CN110261495A - A kind of method and its detection kit for identifying colorectal cancer biomarker - Google Patents
A kind of method and its detection kit for identifying colorectal cancer biomarker Download PDFInfo
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Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying colorectal cancer.The biomarker is the free mannose and glucose obtained 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 colorectal 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 mannose and glucose and colorectal cancer, the novel colorectal cancer clinical detection marker of searching with mannose and glucose free in fast quantification serum in patients with colorectal.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to it is a kind of identify colorectal cancer biomarker method and its
Detection kit.
Background technique
Colorectal cancer (CRC) is the malignant tumour that the third is common in the world and the second largest original for leading to cancer mortality
Cause.It is estimated that there are about 1,400,000 new cases and 700,000 deaths for the whole world in 2012.The postoperative 5 years survival rates of early stage patient
It more than 90%, but has been at present advanced stage the death rate to be caused to increase since Most patients lack when apparent clinical symptoms are found
Add.It the use of relatively broad diagnostic method include: clinically at present colonoscopy, ultrasonic examination, computed tomography
(CT), MRI, occult blood test (FOBT), the traditional serologicals biomarker such as CEA, CA19-9.Colonoscopy conduct
The standard detecting method of diagnosis of colorectal carcinoma has highly sensitive and specificity, is able to detect and cuts off lesion, or even in cancer
In preceding lesion be also in this way, but due to it be it is invasive inspection usually sense of discomfort can be brought to patient and also its accuracy also by
The surgical skills experience of doctor and the influence of patient compliance.Ultrasound, CT, MRI can be used for the inspection of tumour, but when discovery is swollen
It still needs to make a definite diagnosis by colonoscopy when block.Occult blood test (FOBT) can be used as the use of wide spectrum Screening tests, but its is sensitive
The feature of degree difference hinders extensive use in clinical practice.And blood analysis be it is non-invasive, can be to the maximum extent
It reduces the risk of patient and easy to operate can be repeated several times to reduce diagnostic error rate, but traditional serum biology blood marker
Diagnosis performance it is bad cannot diagnose colorectal cancer in early detection, therefore find a kind of novel high sensitivity and specificity
Marker is particularly significant.
Reductive monosaccharide seems to be only limitted to mannose and grape in serum.The content of glucose in human serum free monosaccharide
Highest, the range of normal person are 3.90~6.16mmol/L.Currently, hospital laboratory can be directly by surveying biochemical indicator detection
Concentration of glucose temporarily cannot directly detect mannose concentration.The concentration range of free mannose is in health adult's blood plasma
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
The object of the present invention is to provide for identifying biomarker and its application of colorectal cancer, the present invention uses column
High performance liquid chromatography derived from preceding PMP (HPLC) detects monosaccharide more particularly to serum middle reaches in serum in patients with colorectal
Detection from mannose and glucose.Technical solution of the present invention can be used for detecting colorectal cancer patients or normal human serum trip
From mannose and concentration of glucose, can be used for identifying colorectal 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 colorectal cancer, the biomarker are serum by height derived from PMP before column
The free mannose and glucose that effect liquid phase chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of colorectal cancer, 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 drawn；
(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 colorectal cancer, it is characterised in that: the reagent
It include the titer (carcinoma of the rectum) for the glucose that concentration is 4163 μm of ol/L in box.
The titer and concentration for the mannose for being 81.09 μm of ol/L including concentration in the kit are 3885 μm of ol/L
Glucose standard (colon cancer).
A kind of kit for identifying colorectal cancer, 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 the titer (carcinoma of the rectum) for the glucose that concentration is 4163 μm of ol/L in the kit.
The titer and concentration for the mannose for being 81.09 μm of ol/L including concentration in the kit are 3885 μm of ol/L
Glucose standard (colon cancer).
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for colorectal cancer patients 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 statistics of the ratio (G/M ratio) to free serum mannose, glucose and glucose and mannose concentration
Data analysis, establishes the relationship between colorectal cancer and three.It can quickly be distinguished normally using technical solution of the present invention
People and colorectal 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 colorectal cancer patients and the free mannose of normal human serum, glucose and glucose and mannose concentration
Ratio scatter plot；
Fig. 4 is the ROC curve of serum in patients with colorectal free mannose and glucose；
Fig. 5 is normal person and the free mannose of serum in patients with colorectal, glucose and glucose and mannose concentration
The 3 dimensional drawing of 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 Hospital Attached to Medical College, Qingdao Univ., 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,
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 L0.3mol/L hydrogen-oxygens are added
Change sodium, is vortexed and mixes；
(3) PMP is derivative: 60 μ L0.5mol/LPMP are added in each sample, are vortexed and mix, and 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 L0.3mol/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,2.7μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Mobile phase variable gradient A:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient B:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 20% | 80% |
| 20min | 20% | 80% |
Mobile phase variable gradient C:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 22% | 78% |
| 20min | 22% | 78% |
Mobile phase variable gradient D:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 15% | 85% |
| 20min | 15% | 85% |
Mobile phase variable gradient E:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
Figure it is seen that only Man is eluted out under the conditions of gradient A；Under the conditions of gradient B, although three
Can be separated and, but the peak of solvent PMP and Man from it is relatively close；And under the conditions of gradient C, solvent peak and the peak Man weight
It closes；Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient D 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,
0.25 mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL, 0.0025mg/mL, 0.001 mg/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×100 mm,2.7μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Mobile phase variable gradient:
| Time | Acetonitrile | 0.1mol/L ammonium acetate pH 5.5 |
| 0min | 15% | 85% |
| 10min | 22% | 78% |
| 15min | 24% | 76% |
| 15.1min | 15% | 85% |
| 20min | 15% | 85% |
(7) standard curve of mannose and glucose is drawn.
Embodiment 4
(1) it creates alkaline environment: taking 242 normal human sera samples and 360 colorectal cancer patients made a definite diagnosis respectively
10 μ L of blood serum sample adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L0.3mol/L sodium hydroxides are added, and is vortexed and mixes；
(2) for remaining step with embodiment 3, analytical calculation mannose, the concentration of glucose and glucose and mannose are dense
The ratio of degree.Statistic mixed-state is as a result, be shown in Fig. 3.
Fig. 3 result is summarized to Tables 1 and 2.
The comparable trend of table 1. rectal cancer patient free serum monosaccharide and G/M ratio and normal person
| Glucose | Mannose | G/M ratio | |
| The carcinoma of the rectum | ↓*** | ↑NS | ↓*** |
Annotation: " NS " representative is compared with normal people that there was no significant difference, " p < 0.001 * * * ", " p < 0.01 * * " and " * p <
0.05 " respectively represents and has been compared with normal people significant difference, and " ↑ " and " ↓ " respectively represents to be compared with normal people and rise or fall.
2 kinds of free monosaccharide concentration (μm ol/L) and its ratio result in 2. rectal cancer patient of table and normal human serum
| Glucose | Mannose | G/M ratio | |
| Normal person | 5660±1891 | 69.95±36.14 | 83.83±16.56 |
| The carcinoma of the rectum | 5293±31143 | 80.32±58.03 | 74.93±30.67 |
In addition, thering is the glucose of significant difference to carry out ROC curve analysis with normal person in table 1, found with expectation
The higher index of area under the curve (AUC), sensitivity and specificity, to determine best screening positive critical value (cutoff
Value), AUC illustrates that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized to table 3.
In 3. rectal cancer patient serum of table the cutoff value (μm ol/L) and its sensitivity (%) of mannose and glucose and
Specific (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Glucose | 0.5963 | 4163 | 43.44 | 88.69 |
From Fig. 3, table 1, table 2 is as can be seen that rectal cancer patient free serum glucose significantly rises.In addition, table 3 is shown,
The specificity of glucose is higher, therefore can detect rectal cancer patient using free glucose as biomarker.
Embodiment 5
(1) it creates alkaline environment: 137 colorectal cancer patients blood serum samples, 10 μ L being taken to add 30 μ L ultrapure waters in 1.5mL respectively
In EP pipe, 40 μ L0.3mol/L sodium hydroxides are added, is vortexed and mixes；
(2) for remaining step with embodiment 2, analytical calculation glucose, the concentration of mannose and glucose and mannose are dense
The ratio of degree.Statistic mixed-state is as a result, be shown in Fig. 1, Fig. 2.Fig. 1 result is summarized to table 1,2 kinds of free monosaccharide concentration and concentration proportion
Analysis the results are shown in Table 2.
The comparable trend of table 1. colorectal cancer patients free serum monosaccharide and G/M ratio and normal person
| Glucose | Mannose | G/M ratio | |
| Colon cancer | ↓*** | ↑* | ↓*** |
Annotation: " NS " representative is compared with normal people that there was no significant difference, " p < 0.001 * * * ", " p < 0.01 * * " and " * p <
0.05 " respectively represents and has been compared with normal people significant difference, and " ↑ " and " ↓ " respectively represents to be compared with normal people and rise or fall.
2 kinds of free monosaccharide concentration (μm ol/L) and its ratio result in 2. colorectal cancer patients of table and normal human serum
| Glucose | Mannose | G/M ratio | |
| Normal person | 5516±2295 | 68.70±31.71 | 83.52±16.20 |
| Colon cancer | 4788±2621 | 77.63±39.44 | 66.49±28.37 |
In addition, there is the mannose of significant difference and glucose to carry out ROC curve analysis with normal person in table 1, with
It is expected that the higher index of area under the curve (AUC), sensitivity and specificity is found, to determine best screening positive critical value
(cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized to table 3.
In 3. colorectal cancer patients serum of table the cutoff value (μm ol/L) and its sensitivity (%) of mannose and glucose and
Specific (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Mannose | 0.5798 | 81.09 | 41.01 | 78.42 |
| Glucose | 0.6247 | 3885 | 43.17 | 91.37 |
From Fig. 3, table 1, table 2 is as can be seen that colorectal cancer patients free serum mannose significantly rises, under glucose is significant
Drop.In addition, table 3 is shown, the specificity of mannose and glucose is higher, therefore can be made a living with free mannose and glucose
Object marker detects colorectal cancer patients.
Embodiment 6
It is used to detect the application of colorectal cancer 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 L0.3mol/L sodium hydroxides are vortexed and mix；
(2) PMP is derivative: 60 μ L0.5mol/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:
| 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% |
(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, 0.005 mg/ containing the above monosaccharide
The hybrid standard product solution of mL, 0.0025mg/mL, 0.001mg/mL, 0.0005mg/mL, matching while using；Take 40 μ L mixing single
Saccharide is managed in 1.5mL EP, and 40 μ L 0.3mol/L sodium hydroxides are added, and is vortexed and is mixed；Remaining step with (2), (3),
(4)、(5)。
(7) standard curve in the chromatogram for obtaining step (5) and step (5) compares simultaneously analytical calculation, determines sweet dew
The ratio of sugar, the concentration of glucose and glucose and mannose concentration.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and colorectal cancer patients more intuitively to observe triple combination, see Fig. 5.
If the free mannose concentration in test serum sample is greater than 64.71 μm of ol/L and free concentration of glucose is greater than
5149 μm of ol/L then determine test serum sample for colorectal 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 mannose, glucose and the glucose and mannose concentration that dissociate in serum can distinguish normal person and colorectal cancer
Patient is suitble to clinically be used for the diagnosis of colorectal cancer patients.
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 colorectal cancer, it is characterised in that: the biomarker is serum by 1- before column
The free mannose and glucose that high performance liquid chromatography derived from phenyl -5- methylpyrazole quinoline ketone (PMP) obtains.
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of colorectal cancer, feature exists
In: 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 drawn；
(7) chromatogram for obtaining step (5) and the standard curve of step (6) compare simultaneously analytical calculation, determine mannose, grape
The concentration of sugar and the ratio of glucose and mannose concentration.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of colorectal cancer, feature exists
In: 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 colorectal cancer, feature exists
In: 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 colorectal cancer, feature exists
In: 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 colorectal cancer, feature exists
In: 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 colorectal cancer is in colorectal cancer disease described in claim 2-6
Application, it is characterised in that the blood serum sample be colorectal cancer patients serum.
8. a kind of kit for identifying colorectal cancer, 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 colorectal cancer according to claim 8, which is characterized in that include concentration in the kit
For the titer (carcinoma of the rectum) of the glucose of 4163 μm of ol/L.
It include the titer for the mannose that concentration is 81.09 μm of ol/L and the Glucose standards that concentration is 3885 in the kit
Liquid (colon cancer).
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and Portugal in serum in patients with colorectal
Purposes in the quantitative kit of grape sugar.
11. a kind of kit for identifying colorectal cancer, it is characterised in that identified by mannose in serum and glucose quantitation
Colorectal cancer.
12. the purposes that the kit of the described in any item identification colorectal cancers of claim 8-11 detects colorectal cancer in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Colorectal cancer.
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| CN114032283A (en) * | 2021-09-15 | 2022-02-11 | 陈翠英 | Intestinal cancer detection reagent and application thereof in intestinal cancer detection |
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