquid chromatography derived from PMP before column.Technical solution of the present invention has pre-treatment simple, and analysis time is short, and instrument price is reasonable, meet conventional use, operating procedure is easy to learn, and testing result accuracy is high, the advantages that need to only take a blood sample can distinguish normal person and patients with renal cell carcinoma, and required serum amount is few, and blood sampling volume is less than 1mL.Acquired results show that this analysis method can have very important meaning for the relationship between research free serum mannose and glucose and kidney, the novel kidney clinical detection marker of searching with mannose and glucose free in fast quantification patients with renal cell carcinoma serum.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of method and its detection of the biomarker for identifying kidney
Kit.
Background technique
Kidney is unified important organ of urinary system, and major function is generation and draining urine, and drains human body with this
Metabolic waste plays an important role to the stabilization for maintaining organismic internal environment；In addition, kidney is also an endocrine organ, it is main to adjust
Save blood pressure, RBC acceptor garland rate and bone growth etc..Therefore, kidney generates disease and produces a very large impact to the function of human body.Kidney
The diagnosis of disease, which generally requires, carries out uroscopy, blood test, imageological examination and pathological examination, and combines clinical manifestation
To be diagnosed.These diagnostic methods can bring certain drawbacks.For example, imageological examination is high to the accuracy rate of Diagnosis of Renal Cell Carcinoma
Up to 90% or more, but imageological examination has radiation, can have an impact to human body.In addition, currently, that generally acknowledges not yet can
Tumor biomarkers for clinical diagnosis kidney.The clinical diagnosis of kidney relies primarily on imageological examination, makes a definite diagnosis, and needs
The pathological examination of Renal biospy.Therefore, a kind of convenience, hurtless measure or minimally invasive diagnosis and Prognosis scoveillance method are found to patient's
Situation real-time monitoring, it is very necessary for formulating the therapeutic scheme of individuation.And optimal early detection is exactly to be not required to biopsy,
And the serum analysis that sensitivity is high with specificity.
The content highest of glucose in human serum free monosaccharide, the range of normal person are 3.90~6.16mmol/L.Mesh
Before, hospital laboratory directly can detect concentration of glucose by surveying biochemical indicator, and it is dense temporarily cannot directly to detect mannose
Degree.The concentration range of free mannose is 20~80 μm of ol/L in health adult's blood plasma.The content of mannose is about that glucose contains
The 1% of amount.Its major part is considered as from the glucose isomerization in cell.And some researches show that flow in cell recently
Free mannose out is the main source of free mannose in mammalian.The free mannose in this two parts source
Maintain the stabilization of mannose in serum.D-MANNOSE is glycoprotein, cell surface glycoconjugates and glycolsyl-phosphatidylinositol
Required monosaccharide in the structures such as anchorin.Wherein, mannose is the important component of N- sugar chain in various polysaccharide compounds, and N- is poly-
Glucose or mannose of the mannose residue in blood in sugar.Free mannose is normal plasma composition.Currently,
The measuring method of serum mannose mainly has enzyme process, gas-liquid chromatography, high resolution liquid chromatography, gas-liquid chromatography-mass spectrography and hair
Cons electrophoresis method etc..But there is certain shortcoming in these methods.For example, using enzyme process, gas-liquid chromatography and high-resolution
When liquid chromatography, to avoid the influence needs of glucose of high concentration from removing it, cause pretreatment process comparatively laborious；Gas-liquid
Chromatography mass spectrometry instrument price is expensive, is not suitable for routine purpose；Required serum sample amount is big.Before document report column
High performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) simultaneously detect in patients with renal cell carcinoma serum dissociate it is sweet
Dew sugar and glucose.
Summary of the invention
The object of the present invention is to provide for identifying biomarker and its application of kidney, the present invention uses PMP before column
Dissociate in monosaccharide more particularly to serum in derivative high performance liquid chromatography (HPLC) detection patients with renal cell carcinoma serum mannose and
The detection of glucose.Technical solution of the present invention can be used for detecting patients with renal cell carcinoma or the free mannose of normal human serum and grape
Sugared concentration can be used for identifying patients with renal cell carcinoma.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of kidney, the biomarker are serum by efficient liquid derived from PMP before column
The free mannose that phase chromatography obtains and the ratio (G/M concentration ratio) with glucose and mannose concentration.
For identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: this method is derivative for PMP before column
High performance liquid chromatography, comprising the following specific steps
(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in EP pipe, sodium hydroxide is added, be vortexed
It mixes；
(2) PMP is derivative: PMP is added in each sample, is vortexed and mixes, and centrifugation is placed on baking oven reaction；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and hydrochloric acid is added in each sample, is vortexed mixed
It is even；
(4) extract: organic solvent is added in every pipe, is vortexed, and centrifugation removes lower layer's organic solvent, retains supernatant；
(5) sample is centrifuged, takes supernatant in sample bottle, covering tightly lid in the brown equipped with interpolation pipe, is centrifuged, is ready to use in
High-efficient liquid phase analysis；
(6) standard curve of mannose and glucose is drawn；
(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares simultaneously analytical calculation, determines sweet dew
Sugar, the concentration of glucose and G/M concentration ratio.
The high performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 color
It composes column (4.6mm × 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.
The change of gradient of mobile phase in the step (5) are as follows:
0min, A phase 15%, B phase 85%；
10min, A phase 22%, B phase 78%；
15min, A phase 24%, B phase 76%；
15.1min, A phase 15%, B phase 85%；
20min, A phase 15%, B phase 85%.
The concentration of hydrochloric acid is 0.3mol/L in the step (3).
Organic solvent is at least one in chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4)
Kind.
Biomarker is preparing the purposes in the kit for detecting kidney, it is characterised in that: in the kit
It is 82.66 μm of ol/L mannoses and G/M concentration than the titer for 71.70 including concentration.
A kind of kit for identifying kidney, which is characterized in that the kit contains biomarker, i.e., serum is by before column
The free mannose and G/M concentration ratio that high performance liquid chromatography derived from PMP obtains.
In the kit include concentration be 82.66 μm of ol/L mannoses and with G/M concentration than the titer for 71.70.
Identify that the kit of kidney is preparing the purposes in the kit for detecting kidney.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for the trip of patients with renal cell carcinoma serum by the present invention
Detection from monosaccharide, advantageous effects are:
1, pre-treatment is simple.
2, analysis time is short.
3, required amount of serum is few, simply needs to be less than the blood volume of 1mL every time.
4, instrument price is reasonable, meets conventional use.
5, operating procedure is easy to learn.
6, detection is accurate.
The present invention uses hplc simultaneous determination free serum mannose and glucose derived from PMP before column,
Simplify operation, and amount of serum is few, and the measurement for having proven to the method mannose concentration will not be by high concentration glucose
Influence.By the statistical data analysis to free serum mannose, glucose and G/M concentration ratio, kidney and three are established
Relationship between person.Normal person and patients with renal cell carcinoma can be quickly distinguished using technical solution of the present invention.
Detailed description of the invention
Fig. 1 is two types column chromatography figure；
Fig. 2 is the chromatogram of different eluent gradients；
Fig. 3 is that patients with renal cell carcinoma and the free mannose of normal human serum, glucose and G/M concentration compare scatter plot；
Fig. 4 is the ROC curve of patients with renal cell carcinoma free serum mannose and G/M concentration ratio；
Fig. 5 is the 3 D stereo of normal person and patients with renal cell carcinoma free serum mannose, glucose and G/M concentration than building
Figure.
Specific embodiment
Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.
All blood samples involved in embodiment are provided by affiliated hospital, University Of Qingdao, Qingdao City.
Technical solution of the present invention the following steps are included:
Embodiment 1
(1) precision weighs mannose (Man), gucosamine (GlcN), galactosamine (GalN), glucuronic acid
(GlcUA), glucose (Glc), galactolipin (Gal), xylose (Xyl), fucose (Fuc) in right amount, add deionized water to prepare two parts
Containing the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added
Sodium oxide molybdena is vortexed and mixes；
(3) PMP is derivative: 60 μ L 0.5mol/L 1- phenyl -5- methylpyrazole quinoline ketone (PMP) are added in each sample, are vortexed
It mixes, 70 DEG C of baking ovens react 1h after centrifugation；
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, two parts of samples carry out high-efficient liquid phase analysis, efficient liquid phase using different chromatographic column A and B respectively
Chromatography is 1260 efficient liquid phase system of Agilent, and gained chromatogram is shown in Fig. 1.
Chromatographic column A:Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm) chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
Chromatographic column B:Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm)；
Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm) chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 40min | 22% | 78% |
| 40.1min | 15% | 85% |
| 55min | 15% | 85% |
It will be seen from figure 1 that the Agilent ZORBAX XDB-C18 chromatogram column analysis time is long, chromatographic peak separating degree is poor,
The Agilent Poroshell EC-C18 chromatogram column analysis time is the 1/3 of ZORBAX XDB-C18 chromatographic column, and chromatographic peak is good.
Abscissa is time (min), and ordinate is DAD signal strength.1,2,3,4,5,6,7,8 successively Man, GlcN, GalN, GlcUA,
The chromatographic peak of Glc, Gal, Xyl, Fuc.
Embodiment 2
(1) precision weighs mannose (Man), rhamnose (Rha) and glucose (Glc) in right amount, and deionized water is added to prepare phase
Same 5 parts contain the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added
Sodium oxide molybdena is vortexed and mixes；
(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation
1h；
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, 5 parts of samples are used for high-efficient liquid phase analysis, gained chromatography under different mobile phase variable gradients respectively
Figure, is shown in Fig. 2.
High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column
(4.6mm × 100mm, 2.7 μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Mobile phase variable gradient A:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient B:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 20% | 80% |
| 20min | 20% | 80% |
Mobile phase variable gradient C:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 22% | 78% |
| 20min | 22% | 78% |
Mobile phase variable gradient D:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient E:
Figure it is seen that only Man is eluted out under the conditions of gradient A；Under the conditions of gradient B, although three
Can be separated and, but the peak of solvent PMP and Man from it is relatively close；And under the conditions of gradient C, solvent peak and the peak Man weight
It closes；Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient E item
Under part, appearance is symmetrical, and separating degree is good.Abscissa is time (min), and ordinate is DAD signal strength.1,2 and 3 is successively
The chromatographic peak of Man, Rha and Glc.
Embodiment 3
(1) precision weighs mannose and proper amount of glucose, and deionized water is added to be configured to each 0.5mg/mL containing the above monosaccharide,
0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL, 0.0025mg/mL, 0.001mg/mL,
The hybrid standard product solution of 0.0005mg/mL, matching while using；
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added
Sodium oxide molybdena is vortexed and mixes；
(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation
1h；
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.
High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column
(4.6mm × 100mm, 2.7 μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Mobile phase variable gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
(7) standard curve of mannose and glucose is drawn.
Embodiment 4
(1) it creates alkaline environment: taking 242 normal human sera samples and 88 patients with renal cell carcinoma serum samples made a definite diagnosis respectively
10 μ L of product adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L 0.3mol/L sodium hydroxides are added, and is vortexed and mixes；
(2) remaining step is the same as embodiment 3, analytical calculation mannose, the concentration of glucose and G/M concentration ratio.Statistics inspection
It surveys as a result, seeing Fig. 3.
Fig. 3 result is summarized to Tables 1 and 2.
1. patients with renal cell carcinoma free serum monosaccharide of table and G/M concentration are than the comparable trend with normal person
| Mannose | Glucose | G/M concentration ratio | |
| Kidney | ↑*** | ↑NS | ↓*** |
Annotation: " NS " representative is compared with normal people that there was no significant difference, " * * * p < 0.001 ", " * * p < 0.01 " and " * p
< 0.05 ", which is respectively represented, has been compared with normal people significant difference, " ↑ " and " ↓ " respectively represent be compared with normal people rising or under
Drop.
2 kinds of free monosaccharide concentration (μm ol/L) and its ratio result in 2. patients with renal cell carcinoma of table and normal human serum
| Mannose | Glucose | G/M concentration ratio | |
| Normal person | 53.17±10.61 | 4629±642.3 | 89.18±14.31 |
| Kidney | 96.89±49.93 | 4710±1513 | 64.81±36.94 |
In addition, ROC curve analysis has been carried out to the mannose and G/M concentration ratio that have significant difference in table 1 with normal person,
The higher index of area under the curve (AUC), sensitivity and specificity is found with expectation, to determine best screening positive critical value
(cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized to table 3.
In 3. patients with renal cell carcinoma serum of table the cutoff value (μm ol/L) and its sensitivity (%) of mannose and G/M concentration ratio and
Specific (%)
From Fig. 3, table 1, table 2 is as can be seen that patients with renal cell carcinoma free serum mannose concentration significantly rises with G/M concentration than aobvious
Write decline.In addition, table 3 is shown, the specificity of mannose and G/M concentration ratio is higher, and sweet dew sugar detection kidney is special
Property reach 99.49%, therefore patients with renal cell carcinoma can be detected using free mannose and G/M concentration ratio as biomarker.
Embodiment 5
It is used to detect the application of kidney using biomarker of the present invention, specifically includes the following steps:
(1) it creates alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mL EP pipe, be added
40 μ L 0.3mol/L sodium hydroxides are vortexed and mix；
(2) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation
1h；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(4) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(5) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.High performance liquid chromatography is 1260 efficient liquid phase system of Agilent,
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
(6) draw the standard curve of mannose and glucose: precision weighs mannose and proper amount of glucose, adds deionized water
It is configured to each 0.5mg/mL, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL containing the above monosaccharide,
The hybrid standard product solution of 0.0025mg/mL, 0.001mg/mL, 0.0005mg/mL, matching while using；Take 40 μ L mixing monosaccharide marks
Quasi- product are managed in 1.5mL EP, and 40 μ L 0.3mol/L sodium hydroxides are added, and are vortexed and are mixed；Remaining step with (2), (3), (4),
(5)。
(7) standard curve in the chromatogram for obtaining step (5) and step (5) compares simultaneously analytical calculation, determines sweet dew
Sugar, the concentration of glucose and G/M concentration ratio.And the 3 dimensional drawing between three is made by 9 software of Origin, so as to
More intuitively can observation triple combination distinguish normal person and patients with renal cell carcinoma, see Fig. 5.
If the free mannose concentration in test serum sample is greater than 82.66 μm of ol/L and G/M concentration ratios and is less than
71.70, then determine test serum sample for patients with renal cell carcinoma.
Result above shows: this method pre-treatment is simple；Analysis time is short；Required amount of serum is few, every time only
The blood volume of 1mL need to be less than；Instrument price is reasonable, meets conventional use；Operating procedure is easy to learn；Detection is accurate.It detects and counts
Normal person and patients with renal cell carcinoma can be distinguished by calculating mannose, glucose and the G/M concentration ratio to dissociate in serum, be suitble to clinically be used for
The diagnosis of patients with renal cell carcinoma.
The above examples are only used to illustrate the technical scheme of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (13)
1. for identifying the biomarker of kidney, it is characterised in that: the biomarker is serum by 1- phenyl-before column
The ratio of free mannose and glucose that high performance liquid chromatography derived from 5- methylpyrazole quinoline ketone (PMP) obtains and mannose concentration
It is worth (G/M concentration ratio).
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: should
Method is high performance liquid chromatography derived from PMP before column, comprising the following specific steps
(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in EP pipe, sodium hydroxide is added, be vortexed and mix；
(2) PMP is derivative: PMP is added in each sample, is vortexed and mixes, and centrifugation is placed on baking oven reaction；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and hydrochloric acid is added in each sample, is vortexed and is mixed；
(4) extract: organic solvent is added in every pipe, is vortexed, and centrifugation removes lower layer's organic solvent, retains supernatant；
(5) sample is centrifuged, takes supernatant in sample bottle, covering tightly lid in the brown equipped with interpolation pipe, is centrifuged, is ready to use in efficiently
Liquid phase analysis；
(6) standard curve of mannose and glucose is drawn；
(7) chromatogram for obtaining step (5) and the standard curve of step (6) compare simultaneously analytical calculation, determine mannose, grape
The concentration and G/M concentration ratio of sugar.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: institute
Stating high performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm
× 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.
4. according to claim 3 for identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: institute
State the change of gradient of mobile phase in step (5) are as follows:
0min, A phase 15%, B phase 85%；
10min, A phase 22%, B phase 78%；
15min, A phase 24%, B phase 76%；
15.1min, A phase 15%, B phase 85%；
20min, A phase 15%, B phase 85%.
5. according to claim 2 for identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: institute
The concentration for stating hydrochloric acid in step (3) is 0.3mol/L.
6. according to claim 2 for identifying the quantitative analysis method of the biomarker of kidney, it is characterised in that: institute
Stating organic solvent in step (4) is at least one of chloroform, methylene chloride, ethyl acetate and n-hexane.
7. application of the quantitative analysis method of the biomarker of identification kidney in kidney disease described in claim 2-6,
It is characterized in that the blood serum sample is the serum of patients with renal cell carcinoma.
8. a kind of kit for identifying kidney, which is characterized in that the kit contains biomarker described in claim 1,
That is the free mannose and G/M concentration ratio that are obtained by high performance liquid chromatography derived from PMP before column of serum.
9. the kit of identification kidney according to claim 8, which is characterized in that be including concentration in the kit
82.66 μm of ol/L mannoses and G/M concentration are than the titer for 71.70.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and G/M concentration in patients with renal cell carcinoma serum
Than the purposes in quantitative kit.
11. a kind of kit for identifying kidney, it is characterised in that by mannose in serum and G/M concentration than Quantitative measurement kidney
Cancer.
12. the purposes that the kit of the described in any item identification kidneys of claim 8-11 detects kidney in vitro.
13. purposes according to claim 12, it is characterised in that pass through the mannose and the quantitative mirror of G/M concentration ratio in serum
Determine kidney.
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Non-Patent Citations (5)
| Title |
|---|
| 丁克祥编: "《简明临床生化参考值手册》", 31 December 1987 * |
| 中国科协学会学术部: "《网络药理学一中药现代化的新思路与新方法》", 30 November 2014 * |
| 吴梧桐等: "《生物化学》", 31 August 2015 * |
| 胡智祥: "《医院临床检验技术操作规范与实（化）验室管理全书》", 31 August 2004 * |
| 董吁钢等: "《心血管疾病预防与康复》", 28 February 2013 * |
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