CN109470787A - A kind of method and its detection kit for identifying breast Cancer Biomarkers object - Google Patents
A kind of method and its detection kit for identifying breast Cancer Biomarkers object Download PDFInfo
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Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying breast 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, 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 can distinguish normal person and patient with breast cancer after efficient liquid phase chromatographic analysis, 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 have very important meaning for the relationship between research free serum mannose and glucose and breast cancer, searching Novel breast gland cancer clinical detection marker with mannose and glucose free in fast quantification blood serum of patients with human breast carcinoma.
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 breast cancer
Test agent box.
Background technique
Breast cancer occupies first of female cancer morbidity, the global cancer report in 2012 of international cancer research institute (IARC) publication
It accuses (Globocan 2012) data show, global women new breast cancer case about 167.6 ten thousand every year, death about 52.2 ten thousand.
At present the common breast cancer screening means in the whole world include clinical breast examination (clinical breast examination,
CBE), mastography (mammography, MAM), ultrasonic imaging (ultrasonography, US) and magnetic resonance imaging
(magnetic resonance imaging, MRI) etc..Although Death Rate of Breast Cancer can be reduced, there is also certain simultaneously
Limitation.Most common clinical detection marker CA125, CA19-9 and CEA in tumor markers measurement, blood test
Because the characteristics of its is easy to operate, hurtless measure is as ideal detection method, so screening suitable serum biomarker object
It is the key that breast cancer clinical diagnosis.
The content highest of glucose in human serum free monosaccharide, the range of normal person are 3.90~6.16mmol/L.Mesh
Before, hospital laboratory directly can detect concentration of glucose by surveying biochemical indicator, and it is dense temporarily cannot directly to detect mannose
Degree.The concentration range of free mannose is 20~80 μm of ol/L in health adult's blood plasma.The content of mannose is about that glucose contains
The 1% of amount.Its major part is considered as from the glucose isomerization in cell.And some researches show that flow in cell recently
Free mannose out is the main source of free mannose in mammalian.The free mannose in this two parts source
Maintain the stabilization of mannose in serum.D-MANNOSE is glycoprotein, cell surface glycoconjugates and glycolsyl-phosphatidylinositol
Required monosaccharide in the structures such as anchorin.Wherein, mannose is the important component of N- sugar chain in various polysaccharide compounds, and N- is poly-
Glucose or mannose of the mannose residue in blood in sugar.Free mannose is normal plasma composition.Currently,
The measuring method of serum mannose mainly has enzyme process, gas-liquid chromatography, high resolution liquid chromatography, gas-liquid chromatography-mass spectrography and hair
Cons electrophoresis method etc..But there is certain shortcoming in these methods.For example, using enzyme process, gas-liquid chromatography and high-resolution
When liquid chromatography, to avoid the influence needs of glucose of high concentration from removing it, cause pretreatment process comparatively laborious；Gas-liquid
Chromatography mass spectrometry instrument price is expensive, is not suitable for routine purpose；Required serum sample amount is big.Before document report column
High performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) simultaneously detect in blood serum of patients with human breast carcinoma dissociate
Mannose and glucose.
Summary of the invention
Before for identifying that biomarker and its application of breast cancer, the present invention use column
Dissociate in monosaccharide more particularly to serum in high performance liquid chromatography derived from PMP (HPLC) detection blood serum of patients with human breast carcinoma sweet
The detection of dew sugar and glucose.Technical solution of the present invention can be used for detecting patient with breast cancer or the free sweet dew of normal human serum
Sugar and concentration of glucose, can be used for identifying patient with breast cancer.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of breast cancer, the biomarker are serum by efficient derived from PMP before column
The free mannose and glucose that liquid chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of breast cancer, 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 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 (4.6mm × 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 breast cancer, it is characterised in that: the kit
In include concentration is 64.71 μm of ol/L mannoses and concentration is 5149 μm of ol/L glucose titer.
A kind of kit for identifying breast cancer, which is characterized in that the kit contains biomarker, i.e. serum passes through column
The free mannose and glucose that high performance liquid chromatography derived from preceding PMP obtains.
It include the mark for the glucose that concentration is 64.71 μm of ol/L mannoses and concentration is 5149 μm of ol/L in the kit
Quasi- liquid.
Identify that the kit of breast cancer is preparing the purposes in the kit for detecting breast cancer.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for blood serum of patients with human breast carcinoma 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 concentration
Data analysis, establishes the relationship between breast cancer and three.Normal person can be quickly distinguished using technical solution of the present invention
And patient with breast cancer.
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 the ratio of patient with breast cancer and normal human serum free mannose, glucose and glucose and mannose concentration
It is worth scatter plot；
Fig. 4 is the ROC curve of blood serum of patients with human breast carcinoma free mannose and glucose；
Fig. 5 is the ratio of normal person and blood serum of patients with human breast carcinoma free mannose, glucose and glucose and mannose concentration
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 310 normal human sera samples and 310 blood of patients with breast cancer 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 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. blood serum of patients with human breast carcinoma free monosaccharide and G/M ratio and normal person
| Mannose | Glucose | G/M ratio | |
| Breast cancer | ↑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 ", which is respectively represented, has been compared with normal people significant difference, " ↑ " and " ↓ " respectively represent be compared with normal people rising or under
Drop.
2 kinds of free monosaccharide concentration (μm ol/L) and its ratio result in 2. patient with breast cancer of table and normal human serum
| Mannose | Glucose | G/M ratio | |
| Normal person | 59.68±22.88 | 4927±1370 | 86.08±16.51 |
| Breast cancer | 64.66±31.85 | 7207±2817 | 121.0±41.31 |
In addition, glucose and mannose concentration ratio carry out to the glucose for having significant difference in table 1 with normal person
ROC curve analysis finds the higher index of area under the curve (AUC), sensitivity and specificity with expectation, to determine best sieve
Positive critical value (cutoff value) is looked into, AUC illustrates that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, by Fig. 4 result
It summarizes to table 3.
In 3. blood serum of patients with human breast carcinoma of table the cutoff value (μm ol/L) of glucose and glucose and mannose concentration ratio and
Its sensitivity (%) and specificity (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Glucose | 0.8099 | 5486 | 74.52 | 79.03 |
| G/M | 0.7922 | 109.2 | 58.39 | 91.94 |
From Fig. 3, table 1, the glucose as can be seen that blood serum of patients with human breast carcinoma dissociates of table 2, glucose and mannose concentration ratio are aobvious
It writes and rises.In addition, the specificity of the display of table 3, glucose and glucose and the ratio between mannose concentration is higher, therefore can be with trip
From glucose, glucose and mannose concentration ratio are biomarker to detect patient with breast cancer.
Embodiment 5
It is used to detect the application of breast 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 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: 350mm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
(6) draw the standard curve of mannose and glucose: precision weighs mannose and proper amount of glucose, adds deionized water
It is configured to each 0.5mg/mL, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL containing the above monosaccharide,
The hybrid standard product solution of 0.0025mg/mL, 0.001mg/mL, 0.0005mg/mL, matching while using；Take 40 μ L mixing monosaccharide marks
Quasi- product are managed in 1.5mL EP, and 40 μ L 0.3mol/L sodium hydroxides are added, and are vortexed and are mixed；Remaining step with (2), (3), (4),
(5)。
(7) standard curve in the chromatogram for obtaining step (5) and step (5) compares simultaneously analytical calculation, determines sweet dew
The ratio of sugar, the concentration of glucose and glucose and mannose concentration.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and patient with breast cancer more intuitively to observe triple combination, see Fig. 5.
If the free concentration of glucose in test serum sample is greater than 5486 μm of ol/L, G/ between glucose and mannose
M ratio is greater than 109.2, then determines that test serum sample very likely comes from patient with breast cancer.
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 breast cancer trouble
Person is suitble to clinically be used for the diagnosis of patient with breast cancer.
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 breast cancer, it is characterised in that: the biomarker is serum by 1- benzene before column
The free mannose and glucose 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 breast cancer, 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 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 breast cancer, 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 breast cancer, 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 breast cancer, 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 breast cancer, 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 breast cancer answering in breast cancer disease described in claim 2-6
With, it is characterised in that the blood serum sample is the serum of patient with breast cancer.
8. a kind of kit for identifying breast 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 breast cancer according to claim 8, which is characterized in that be including concentration in the kit
The titer for the glucose that 64.71 μm of ol/L mannoses and concentration are 5149 μm of ol/L.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and grape in blood serum of patients with human breast carcinoma
Purposes in the quantitative kit of sugar.
11. a kind of kit for identifying breast cancer, it is characterised in that pass through the mannose and glucose quantitation identification cream in serum
Gland cancer.
12. the purposes that the kit of the described in any item identification breast cancer of claim 8-11 detects breast cancer in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Breast cancer.
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| 中国科协学会学术部: "《网络药理学一中药现代化的新思路与新方法》", 30 November 2014 * |
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