CN109187813A - A kind of method and its detection kit for identifying pleural effusion biomarker - Google Patents
A kind of method and its detection kit for identifying pleural effusion biomarker Download PDFInfo
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- CN109187813A CN109187813A CN201811324086.8A CN201811324086A CN109187813A CN 109187813 A CN109187813 A CN 109187813A CN 201811324086 A CN201811324086 A CN 201811324086A CN 109187813 A CN109187813 A CN 109187813A
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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
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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 for identifying biomarker and its application of pleural effusion.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 pleural effusion Disease Clinical detection of searching.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to biomarker and its detection for identifying pleural effusion are answered
With.
Background technique
Pleural effusion (pleural effusion) is that one kind characterized by pathological liquid accumulation in pleural cavity is common
Clinical disease.This extra liquid can damage breathing by limiting the expansion of lung.Various pleural effusions depend on liquid
Property and the reason of cause it to enter pleural cavity, be pleural effusion (slurries), hemothorax (blood) is urinated chest (urine), chylothorax
(chyle) or pyothorax (purulence).Hydrostatic pressing increases (such as congestive heart failure) in pleura capillary, pleura permeability increases
Colloid osmotic pressure reduces (such as Hypoproteinemia, cirrhosis) in (such as pleura inflammation, tumour), pleura capillary, partial pleura
Lymphostasis (such as carcinous lymph pipe choking) and thoracic injury, can cause pleural effusion.The identification of pleural effusion
The problem always being in clinical position is diagnosed, being just reported early in PET/CT in 1997 can be used for diagnosing MPE, but due to sensitivity
Degree and specificity are all barely satisfactory, do not obtain recommendation so far and are applied to actual clinical position.Thoracoscope is for Diagnosis of malignant thoracic cavity
The value of hydrops obtains division of respiratory disease and the extensive approval of oncology doctor already, small in conjunction with that can detect using fluorescent technique
Pleural Lesions simultaneously accurately recognize extent of disease, greatly improve the susceptibility and negative predicted value of diagnosis.Diagnosis of malignant thoracic cavity product
The goldstandard of liquid is that malignant cell is found in hydrothorax or biopsy of pleura tissue, and problem is and not all malignant pleural effusion
Malignant cell can be found.Therefore, scientists, which wish to find, noninvasive improves diagnosis efficiency.How thoracic cavity is accurately identified
The Susceptible population of hydrops so as to early intervention, or can screen effect that suitable biomarker is treated with predictive disease or
The progress etc. of predictive disease has 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 pleural effusion
The mannose and glucose to dissociate in patients serum.
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 detection of the report by this method applied to dissociate in the serum of patients with pleural mannose and glucose at present.
Summary of the invention
The object of the present invention is to provide for identifying biomarker and its application of pleural effusion, the present invention uses column
High performance liquid chromatography derived from preceding PMP (HPLC) detects monosaccharide more particularly to serum middle reaches in patients with pleural serum
Detection from mannose and glucose.Technical solution of the present invention can be used for detecting patients with pleural or normal Check-up crowd
Free serum glucose and mannose concentration and to the two calculated by peak area ratio, can be used for identifying patients with pleural.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of pleural effusion, the biomarker are that patients serum is derivative by PMP before column
High performance liquid chromatography obtained free mannose and glucose.
For identifying the quantitative analysis method of the biomarker of pleural effusion, it is characterised in that: this method is PMP before column
Derivative high performance liquid chromatography, comprising the following specific steps
(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in EP pipe, sodium hydroxide is added, be vortexed
It mixes；
(2) PMP is derivative: PMP is added in each sample, is vortexed and mixes, and centrifugation is placed on baking oven reaction；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and hydrochloric acid is added in each sample, is vortexed mixed
It is even；
(4) extract: organic solvent is added in every pipe, is vortexed, and centrifugation removes lower layer's organic solvent, retains supernatant；
(5) sample is centrifuged, takes supernatant in sample bottle, covering tightly lid in the brown equipped with interpolation pipe, is centrifuged, is ready to use in
High-efficient liquid phase analysis；
(6) standard curve of mannose and glucose is 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 pleural effusion.
Biomarker is preparing the purposes in the kit for detecting pleural effusion, it is characterised in that: the reagent
It include concentration in box is 82.42 μm of ol/L mannoses and concentration is the titer of 4474 μm of ol/L glucose.
A kind of kit for identifying pleural effusion, which is characterized in that the kit contains biomarker, i.e. serum passes through
The free mannose and glucose that high performance liquid chromatography derived from PMP obtains before column.
It include concentration in the kit is 82.42 μm of ol/L mannoses and concentration is 4474 μm of ol/L glucose
Titer.
Identify that the kit of pleural effusion is preparing the purposes in the kit for detecting pleural effusion.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for patients with pleural blood by the present invention
The detection of clear 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 ratio (G/M ratio) to free serum glucose, mannose concentration and glucose and mannose peak area
Statistical data analysis, establish the relationship with pleural effusion.Using technical solution of the present invention, by detecting serum middle reaches
From mannose and glucose, calculate the ratio of mannose and concentration of glucose and glucose and mannose peak area, can be with
Quickly distinguish normal population and patients with pleural.
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 pleural and normal Check-up crowd free serum concentration of glucose, mannose concentration and glucose
With the scatter plot of mannose peak area ratio
Fig. 4 is patients with pleural and normal Check-up crowd glucose and mannose peak area ratio, as the life of serum sugar
The ROC curve figure of object marker.
Fig. 5 is patients with pleural and normal Check-up crowd free serum concentration of glucose, mannose concentration and glucose
With the 3 dimensional drawing of mannose peak area ratio 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.
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/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 × 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:
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 pleural made a definite diagnosis and 95 age-sexes matched just respectively
Normal 10 μ L of Check-up crowd blood serum sample adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L 0.3mol/L sodium hydroxides, whirlpool is added
Rotation 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 comparable trend of table 1. patients with pleural free serum monosaccharide and G/M peak area ratio and normal Check-up crowd
| Glucose | Mannose | G/M ratio | |
| Pleural effusion | ↓*** | ↑NS | ↓*** |
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.
2 kinds of free monosaccharide concentration (μm ol/L) and its peak area in 2. patients with pleural of table and normal Check-up crowd serum
Ratio result
| Glucose | Mannose | G/M ratio | |
| Normal Check-up crowd | 6332±2669 | 82.42±40.12 | 79.16±16.68 |
| Pleural effusion | 4474±1866 | 91.18±56.24 | 41.29±15.55 |
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 | 0.9554 | 60.54 | 91.89% | 91.89% |
From Fig. 3, table 1, table 2 can be seen that the matched normal Check-up crowd of age-sex and compare, patients with pleural serum
Free glucose is remarkably decreased, and mannose slightly rises, and glucose is remarkably decreased with mannose peak area ratio.In addition, table 3 is aobvious
Show, the specificity and sensitivity of mannose and glucose peaks area ratio are higher, illustrate free serum glucose and mannose
Peak area ratio and pleural effusion disease have relationship, and G/M ratio can distinguish normal person and patients with pleural.It therefore, can be with
Patients with pleural is detected as biomarker using free serum glucose and mannose peak area ratio.
Embodiment 5
It is used to detect the application of pleural effusion using biomarker of the present invention, specifically includes the following steps:
(1) it creates alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mL EP pipe, be added
40 μ L 0.3mol/L sodium hydroxides are vortexed and mix；
(2) PMP is derivative: 60 μ L 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 pleural 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 91.89%, and specificity is 91.89%, sensibility and specificity with higher.
If the free glucose and mannose peak area ratio in test serum sample determine to be measured less than 41.29
Blood serum sample is patients with pleural.
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 pleural effusion
Very important meaning will be suitble to clinically be used for the diagnosis of patients with pleural.
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 pleural effusion, it is characterised in that: the biomarker is serum by 1- before column
The free glucose and mannose that high performance liquid chromatography (HPLC) derived from phenyl -5- methylpyrazole quinoline ketone (PMP) obtains.
2. according to claim 1 for identifying the biomarker of pleural effusion, it is characterised in that: this method is before column
High performance liquid chromatography derived from PMP, 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 pleural effusion, it is characterised in that: the efficient liquid phase
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 pleural effusion, it is characterised in that: the step (5)
The change of gradient of middle mobile phase 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 pleural effusion, it is characterised in that: the step (3)
The concentration of middle hydrochloric acid is 0.3mol/L.
6. according to claim 2 for identifying the quantitative analysis method of pleural effusion biomarker, 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 the quantitative analysis method of pleural effusion biomarker in pleural effusion disease described in claim 2-6
Using, it is characterised in that the blood serum sample is the serum of patients with pleural.
8. a kind of kit for identifying pleural effusion, 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 pleural effusion according to claim 8, which is characterized in that include concentration in the kit
For the titer for the glucose that 82.42 μm of ol/L mannoses and concentration are 4474 μm of ol/L.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and Portugal in patients with pleural serum
Purposes in the quantitative kit of grape sugar.
11. a kind of kit for identifying pleural effusion, it is characterised in that identified by mannose in serum and glucose quantitation
Pleural effusion.
12. the purposes that the kit of the described in any item identification pleural effusions of claim 8-11 detects pleural effusion in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Pleural effusion.
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Non-Patent Citations (5)
| Title |
|---|
| 丁克祥 编: "《简明临床生化参考值手册》", 31 December 1987, 湖北科学技术出版社 * |
| 中国科协学会学术部: "《网络药理学-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 * |
| 吴梧桐 等: "《生物化学》", 31 August 2015, 中国医药科技出版社 * |
| 胡智祥: "《医院临床检验技术操作规范与实(化)验室管理全书第一卷》", 31 August 2004, 银声音像出版社 * |
| 董吁钢 等: "《心血管疾病预防与康复》", 28 February 2013, 中山大学出版社 * |
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