CN109596724A - A kind of method and its detection kit for identifying cancer of the esophagus biomarker - Google Patents
A kind of method and its detection kit for identifying cancer of the esophagus biomarker Download PDFInfo
- Publication number
- CN109596724A CN109596724A CN201811323456.6A CN201811323456A CN109596724A CN 109596724 A CN109596724 A CN 109596724A CN 201811323456 A CN201811323456 A CN 201811323456A CN 109596724 A CN109596724 A CN 109596724A
- Authority
- CN
- China
- Prior art keywords
- esophagus
- mannose
- cancer
- phase
- glucose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying cancer of the esophagus.The biomarker is the ratio of the free glucose and mannose that obtain in serum by high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.Detection method is high performance liquid chromatography derived from PMP before column.Technical solution of the present invention has pre-treatment simple, and analysis time is short, and instrument price is reasonable, meet conventional use, operating procedure is easy to learn, and testing result accuracy is high, the advantages that need to only take a blood sample can distinguish normal person and esophageal cancer patients, 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 glucose and mannose and cancer of the esophagus, the novel cancer of the esophagus clinical detection marker of searching with mannose and glucose free in fast quantification esophageal cancer patients serum.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of method and its inspection of the biomarker for identifying cancer of the esophagus
Test agent box.
Background technique
Esophagus belongs to digestive organ, and connection throat to stomach is close to the veutro of backbone, and help transports food and enters stomach
Portion is the key that food enters digestive system, therefore esophagus generates disease and can seriously affect to body generation.The diagnosis of cancer of the esophagus
It generally requires and carries out imageological examination, esophoagoscopy and blood test etc., and diagnosed in conjunction with clinical manifestation.These are examined
Disconnected method can bring certain drawbacks.Therefore, a kind of convenience is found, the detection method of hurtless measure is very necessary.Blood
It checks because the characteristics of its is easy to operate, hurtless measure is as ideal detection method, it is most common in blood test to face
Bed detection marker is carcinomebryonic antigen (CEA), cytokeratin 19 fragment (Cyfra21-1) and Squamous intraepithelial lesion
(SCC), but its sensitivity is lower, is not easy effective screening patient, so screening suitable blood biomarker is that cancer of the esophagus is faced
The key of bed diagnosis.
Reductive monosaccharide seems to be only limitted to mannose and glucose in serum.Glucose contains in human serum free monosaccharide
Highest is measured, the range of normal person is 3.90~6.16mmol/L.Currently, hospital laboratory can be directly by surveying biochemical indicator inspection
Concentration of glucose is surveyed, mannose concentration temporarily cannot be directly detected.The concentration range of free mannose in health adult's blood plasma
For 20~80 μm of ol/L.The content of mannose is about the 1% of glucose content.It is considered as by the grape in cell that it is most of
Sugared isomerization.And recently some researches show that, the free mannose flowed out in cell be dissociate in mammalian it is sweet
Reveal the main source of sugar.The free mannose in this two parts source maintains the stabilization of mannose in serum.D-MANNOSE is sugar
Required monosaccharide in the structures such as albumen, cell surface glycoconjugates and glycosylphosphatidylinositol-anchored proteins.Wherein, mannose
The important component of N- sugar chain in various polysaccharide compounds, glucose of the mannose residue in N- glycan in the blood or
Mannose.Free mannose is normal plasma composition.Currently, the measuring method of serum mannose mainly has enzyme process, gas-liquid color
Spectrometry, high resolution liquid chromatography, gas-liquid chromatography-mass spectrography and capillary electrophoresis etc..But these methods exist it is certain not
Foot place.For example, when using enzyme process, gas-liquid chromatography and high resolution liquid chromatography, for avoid high concentration glucose influence
It needs to remove it, causes pretreatment process comparatively laborious；Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for routine mesh
's；Required serum sample amount is big.
Summary of the invention
Before for identifying that biomarker and its application of cancer of the esophagus, the present invention use column
Dissociate in monosaccharide more particularly to serum in high performance liquid chromatography derived from PMP (HPLC) detection esophageal cancer patients serum sweet
The detection of dew sugar and glucose.Technical solution of the present invention can be used for detecting esophageal cancer patients or the free grape of normal human serum
The ratio of sugar and mannose, can be used for identifying esophageal cancer patients.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of cancer of the esophagus, the biomarker are serum by efficient derived from PMP before column
The ratio of free glucose and mannose that liquid chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of cancer of the esophagus, it is characterised in that: this method is spread out for PMP before column
Raw high performance liquid chromatography, comprising the following specific steps
(1) adjusting pH is 7-14: taking blood serum sample to be detected to add ultrapure water in 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.
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 cancer of the esophagus.
Biomarker is preparing the purposes in the kit for detecting cancer of the esophagus, it is characterised in that: the kit
In include titer that glucose and mannose ratio are 63.89.
A kind of kit for identifying cancer of the esophagus, which is characterized in that the kit contains biomarker, i.e. serum passes through column
The ratio of free glucose and mannose that high performance liquid chromatography derived from preceding PMP obtains.
It include the titer that glucose is 63.89 with mannose ratio in the kit.
Identify that the kit of cancer of the esophagus is preparing the purposes in the kit for detecting cancer of the esophagus.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for esophageal cancer patients serum by the present invention
The detection of free monosaccharide, advantageous effects are:
1, pre-treatment is simple.
2, analysis time is short.
3, required amount of serum is few, simply needs to be less than the blood volume of 1mL every time.
4, instrument price is reasonable, meets conventional use.
5, operating procedure is easy to learn.
6, detection is accurate.
The present invention uses hplc simultaneous determination free serum mannose and glucose derived from PMP before column,
Simplify operation, and amount of serum is few, and the measurement for having proven to the method mannose concentration will not be by high concentration glucose
Influence.Pass through the statistics of the ratio (G/M ratio) to free serum mannose, glucose and glucose and mannose total amount
Data analysis, establishes the relationship between cancer of the esophagus and three.Normal person can be quickly distinguished using technical solution of the present invention
And esophageal cancer patients.
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 esophageal cancer patients and the free mannose of normal human serum, glucose and glucose and mannose total amount ratio
Scatter plot；
Fig. 4 is the ROC curve of esophageal cancer patients free serum mannose, glucose and glucose and mannose ratio；
Fig. 5 is the ratio of normal person and esophageal cancer patients free serum mannose, glucose and glucose and mannose total amount
It is worth the 3 dimensional drawing of building.
Specific embodiment
Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.
All blood samples involved in embodiment are provided by affiliated hospital, University Of Qingdao, Qingdao City.
Technical solution of the present invention the following steps are included:
Embodiment 1
(1) precision weighs mannose (Man), gucosamine (GlcN), galactosamine (GalN), glucuronic acid
(GlcUA), glucose (Glc), galactolipin (Gal), xylose (Xyl), fucose (Fuc) in right amount, add deionized water to prepare two parts
Containing the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added
Sodium oxide molybdena is vortexed and mixes；
(3) PMP is derivative: 60 μ L 0.5mol/L 1- phenyl -5- methylpyrazole quinoline ketone (PMP) are added in each sample, are vortexed
It mixes, 70 DEG C of baking ovens react 1h after centrifugation；
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, two parts of samples carry out high-efficient liquid phase analysis, efficient liquid phase using different chromatographic column A and B respectively
Chromatography is 1260 efficient liquid phase system of Agilent, and gained chromatogram is shown in Fig. 1.
Chromatographic column A:Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm) chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate 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:
| 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 pH5.5 |
| 0min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient B:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 20% | 80% |
| 20min | 20% | 80% |
Mobile phase variable gradient C:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 22% | 78% |
| 20min | 22% | 78% |
Mobile phase variable gradient D:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient E:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
Figure it is seen that only Man is eluted out under the conditions of gradient A；Under the conditions of gradient B, although three
Can be separated and, but the peak of solvent PMP and Man from it is relatively close；And under the conditions of gradient C, solvent peak and the peak Man weight
It closes；Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient E item
Under part, appearance is symmetrical, and separating degree is good.Abscissa is time (min), and ordinate is DAD signal strength.1,2 and 3 is successively
The chromatographic peak of Man, Rha and Glc.
Embodiment 3
(1) precision weighs mannose and proper amount of glucose, and deionized water is added to be configured to each 0.5mg/mL containing the above monosaccharide,
0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL, 0.0025mg/mL, 0.001mg/mL,
The hybrid standard product solution of 0.0005mg/mL, matching while using；
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard 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 282 normal human sera samples and 282 esophageal cancer patients blood made a definite diagnosis respectively
10 μ L of final proof product adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L 0.3mol/L sodium hydroxides are added, and is vortexed and mixes；
(2) for remaining step with embodiment 3, analytical calculation mannose, the concentration of glucose and glucose and mannose are total
The ratio of amount.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. esophageal cancer patients free serum monosaccharide and G/M ratio and normal person
| Mannose | Glucose | G/M ratio | |
| Cancer of the esophagus | ↑*** | ↓** | ↓*** |
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 total amount ratio result in 2. esophageal cancer patients of table and normal human serum
| Mannose | Glucose | G/M ratio | |
| Normal person | 74.69±40.35 | 5755±2122 | 80.35±15.96 |
| Cancer of the esophagus | 88.21±43.40 | 5445±2742 | 46.09±16.51 |
In addition, to free mannose, glucose and the glucose and mannose for having significant difference in table 1 with normal person
Ratio (G/M) has carried out ROC curve analysis, finds the higher finger of area under the curve (AUC), sensitivity and specificity with expectation
Mark, to determine best screening positive critical value (cutoff value), AUC illustrates that diagnosis effect is better closer to 1.Testing result
See Fig. 4, Fig. 4 result is summarized to table 3.
Dissociate the ratio (G/M) of mannose, glucose and glucose and mannose in 3. esophageal cancer patients serum of table
Cutoff value (μm ol/L) and its sensitivity (%) and specificity (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Mannose | 0.6061 | 69.63 | 60.99 | 60.45 |
| Glucose | 0.5660 | 4536 | 44.33 | 73.13 |
| G/M | 0.9242 | 63.89 | 86.52 | 89.55 |
From Fig. 3, table 1, table 2 is as can be seen that esophageal cancer patients free serum glucose and mannose ratio (G/M) significantly drop
It is low.In addition, table 3 is shown, the specificity of the ratio (G/M) of glucose and mannose and sensitivity are higher, therefore can be with trip
Esophageal cancer patients are detected from the ratio (G/M) of glucose and mannose is biomarker.
Embodiment 5
It is used to detect the application of cancer of the esophagus using biomarker of the present invention, specifically includes the following steps:
(1) it creates alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mL EP pipe, be added
40 μ L 0.3mol/L sodium hydroxides are vortexed and mix；
(2) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation
1h；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(4) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(5) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.High performance liquid chromatography is 1260 efficient liquid phase system of Agilent,
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
(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 total amount.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and esophageal cancer patients more intuitively to observe triple combination, see Fig. 5.
If the ratio (G/M) of free glucose and mannose in test serum sample determines to be measured less than 63.89
Blood serum sample is esophageal cancer patients.
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 dissociate in serum glucose and mannose can distinguish normal person and esophageal cancer patients, be suitble to clinically be used for esophagus
The diagnosis of cancer 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 cancer of the esophagus, it is characterised in that: the biomarker is serum by 1- benzene before column
The ratio of free glucose and mannose that high performance liquid chromatography derived from base -5- methylpyrazole quinoline ketone (PMP) obtains.
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of cancer of the esophagus, 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 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 total amount.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of cancer of the esophagus, it is characterised in that:
The high performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column
(4.6mm × 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.
4. according to claim 3 for identifying the quantitative analysis method of the biomarker of cancer of the esophagus, it is characterised in that:
The change of gradient of mobile phase in the step (5) are as follows:
0min, A phase 15%, B phase 85%；
10min, A phase 22%, B phase 78%；
15min, A phase 24%, B phase 76%；
15.1min, A phase 15%, B phase 85%；
20min, A phase 15%, B phase 85%.
5. according to claim 2 for identifying the quantitative analysis method of the biomarker of cancer of the esophagus, it is characterised in that:
The concentration of hydrochloric acid is 0.3mol/L in the step (3).
6. according to claim 2 for identifying the quantitative analysis method of the biomarker of cancer of the esophagus, 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. the quantitative analysis method of the biomarker of identification cancer of the esophagus answering in cancer of the esophagus disease described in claim 2-6
With, it is characterised in that the blood serum sample is the serum of esophageal cancer patients.
8. a kind of kit for identifying cancer of the esophagus, which is characterized in that the kit contains biomarker described in claim 1
The ratio of free glucose and mannose that object, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification cancer of the esophagus according to claim 8, which is characterized in that include glucose in the kit
With mannose total amount than the titer for 63.89.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and grape in esophageal cancer patients serum
Purposes in the quantitative kit of sugar.
11. a kind of kit for identifying cancer of the esophagus, it is characterised in that pass through the mannose and glucose quantitation identification food in serum
Road cancer.
12. the purposes that the kit of the described in any item identification cancers of the esophagus of claim 8-11 detects cancer of the esophagus in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Cancer of the esophagus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811323456.6A CN109596724A (en) | 2018-11-01 | 2018-11-01 | A kind of method and its detection kit for identifying cancer of the esophagus biomarker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811323456.6A CN109596724A (en) | 2018-11-01 | 2018-11-01 | A kind of method and its detection kit for identifying cancer of the esophagus biomarker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109596724A true CN109596724A (en) | 2019-04-09 |
Family
ID=65958193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811323456.6A Pending CN109596724A (en) | 2018-11-01 | 2018-11-01 | A kind of method and its detection kit for identifying cancer of the esophagus biomarker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109596724A (en) |
-
2018
- 2018-11-01 CN CN201811323456.6A patent/CN109596724A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 丁克祥: "《简明临床生化参考值手册》", 31 December 1987 * |
| 中国科协学会学术部: "《网络药理学一中药现代化的新思路与新方法》", 30 November 2014 * |
| 吴梧桐 等: "《生物化学》", 31 August 2015 * |
| 胡智祥: "《医院临床检验技术操作规范与实（化）验室管理全书第一卷》", 31 August 2004 * |
| 董吁钢 等: "《心血管疾病预防与康复》", 28 February 2013 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Serum unsaturated free fatty acids: a potential biomarker panel for early-stage detection of colorectal cancer | |
| CN109342597A (en) | A kind of method and its detection kit for identifying cerebral infarction biomarker | |
| CN112162050B (en) | Application of MIT and/or DIT as thyroid cancer marker and kit | |
| CN109342593A (en) | A kind of method and its detection kit for identifying carcinoma of endometrium biomarker | |
| CN109613127A (en) | A kind of method and detection kit for identifying urarthritis biomarker | |
| CN109490436A (en) | A kind of method and its detection kit for identifying coronary heart disease biomarker | |
| CN109187814A (en) | A kind of method and its detection kit for identifying kidney transplant prognosis biomarker | |
| CN110261495A (en) | A kind of method and its detection kit for identifying colorectal cancer biomarker | |
| CN109212099A (en) | A kind of method and its detection kit for identifying gastric ulcer biomarker | |
| CN109342594A (en) | A kind of method and its detection kit for identifying gastric cancer biomarker | |
| CN109406656A (en) | A kind of method and its detection kit for identifying psoriasis biomarker | |
| CN109187816A (en) | A kind of method and its detection kit for identifying pancreatitis biomarker | |
| CN109406667A (en) | A kind of method and its detection kit for identifying dry syndrome biomarker | |
| CN109342598A (en) | A kind of method and its detection kit for identifying cerebral hemorrhage biomarker | |
| CN109212101A (en) | A kind of method and its detection kit for identifying asthma biomarker | |
| CN109406671A (en) | A kind of method and its kit for identifying rheumatoid arthritis biomarker | |
| CN110261494A (en) | A kind of method and its detection kit for identifying thyroid malignancy biomarker | |
| CN109187818A (en) | A kind of method and its detection kit for identifying chronic obstructive pulmonary disease biomarker | |
| CN109596724A (en) | A kind of method and its detection kit for identifying cancer of the esophagus biomarker | |
| CN109406670A (en) | A kind of method and its detection kit for identifying bladder cancer biomarkers object | |
| CN109521107A (en) | A kind of method and its detection kit for identifying kidney biomarker | |
| CN109342595A (en) | A kind of method and its detection kit for identifying gastritis biomarker | |
| CN109239234A (en) | A kind of method and its detection kit for identifying cancer of pancreas biomarker | |
| CN109406658A (en) | A kind of method and its detection kit for identifying anaemia biomarker | |
| CN109470787A (en) | A kind of method and its detection kit for identifying breast Cancer Biomarkers object |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190409 |