CN109187818A - A kind of method and its detection kit for identifying chronic obstructive pulmonary disease biomarker - Google Patents
A kind of method and its detection kit for identifying chronic obstructive pulmonary disease biomarker Download PDFInfo
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- CN109187818A CN109187818A CN201811324136.2A CN201811324136A CN109187818A CN 109187818 A CN109187818 A CN 109187818A CN 201811324136 A CN201811324136 A CN 201811324136A CN 109187818 A CN109187818 A CN 109187818A
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Abstract
The present invention provides for identifying biomarker and its application of Chronic Obstructive Pulmonary Disease.The biomarker is the free glucose and mannose that patients serum obtains by high performance liquid chromatography (HPLC) derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.Technical solution of the present invention has pre-treatment simple, analysis time is short, instrument price is reasonable, meet conventional use, operating procedure is easy to learn, and testing result accuracy is high, and need to only take a blood sample carries out the analysis of HPLC instrument and can distinguish normal person and patient, and the advantages that required serum amount is few, and blood sampling volume is less than 1mL.Acquired results show that this analysis method can quickly analyze the mannose and glucose to dissociate in serum, and mannose, glucose sugar concentration and the two peak area can be calculated, there is very important meaning for the marker of the relationship between research free serum mannose and glucose and disease, the novel Chronic Obstructive Pulmonary Disease Disease Clinical detection of searching.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to for identify Chronic Obstructive Pulmonary Disease biomarker and
It detects application.
Background technique
Chronic Obstructive Pulmonary Disease (COPD is also chronic obstructive pulmonary disease) is that one kind can prevent the chronic respiratory disease that can be controlled, with
Aging of population, global incidence show a increasing trend.COPD is defined as a kind of common disease that can be prevented and treated by 2017 editions GOLD,
It is characterized in that " continue existing respiratory symptom and flow limitation, usually caused by deleterious particle or gas exposure and/(or) lung
Bubble is abnormal and causes ", increase " duration respiratory symptom ".Currently, China mainly receives patient COPD for the treatment of, it is most of
Or even the overwhelming majority is all the patient of middle severe, and according to statistics, the China COPD patient very slight without symptom or symptom
70.3% is accounted for, the U.S. then accounts for 76%.For COPD patient, exactly when more slight, asymptomatic, under lung function
Drop be it is most fast, if be not effectively treated at this time, his lung function just will receive very big damage.Chronic obstructive pulmonary disease is
A kind of different substantiality disease shows as different patients with different Disease Clinical features.How chronic obstructive pulmonary disease easy is accurately identified
Can touching group be so as to early intervention, or screen effect or predictive disease that suitable biomarker is treated with predictive disease
Progress etc. have become hot issue.
The content highest of glucose in human serum free monosaccharide, the range of normal Check-up crowd is 3.90~
6.16mmol/L.Currently, hospital laboratory directly can detect concentration of glucose by surveying biochemical indicator, it temporarily cannot be direct
Detect mannose concentration.The concentration range of free mannose is 20~80 μm of ol/L in health adult's blood plasma.The content of mannose
About the 1% of glucose content.Its major part is considered as from the glucose isomerization in cell.And there is research recently
Show that the free mannose flowed out in cell is the main source of free mannose in mammalian.This two parts comes
The free mannose in source maintains the stabilization of mannose in serum.D-MANNOSE is glycoprotein, cell surface glycoconjugates and sugar
Required monosaccharide in the structures such as base phosphatidylinositols anchorin.Wherein, mannose is N- sugar chain in various polysaccharide compounds
Important component, glucose or mannose of the mannose residue in blood in N- glycan.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 spectrometry and capillary electrophoresis etc..But there is certain shortcoming in these methods.For example, using enzyme process, gas-liquid
When chromatography and high resolution liquid chromatography, to avoid the influence needs of glucose of high concentration from removing it, cause pre-treatment
Journey is comparatively laborious；Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for routine purpose；Required serum sample amount is big.Do not have at present
There is high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before document report column while detecting chronic obstruction
Property lung disease patients serum in dissociate mannose and glucose.
Therefore, establish in the serum of efficiently and accurately dissociate mannose detection method, for research free serum monosaccharide with
The marker of relationship, searching Disease Clinical detection between disease has very important meaning.
The PMP derivatization method of monosaccharide is current more common monosaccharide detection method.This method including the following steps:
(1) alkaline condition is created for PMP, (2) are added PMP, are derived at 70 DEG C, and (3) adjust the pH value of solution after deriving, will be remaining
PMP is extracted.103969371 B of patent of invention CN discloses that " a kind of blood degrades to obtain and detect the method for monosaccharide in cancer
Application in disease detection ".Polysaccharide therein and glycoprotein are degraded to by the patent of invention first by blood serum sample by acid degradation
Monosaccharide component；Then the 8 kinds of monosaccharide obtained after PMP is derivative and efficient liquid phase is to degradation detect.Before degradation
Serum, sample component and property have occurred and that great variety.Although the PMP of monosaccharide spreads out, method is wider in every field application
It is general, but there is no report that this method is applied to dissociate in the serum of Patients with Chronic Obstructive Pulmonary Disease mannose and glucose at present
Detection.
Summary of the invention
The object of the present invention is to provide for identifying biomarker and its application of Chronic Obstructive Pulmonary Disease, this hair
The bright monosaccharide using in the detection Patients with Chronic Obstructive Pulmonary Disease serum of high performance liquid chromatography (HPLC) derived from PMP before column,
More particularly to the detection of dissociate in serum mannose and glucose.Technical solution of the present invention can be used for detecting chronic obstructive
Lung disease patient or normal Check-up crowd free serum glucose and mannose concentration and to the two calculated by peak area ratio, can be with
For identifying Patients with Chronic Obstructive Pulmonary Disease.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of Chronic Obstructive Pulmonary Disease, the biomarker are patients serum by before column
The free mannose and glucose that high performance liquid chromatography derived from PMP obtains.
For identifying the quantitative analysis method of the biomarker of Chronic Obstructive Pulmonary Disease, it is characterised in that: this method
For 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 (4.6 mm × 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 Chronic Obstructive Pulmonary Disease.
Biomarker is preparing the purposes in the kit for detecting Chronic Obstructive Pulmonary Disease, it is characterised in that:
It include the titer for the glucose that concentration is 94.7 μm of ol/L mannoses and concentration is 4671 μm of ol/L in the kit.
A kind of kit for identifying Chronic Obstructive Pulmonary Disease, which is characterized in that the kit contains biomarker, i.e.,
The free mannose and glucose that serum is obtained by high performance liquid chromatography derived from PMP before column.
It include the mark for the glucose that concentration is 94.7 μm of ol/L mannoses and concentration is 4671 μm of ol/L in the kit
Quasi- liquid.
Identify the kit of Chronic Obstructive Pulmonary Disease in preparing the kit for detecting Chronic Obstructive Pulmonary Disease
Purposes.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for Chronic Obstructive Pulmonary Disease by the present invention
The detection of patient's free serum 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 ratio (G/M ratio) to free serum glucose, mannose concentration and glucose and mannose peak area
Statistical data analysis, establish the relationship with Chronic Obstructive Pulmonary Disease.Using technical solution of the present invention, pass through detection
The mannose and glucose to dissociate in serum calculates mannose and concentration of glucose and glucose and mannose peak area
Ratio can quickly distinguish normal population and Patients with Chronic Obstructive Pulmonary Disease.
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 Patients with Chronic Obstructive Pulmonary Disease and normal Check-up crowd free serum concentration of glucose, mannose concentration
And the scatter plot of glucose and mannose peak area ratio
Fig. 4 is Patients with Chronic Obstructive Pulmonary Disease and normal Check-up crowd glucose and mannose peak area ratio, as
The ROC curve figure of serum sugar biomarker
Fig. 5 is Patients with Chronic Obstructive Pulmonary Disease and normal Check-up crowd free serum concentration of glucose, mannose concentration
And the 3 dimensional drawing of glucose and the building of mannose peak area ratio
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.
Embodiment 1
Technical solution of the present invention the following steps are included:
(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 PMP (1- phenyl -5- methylpyrazole quinoline ketone) is added in each sample, is vortexed and mixes, centrifugation
70 DEG C of baking ovens react 1h afterwards；
(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 pH 5.5 |
| 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:
It will be seen from figure 1 that the AgilentZORBAXXDB-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,
The chromatographic peak of GlcUA, 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/LPMP 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 × 100 mm, 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 0.0005 mg/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/LPMP 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 × 100 mm, 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 95 Patients with Chronic Obstructive Pulmonary Disease made a definite diagnosis and 95 age-sexes respectively
Matched 10 μ L of normal Check-up crowd blood serum sample adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L 0.3mol/L hydrogen are added
Sodium oxide molybdena is vortexed and mixes；
(2) remaining step is the same as embodiment 3, analytical calculation glucose, the concentration of mannose and glucose and mannose peak
The ratio of area.Statistic mixed-state is as a result, be shown in Fig. 3.Fig. 3 result is summarized to Tables 1 and 2.
The phase of table 1. Patients with Chronic Obstructive Pulmonary Disease free serum monosaccharide and G/M peak area ratio and normal Check-up crowd
To trend
| Glucose | Mannose | G/M ratio | |
| Chronic Obstructive Pulmonary Disease | ↓*** | ↑** | ↓*** |
Annotation: there was no significant difference compared with normal Check-up crowd for " NS " representative, " * * * p < 0.001 ", " * * p < 0.01 "
" * p < 0.05 ", which is respectively represented, has significant difference compared with normal Check-up crowd, and " ↑ " and " ↓ " respectively represents and normal physical examination
Compared to rising or falling, "-" is indicated with normal Check-up crowd without significant change crowd.
In 2. Patients with Chronic Obstructive Pulmonary Disease of table and normal Check-up crowd serum 2 kinds of free monosaccharide concentration (μm ol/L) and
Its peak area ratio result
| Glucose | Mannose | G/M ratio | |
| Normal Check-up crowd | 6116±2431 | 78.45±32.04 | 78.93±14.19 |
| Chronic Obstructive Pulmonary Disease | 4671±2798 | 94.7±46.84 | 39.82±15.72 |
In addition, to thering is the mannose of significant difference and glucose peaks area ratio to carry out ROC song in table 1 with normal person
Line analysis finds the higher index of area under the curve (AUC), sensitivity and specificity with expectation, to determine best screening sun
Property 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.
Table 3
| AUC | Cutoff value | Sensitivity | Specificity | |
| G/M ratio | 0.9588 | 57.51 | 87.5% | 98.21% |
From Fig. 3, table 1, table 2 can be seen that the matched normal Check-up crowd of age-sex and compare, COPD patient free serum
Glucose decline, mannose rise, and glucose is remarkably decreased with mannose peak area ratio.In addition, table 3 show, mannose and
The specificity of glucose peaks area ratio and sensitivity are higher, illustrate free serum glucose and mannose peak area ratio with
Chronic Obstructive Pulmonary Disease disease has relationship, and G/M ratio can distinguish normal person and Patients with Chronic Obstructive Pulmonary Disease.Therefore,
Patients with Chronic Obstructive Pulmonary Disease can be detected using glucose free clearly and mannose peak area ratio as biomarker.
Embodiment 5
It is used to detect the application of Chronic Obstructive Pulmonary Disease using biomarker of the present invention, specifically includes following
Step: (1) creating alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mLEP pipe, is added 40
μ L 0.3mol/L sodium hydroxide is vortexed and mixes；
(2) PMP is derivative: 60 μ L PMP are added in each sample, are vortexed and mix, and 70 DEG C of baking ovens react 1h after centrifugation；
(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 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: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
| Time | A (acetonitrile) | B (0.1mol/L ammonium acetate pH 5.5) |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
(6) draw the standard curve of glucose and mannose: precision weighs glucose and appropriate mannose, 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 (6) compares simultaneously analytical calculation, determines grape
The ratio of sugar, the concentration of mannose and glucose and mannose peak area.And it is made between three by 9 software of Origin
3 dimensional drawing, normal person and patients with COPD can be distinguished more intuitively to observe triple combination, see Fig. 5.
ROC curve is made by Prism software, to determine a possibility that G/M ratio is as marker, as a result, it has been found that quick
Perception is 87.5%, and specificity is 98.2%, sensibility and specificity with higher.
If the free glucose and mannose peak area ratio in test serum sample determine blood to be measured less than 39.8
Final proof product are Patients with Chronic Obstructive Pulmonary Disease.
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.Institute of the present invention
The method stated has the marker of relationship, searching Disease Clinical detection between research free serum monosaccharide and chronic obstructive pulmonary disease non-
Often important meaning will be suitble to clinically be used for the diagnosis of Patients with Chronic Obstructive Pulmonary Disease.
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 Chronic Obstructive Pulmonary Disease, it is characterised in that: the biomarker is serum warp
Cross high performance liquid chromatography (HPLC) derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) obtains before column free glucose and sweet
Dew sugar.
2. according to claim 1 for identifying the biomarker of Chronic Obstructive Pulmonary Disease, it is characterised in that: the party
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 glucose and mannose is prepared；
(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares and analytical calculation, determines glucose, sweet
Reveal the concentration of sugar and the ratio of glucose and mannose peak area.
3. according to claim 1 for identifying the biomarker of Chronic Obstructive Pulmonary Disease, it is characterised in that: described
High performance liquid chromatography be 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 biomarker of Chronic Obstructive Pulmonary Disease, it is characterised in that: described
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 biomarker of Chronic Obstructive Pulmonary Disease, it is characterised in that: described
The concentration of hydrochloric acid is 0.3mol/L in step (3).
6. it is according to claim 2 for identifying the quantitative analysis method of Chronic Obstructive Pulmonary Disease biomarker,
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. the quantitative analysis method of identification Chronic Obstructive Pulmonary Disease biomarker is in chronic obstruction described in claim 2-6
Property lung disease disease in application, it is characterised in that the blood serum sample be Patients with Chronic Obstructive Pulmonary Disease serum.
8. a kind of kit for identifying Chronic Obstructive Pulmonary Disease, which is characterized in that the kit contains described in claim 1
The free mannose and glucose that biomarker, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification Chronic Obstructive Pulmonary Disease according to claim 8, which is characterized in that in the kit
The titer for the glucose that including concentration be 94.7 μm of ol/L mannoses and concentration is 4671 μm of ol/L.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for sweet in Patients with Chronic Obstructive Pulmonary Disease serum
Purposes in dew sugar and the kit of glucose quantitation.
11. a kind of kit for identifying Chronic Obstructive Pulmonary Disease, it is characterised in that pass through the mannose and glucose in serum
Quantitative measurement Chronic Obstructive Pulmonary Disease.
12. the kit of the described in any item identification Chronic Obstructive Pulmonary Disease of claim 8-11 detects chronic obstruction in vitro
The purposes of property lung disease.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Chronic Obstructive Pulmonary Disease.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110687231A (en) * | 2019-11-26 | 2020-01-14 | 四川大学华西医院 | Typing detection kit for chronic obstructive pulmonary disease |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110687231A (en) * | 2019-11-26 | 2020-01-14 | 四川大学华西医院 | Typing detection kit for chronic obstructive pulmonary disease |
| CN110687231B (en) * | 2019-11-26 | 2022-10-18 | 四川大学华西医院 | Typing detection kit for chronic obstructive pulmonary disease |
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