CN109342598A - A kind of method and its detection kit for identifying cerebral hemorrhage biomarker - Google Patents
A kind of method and its detection kit for identifying cerebral hemorrhage biomarker Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying cerebral hemorrhage.The biomarker is the free mannose and glucose obtained in serum by high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (1-Phenyl-3-methyl-5-pyrazolone, 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 cerebral hemorrhage, 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 cerebral hemorrhage, the novel cerebral hemorrhage clinical detection marker of searching with mannose and glucose free in fast quantification serum of patients with intracerebral hemorrhage.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of method and its inspection of the biomarker for identifying cerebral hemorrhage
Test agent box.
Background technique
Cerebral hemorrhage refers to bleeding caused by angiorrhoxis in non-traumatic cerebral essence, account for whole cerebral apoplexies 20%~
30%, acute stage case fatality rate is 30%~40%.The reason of generation, is mainly related with cerebrovascular lesion, i.e., with hyperlipidemia, sugar
It is closely related to urinate disease, hypertension, the aging of blood vessel, smoking etc..The patient of cerebral hemorrhage often due to it is excited, hard firmly when
Sudden onset, Infant Mortality is very high, and there are different degrees of dyskinesia, cognitive disorder, speeches to swallow for majority in survivor
The sequelae such as obstacle.The diagnosis of cerebral hemorrhage generally passes through head CT examination, and is diagnosed in conjunction with clinical manifestation.Due to the disease
Sick rapid onset (symptom for occurring limbs disturbance and increased intracranial pressure in a few minutes or a few houres), therefore find a kind of side
Just, human health can be monitored at any time, the detection method of hurtless measure is very necessary.And optimal 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 serum of patients with intracerebral hemorrhage dissociate
Mannose and glucose.
Summary of the invention
Before for identifying that biomarker and its application of cerebral hemorrhage, the present invention use column
Dissociate in monosaccharide more particularly to serum in high performance liquid chromatography derived from PMP (HPLC) detection serum of patients with intracerebral hemorrhage sweet
The detection of dew sugar and glucose.Technical solution of the present invention can be used for detecting patients with cerebral hemorrhage or the free sweet dew of normal human serum
Sugar and concentration of glucose, can be used for identifying patients with cerebral hemorrhage.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of cerebral hemorrhage, the biomarker are serum by efficient derived from PMP before column
The free mannose and glucose that liquid chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of cerebral hemorrhage, it is characterised in that: this method is spread out for PMP before column
Raw 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 1.5mL centrifuge tube, hydroxide is added
Sodium is vortexed and 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 prepared；
(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares simultaneously analytical calculation, determines sweet dew
The ratio of sugar, the concentration of glucose and glucose and mannose concentration.
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 cerebral hemorrhage.
Biomarker is preparing the purposes in the kit for detecting cerebral hemorrhage, it is characterised in that: the kit
In include concentration is 78.00 μm of ol/L mannoses and concentration is 5621 μm of ol/L glucose titer.
A kind of kit for identifying cerebral hemorrhage, which is characterized in that the kit contains biomarker, i.e. serum passes through column
The free mannose and glucose that high performance liquid chromatography derived from preceding PMP obtains.
It include the mark for the glucose that concentration is 78.00 μm of ol/L mannoses and concentration is 5621 μm of ol/L in the kit
Quasi- liquid.
Identify that the kit of cerebral hemorrhage is preparing the purposes in the kit for detecting cerebral hemorrhage.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for serum of patients with intracerebral hemorrhage by the present invention
The detection of free 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.Pass through the statistics of the ratio (G/M ratio) to free serum mannose, glucose and glucose and mannose concentration
Data analysis, establishes the relationship between cerebral hemorrhage and three.Normal person can be quickly distinguished using technical solution of the present invention
And patients with cerebral hemorrhage.
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 the ratio of patients with cerebral hemorrhage and normal human serum free mannose, glucose and glucose and mannose concentration
It is worth scatter plot；
Fig. 4 is the ROC curve of serum of patients with intracerebral hemorrhage free mannose and glucose and mannose ratio (G/M)；
Fig. 5 is the ratio of normal person and serum of patients with intracerebral hemorrhage free mannose, glucose and glucose and mannose concentration
It is worth the 3 dimensional drawing of building.
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 of University Of Qingdao of 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 in 1.5mL centrifuge tube, 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:
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 pH 5.5 |
| 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 in 1.5mL centrifuge tube, 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:
| 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% |
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 in 1.5mL centrifuge tube, 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 pH 5.5 |
| 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 360 patients with cerebral hemorrhage blood made a definite diagnosis respectively
10 μ L of final proof product adds 30 μ L ultrapure waters in 1.5mL centrifuge tube, and 40 μ L 0.3mol/L sodium hydroxides are added, and is vortexed and mixes；
(2) for remaining step with embodiment 3, analytical calculation mannose, the concentration of glucose and glucose and mannose are dense
The ratio of degree.Statistic mixed-state is as a result, be shown in Fig. 3.
Fig. 3 result is summarized to Tables 1 and 2.
The comparable trend of table 1. serum of patients with intracerebral hemorrhage free monosaccharide and G/M ratio and normal person
| Mannose | Glucose | G/M ratio | |
| Cerebral hemorrhage | ↑*** | 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 cerebral hemorrhage of table and normal human serum
| Mannose | Glucose | G/M ratio | |
| Normal person | 69.08±34.09 | 5720.85±2525.94 | 85.37±16.76 |
| Cerebral hemorrhage | 115.04±131.62 | 5624.26±2506.04 | 60.26±19.15 |
In addition, to the ratio (G/M) of the mannose and mannose and glucose that have significant difference in table 1 with normal person into
It has gone ROC curve analysis, the higher index of area under the curve (AUC), sensitivity and specificity is found with expectation, to determine most
Good screening positive critical value (cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, by Fig. 4
As a result it summarizes to table 3.
The cutoff value (μm ol/L) and its sensitivity (%) of mannose and G/M and special in 3. serum of patients with intracerebral hemorrhage of table
Property (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Mannose | 0.7324 | 78.00 | 65.69 | 73.91 |
| G/M | 0.8452 | 72.07 | 80.39 | 76.09 |
From Fig. 3, table 1, as can be seen that serum of patients with intracerebral hemorrhage dissociates, mannose significantly rises table 2；G/M is remarkably decreased.Separately
Outside, table 3 shows that G/M area under the curve is higher, and its sensitivity and specificity is higher, thus can with free glucose with
The ratio of mannose is biomarker to detect patients with cerebral hemorrhage.
Embodiment 5
It is used to detect the application of cerebral hemorrhage using biomarker of the present invention, specifically includes the following steps:
(1) it creates alkaline environment: taking 10 μ L of blood serum sample to be detected to add 30 μ L ultrapure waters in 1.5mL centrifuge tube, add
Enter 40 μ L 0.3mol/L sodium hydroxides, is vortexed and mixes；
(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:
(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
40 μ L 0.3mol/L sodium hydroxides are added in 1.5mL centrifuge tube in quasi- product, are vortexed and mix；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
The ratio of sugar, the concentration of glucose and glucose and mannose concentration.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and patients with cerebral hemorrhage more intuitively to observe triple combination, see Fig. 5.
If the free glucose and mannose ratio in test serum sample are lower than 72.07, test serum sample is determined
Product are patients with cerebral hemorrhage.
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
The ratio for calculating mannose, glucose and the glucose and mannose concentration that dissociate in serum can distinguish normal person and cerebral hemorrhage trouble
Person is suitble to clinically be used for the diagnosis of patients with cerebral hemorrhage.
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 cerebral hemorrhage, it is characterised in that: the biomarker is serum by 1- benzene before column
The free mannose and glucose that high performance liquid chromatography derived from base -5- methylpyrazole quinoline ketone (PMP) obtains.
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of cerebral hemorrhage, it is characterised in that:
This 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 1.5mL centrifuge tube, sodium hydroxide, whirlpool is added
Rotation 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 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 prepared；
(7) chromatogram for obtaining step (5) and the standard curve of step (6) compare simultaneously analytical calculation, determine mannose, grape
The concentration of sugar and the ratio of glucose and mannose concentration.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of cerebral hemorrhage, it is characterised in that:
The 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 cerebral hemorrhage, it is characterised in that:
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%.
5. according to claim 2 for identifying the quantitative analysis method of the biomarker of cerebral hemorrhage, it is characterised in that:
The concentration of hydrochloric acid is 0.3mol/L in the step (3).
6. according to claim 2 for identifying the quantitative analysis method of the biomarker of cerebral hemorrhage, it is characterised in that:
Organic solvent is at least one of chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4).
7. identifying quantitative analysis method the answering in Cerebral Hemorrhage Disease of the biomarker of cerebral hemorrhage described in claim 2-6
With, it is characterised in that the blood serum sample is the serum of patients with cerebral hemorrhage.
8. a kind of kit for identifying cerebral hemorrhage, which is characterized in that the kit contains biomarker described in claim 1
The free mannose and glucose that object, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification cerebral hemorrhage according to claim 8, which is characterized in that be including concentration in the kit
The titer for the glucose that 78.00 μ rnol/L mannoses and concentration are 5621 μm of ol/L.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and grape in serum of patients with intracerebral hemorrhage
Purposes in the quantitative kit of sugar.
11. a kind of kit for identifying cerebral hemorrhage, it is characterised in that pass through the mannose and glucose quantitation identification brain in serum
Bleeding.
12. the purposes that the kit of the described in any item identification cerebral hemorrhages of claim 8-11 detects cerebral hemorrhage in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Cerebral hemorrhage.
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