CN109406662A - A kind of method and its kit for identifying degenerative osteoarthropathy biomarker - Google Patents
A kind of method and its kit for identifying degenerative osteoarthropathy 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 degenerative osteoarthropathy.The biomarker is the ratio of the free mannose and glucose and glucose and mannose that obtain in serum by high performance liquid chromatography derived from PMP before column.Detection method is high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.The advantages that technical solution of the present invention has pre-treatment simple, and analysis time is short, and instrument price is reasonable, meets conventional use, and operating procedure is easy to learn, and testing result accuracy is high, need to only take a blood sample, 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 degenerative osteoarthropathy, the novel degenerative osteoarthropathy clinical detection marker of searching with mannose and glucose free in fast quantification degenerative osteoarthropathy patients serum.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of side for the biomarker for identifying degenerative osteoarthropathy
Method and its detection kit.
Background technique
Degenerative osteoarthropathy is a kind of chronic, is that the bone as caused by the aging and joint long term wear of human body closes
The joint disease degenerated, also known as osteoarthritis, degenerative arthritis are saved, mid-aged population is more common in.The feature of the disease exists
Running down in hyaline cartilage along with the variation of surrounding tissue includes ligament, synovial membrane and subchondral bone, leads to joint or backbone
Impacted, as aging of population and life expectancy extend, the burden of bone and joint diseases can be weighed increasingly, therefore find a kind of morning
Phase diagnostic method is even more important.
For degenerative osteoarthropathy clinically mainly by the observation of some symptoms and iconography, usually disease has been sent out
It is raw to arrive very serious degree, but clinically diagnostic blood serum designated object is seldom, there is presently no complete exact blood
Clear marker gets up to be accurately used for alone or in combination early diagnosis or accurate response these disease prognosis of these diseases
Therefore assessment treatment condition finds a kind of convenience, the detection method of hurtless measure is very necessary.And optimal early stage inspection
Survey is exactly to be not required to biopsy, and sensitivity and the high blood analysis of 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) detects degenerative osteoarthropathy patients serum simultaneously
In dissociate mannose and glucose.
Summary of the invention
The object of the present invention is to provide for identifying biomarker and its application of degenerative osteoarthropathy, the present invention
Using the monosaccharide in high performance liquid chromatography derived from PMP before column (HPLC) detection degenerative osteoarthropathy patients serum, especially
It is related to the detection of free mannose and glucose in serum.Technical solution of the present invention can be used for detecting degenerative osteoarthropathy
Patient or the free mannose of normal human serum and concentration of glucose, can be used for identifying degenerative osteoarthropathy patient.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of degenerative osteoarthropathy, the biomarker are that serum spreads out by PMP before column
The free mannose and glucose that raw high performance liquid chromatography obtains.
For identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy, 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 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 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 degenerative osteoarthropathy.
Biomarker is preparing the purposes in the kit for detecting degenerative osteoarthropathy, it is characterised in that: institute
State the titer that the ratio including glucose and mannose concentration in kit is 59.33.
A kind of kit for identifying degenerative osteoarthropathy, which is characterized in that the kit contains biomarker, i.e. blood
The clear free mannose and glucose obtained by high performance liquid chromatography derived from PMP before column.
The titer that ratio including glucose and mannose concentration in the kit is 59.33.
Identify that the kit of degenerative osteoarthropathy is preparing the use in the kit for detecting degenerative osteoarthropathy
On the way.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for degenerative osteoarthropathy by the present invention
The detection of patients serum's 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 degenerative osteoarthropathy and three.It can quick area using technical solution of the present invention
Divide normal person and degenerative osteoarthropathy patient.
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 degenerative osteoarthropathy patient and the free mannose of normal human serum, glucose and glucose and mannose
The ratio scatter plot of concentration；
Fig. 4 is the ratio of degenerative osteoarthropathy patients serum free mannose and glucose and glucose and mannose
ROC curve；
Fig. 5 is normal person and the free mannose of degenerative osteoarthropathy patients serum, glucose and glucose and mannose
The 3 dimensional drawing of the ratio building of concentration.
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:
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 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 pH 5.5 |
| 0min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient B:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 20% | 80% |
| 20min | 20% | 80% |
Mobile phase variable gradient C:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 22% | 78% |
| 20min | 22% | 78% |
Mobile phase variable gradient D:
Mobile phase variable gradient E:
| 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% |
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 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 151 normal human sera samples and 155 degeneration Bones and joints made a definite diagnosis respectively
10 μ L of patient's blood serum sample 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 mixed
It is even；
(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. degenerative osteoarthropathy patients serum free monosaccharide and G/M ratio and normal person
| Glucose | Mannose | G/M ratio | |
| Degenerative osteoarthropathy | ↓*** | ↑*** | ↓*** |
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 knot in 2. degenerative osteoarthropathy patient of table and normal human serum
Fruit
| Glucose | Mannose | G/M ratio | |
| Normal person | 6402±2338 | 50.05±37.25 | 84.26±17.57 |
| Degenerative osteoarthropathy | 4513±1294 | 69.16±28.60 | 45.12±15.58 |
In addition, there is the mannose of significant difference and glucose to carry out ROC curve analysis with normal person in table 1, with
It is expected that the higher index of area under the curve (AUC), sensitivity and specificity is found, 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.
The cutoff value (μm ol/L) of mannose and glucose and its sensitive in 3. degenerative osteoarthropathy patients serum of table
It spends (%) and specific (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Mannose | 0.7543 | 50.65 | 73.55 | 73.51 |
| Glucose | 0.822 | 5234 | 75.48 | 76.82 |
| G/M ratio | 0.9468 | 59.33 | 85.16 | 94.04 |
From Fig. 3, table 1, as can be seen that degenerative osteoarthropathy patients serum dissociates, mannose significantly rises table 2, glucose
And the ratio of glucose and mannose is remarkably decreased.In addition, table 3 is shown, the sensitivity of the ratio of mannose and glucose and
Specificity is higher than individual mannose and glucose, therefore can be using the ratio of dissociate mannose and glucose as biological
Marker detects degenerative osteoarthropathy patient.
Embodiment 5
It is used to detect the application of degenerative osteoarthropathy using biomarker of the present invention, specifically includes following step
It is rapid: (1) to create 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, 40 μ L are added
0.3mol/L sodium hydroxide 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
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
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 degenerative osteoarthropathy patient more intuitively to observe triple combination, see
Fig. 5.
If the ratio of glucose and mannose is to have very big risk to suffer from as G/M≤59.33 in test serum sample
There is degenerative osteoarthropathy.
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 degeneration bone
Arthritic patients are suitble to clinically be used for the diagnosis of degenerative osteoarthropathy patient.
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 degenerative osteoarthropathy, it is characterised in that: the biomarker is serum process
High performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) obtains before column free mannose and glucose and
The ratio of glucose and mannose.
2. it is according to claim 1 for identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy,
Be characterized 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 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 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. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy,
Be characterized 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. it is according to claim 3 for identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy,
It is characterized 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. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy,
Be characterized in that: the concentration of hydrochloric acid is 0.3mol/L in the step (3).
6. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy,
Be characterized in that: organic solvent is at least one in chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4)
Kind.
7. identifying the quantitative analysis method of the biomarker of degenerative osteoarthropathy in degeneration bone described in claim 2-6
Application in arthropathy disease, it is characterised in that the blood serum sample is the serum of degenerative osteoarthropathy patient.
8. a kind of kit for identifying degenerative osteoarthropathy, which is characterized in that the kit contains life described in claim 1
Substance markers object, i.e. serum pass through the free mannose that high performance liquid chromatography derived from PMP obtains before column and glucose and grape
The ratio of sugar and mannose.
9. the kit of identification degenerative osteoarthropathy according to claim 8, which is characterized in that wrapped in the kit
The ratio of glucose and mannose concentration is included as 59.33 titer.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for sweet dew in degenerative osteoarthropathy patients serum
Purposes in sugar and the kit of glucose quantitation.
11. a kind of kit for identifying degenerative osteoarthropathy, it is characterised in that fixed by mannose in serum and glucose
Amount identification degenerative osteoarthropathy.
12. the kit of the described in any item identification degenerative osteoarthropathies of claim 8-11 detects degeneration bone pass in vitro
Save the purposes of disease.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Degenerative osteoarthropathy.
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| 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, 中国科学技术出版社 * |
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