CN109212099A - A kind of method and its detection kit for identifying gastric ulcer biomarker - Google Patents
A kind of method and its detection kit for identifying gastric ulcer biomarker Download PDFInfo
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- CN109212099A CN109212099A CN201811324139.6A CN201811324139A CN109212099A CN 109212099 A CN109212099 A CN 109212099A CN 201811324139 A CN201811324139 A CN 201811324139A CN 109212099 A CN109212099 A CN 109212099A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying gastric ulcer.The biomarker is the ratio of the free glucose obtained in serum by high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column, free mannose and glucose and mannose concentration.Detection method is high performance liquid chromatography derived from PMP before column.The advantages that technical solution of the present invention has pre-treatment simple, and analysis time is short, and instrument price is reasonable, meets conventional use, and operating procedure is easy to learn, and testing result accuracy is high, need to only take a blood sample, 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 gastric ulcer, the novel gastric ulcer clinical detection marker of searching with mannose and glucose free in fast quantification patients w ith peptic ulcer disease 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 gastric ulcer
Test agent box.
Background technique
Gastric ulcer is one of the most common type in ulcer in peptic ulcer, refers mainly to stomach lining and is made by peptic digest liquid autodigestion
It is that the inflammatory necrosis venereal disease betided between cardia and pylorus becomes at the tissue damage for being more than muscularis mucosae.In global range,
The disease incidence of gastric ulcer is 5%~10%, and is in rise year by year trend.In the patient that China carries out gastrocopy, digestibility
Ulcer accounts for 15%~35%, and its canceration rate and the death rate are respectively 1.5%, 2.5%, chronic gastric ulcer complication rate
Height seriously endangers patient health.The diagnosis of gastric ulcer is based primarily upon specific symptom and endoscopy or barium meal at present
It is made a definite diagnosis with the help of agent X-ray examination, blood test is still unable to Accurate Diagnosis peptic ulcer at present.In recent years, with life
Tempo increase living, society, work, psychological burden exacerbation and nonfluid anti-inflammatory agent and low-dosage aspirin are widely used,
Apparent ascendant trend is presented in the disease incidence of gastric ulcer.The early diagnosis of gastric ulcer for the state of an illness control, prevention complication with
And prevent that canceration is most important, needing to find preferably facilitates gastric ulcer early diagnosis, identifies and blood that prognosis determines
Clear biological markers.
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 patients w ith peptic ulcer disease serum dissociate
Mannose and glucose.
Summary of the invention
Before for identifying that biomarker and its application of gastric ulcer, the present invention use column
Dissociate in monosaccharide more particularly to serum in high performance liquid chromatography derived from PMP (HPLC) detection patients w ith peptic ulcer disease serum sweet
The detection of dew sugar and glucose.Technical solution of the present invention can be used for detecting patients w ith peptic ulcer disease or the free sweet dew of normal human serum
Sugar and concentration of glucose, can be used for identifying patients w ith peptic ulcer disease.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of gastric ulcer, the biomarker are serum by efficient derived from PMP before column
The ratio (G/M concentration ratio) of free glucose, free mannose and glucose and mannose concentration that liquid chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of gastric ulcer, 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
Sugar, the concentration of glucose and G/M concentration ratio.
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 gastric ulcer.
Biomarker is preparing the purposes in the kit for detecting gastric ulcer, it is characterised in that: the kit
In include concentration be 5286 μm of ol/L glucose, 60.08 μm of ol/L mannoses and G/M concentration than the titer for 53.73.
A kind of kit for identifying gastric ulcer, which is characterized in that the kit contains biomarker, i.e. serum passes through column
Free glucose, free mannose and the G/M concentration ratio that high performance liquid chromatography derived from preceding PMP obtains.
In the kit include concentration be that 5286 μm of ol/L glucose, 60.08 μm of ol/L mannoses and G/M concentration ratio are
53.73 titer.
Identify that the kit of gastric ulcer is preparing the purposes in the kit for detecting gastric ulcer.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for patients w ith peptic ulcer disease 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.By the analysis of statistical data to free serum glucose, mannose and G/M concentration ratio, establish gastric ulcer with
Relationship between three.Normal person and patients w ith peptic ulcer disease can be quickly distinguished using technical solution of the present invention.
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 that patients w ith peptic ulcer disease and the free glucose of normal human serum, mannose and G/M concentration compare scatter plot；
Fig. 4 is the ROC curve of patients w ith peptic ulcer disease free serum glucose, mannose and G/M concentration ratio；
Fig. 5 is the 3 D stereo of normal person's stomach function regulating ulcers free serum glucose, mannose and G/M concentration than building
Figure.
Specific embodiment
Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.
All blood samples involved in embodiment are provided by affiliated hospital of University Of Qingdao of Qingdao City.
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 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 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 hydrochloric acid are added in each sample,
It 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 29 normal human sera samples and 29 patients w ith peptic ulcer disease serum made a definite diagnosis respectively
10 μ L of sample 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. patients w ith peptic ulcer disease free serum monosaccharide and G/M ratio and normal person
| Mannose | Glucose | G/M ratio | |
| Gastric ulcer | ↑*** | ↓*** | ↓*** |
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. patients w ith peptic ulcer disease of table and normal human serum
| Mannose | Glucose | G/M ratio | |
| Normal person | 54.43±19.93 | 6747±2089 | 78.60±16.54 |
| Gastric ulcer | 80.85±30.36 | 4353±1688 | 37.49±18.21 |
In addition, having carried out ROC song to the glucose, mannose and G/M concentration ratio that have significant difference in table 1 with normal person
Line analysis finds the higher index of area under the curve (AUC), sensitivity and specificity with expectation, to determine best screening sun
Property critical value (cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized
To table 3.
The cutoff value (μm ol/L) and its spirit of 3. patients w ith peptic ulcer disease glucose in serum of table, mannose and G/M concentration ratio
Sensitivity (%) and specificity (%)
| AUC | Cutoff value | Sensitivity | Specificity | |
| Glucose | 0.8216 | 5286 | 72.41 | 79.31 |
| Mannose | 0.7640 | 60.08 | 79.31 | 65.52 |
| G/M | 0.9370 | 53.73 | 86.21 | 96.55 |
From Fig. 3, table 1, table 2 is as can be seen that patients w ith peptic ulcer disease free serum mannose concentration significantly rises, dissociate glucose
Concentration and G/M concentration ratio are remarkably decreased.In addition, table 3 is shown, the sensitivity and specificity of glucose, mannose and G/M concentration ratio
It is higher, therefore gastric ulcer can be detected as biomarker using free glucose, free mannose and G/M concentration ratio and suffered from
Person.
Embodiment 5
It is used to detect the application of gastric ulcer 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 concentration.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and patients w ith peptic ulcer disease more intuitively to observe triple combination, see Fig. 5.
If less than 5286 μm ol/L of free concentration of glucose, free mannose concentration in test serum sample are greater than
60.08 μm of ol/L and G/M concentration ratios then determine test serum sample for patients w ith peptic ulcer disease less than 53.73.
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
Normal person and patients w ith peptic ulcer disease can be distinguished by calculating glucose, mannose and the G/M concentration ratio to dissociate in serum, be suitble to clinically use
In the diagnosis of patients w ith peptic ulcer disease.
The above examples are only used to illustrate the technical scheme of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (13)
1. for identifying the biomarker of gastric ulcer, it is characterised in that: the biomarker is serum by 1- benzene before column
Free glucose that high performance liquid chromatography derived from base -5- methylpyrazole quinoline ketone (PMP) obtains, free mannose and glucose with
The ratio (G/M concentration ratio) of mannose concentration.
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of gastric ulcer, 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 and G/M concentration ratio of sugar.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of gastric ulcer, 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 gastric ulcer, 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 gastric ulcer, 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 gastric ulcer, 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 gastric ulcer answering in gastric ulcer described in claim 2-6
With, it is characterised in that the blood serum sample is the serum of patients w ith peptic ulcer disease.
8. a kind of kit for identifying gastric ulcer, which is characterized in that the kit contains biomarker described in claim 1
Free glucose, free mannose and the G/M concentration ratio that object, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification gastric ulcer according to claim 8, which is characterized in that be including concentration in the kit
5286 μm of ol/L glucose, 60.08 μm of ol/L mannoses and G/M concentration are than the titer for 53.73.
10. the described in any item reagent consumptive materials of claim 2-9 are used for patients w ith peptic ulcer disease glucose in serum, mannose in preparation
With G/M concentration than the purposes in quantitative kit.
11. a kind of kit for identifying gastric ulcer, it is characterised in that pass through glucose, mannose and the G/M concentration ratio in serum
Quantitative measurement gastric ulcer.
12. the purposes that the kit of the described in any item identification gastric ulcer of claim 8-11 detects gastric ulcer in vitro.
13. purposes according to claim 12, it is characterised in that pass through glucose, mannose and the G/M concentration in serum
Than Quantitative measurement gastric ulcer.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113466471A (en) * | 2021-09-06 | 2021-10-01 | 中国农业科学院农产品加工研究所 | Gastric mucosa injury related biomarker, screening method and application |
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2018
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| CN113466471A (en) * | 2021-09-06 | 2021-10-01 | 中国农业科学院农产品加工研究所 | Gastric mucosa injury related biomarker, screening method and application |
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