CN109342595A

CN109342595A - A kind of method and its detection kit for identifying gastritis biomarker

Info

Publication number: CN109342595A
Application number: CN201811323692.8A
Authority: CN
Inventors: 张丽娟; 单鸣; 王继纲
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2018-11-01
Filing date: 2018-11-01
Publication date: 2019-02-15

Abstract

The present invention provides the methods and its detection kit of a kind of biomarker for identifying gastritis.The biomarker is the ratio of the free mannose and glucose and mannose concentration that obtain in serum by high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before column.Detection method is high performance liquid chromatography derived from PMP before column.Technical solution of the present invention has pre-treatment simple, and analysis time is short, and instrument price is reasonable, meet conventional use, operating procedure is easy to learn, and testing result accuracy is high, the advantages that need to only take a blood sample can distinguish normal person and gastritis sufferer, 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 gastritis, the novel gastritis clinical detection marker of searching with mannose and glucose free in fast quantification gastritis sufferer's serum.

Description

A kind of method and its detection kit for identifying gastritis biomarker

Technical field

The invention belongs to pharmaceutical technology fields, and in particular to a kind of method and its detection of the biomarker for identifying gastritis Kit.

Background technique

Gastritis is stomach lining inflammation caused by a variety of causes, by acute gastritis and chronic gastritis in sick emergency point, by the cause of disease Difference can be divided into Hp-relative Gastritis, irritability gastritis, autoimmune gastritis etc..Most Patients with Chronic Gastritis without Any symptom is showed only as indigestion symptom, and the diagnosis and differential diagnosis of current various gastritis relies primarily on endoscopy stomach function regulating Mucous membrane biopsy histology inspection.2014, the investigation that Chinese Medical Association's digestive endoscopy is carried out was included in including 10 cities Amount to 8892 Patients with Chronic Gastritis for there are digestive symptoms and making a definite diagnosis through gastrocopy, the results show that endoscopic diagnosis is chronic Non- atrophic gastritis is most common (49.4%), and followed by chronic non-atrophic gastritis accompanied is rotten to the corn (42.3%), atrophic stomach Scorching ratio is 17.7%；Pathological diagnosis atrophy accounts for 25.8%, and intestinal metaplasia accounts for 23.6%, and intraepithelial neoplasia (cin) accounts for 7.3%.It is examined with pathology Break as " goldstandard ", then the sensibility of endoscopic diagnosis atrophy is only 42%, and specificity is 91%.Illustrate China's chronic atrophy at present The illness rate of property gastritis is higher, and the coincidence rate of scope and pathological diagnosis needs to be further increased.In view of most Patients with Chronic Gastritis Without any symptom, specificity is lacked having symptom, and lacks specific sign, therefore be difficult to do according to sings and symptoms The correct diagnosis of chronic gastritis out.The early diagnosis of chronic gastritis and for the symptom that controls inflammation, improve patients ' life quality, drop The causing danger property of low gastric ulcer and gastric cancer is most important.Therefore, it needs preferably to facilitate gastritis early diagnosis, identify and pre- The serum biological indicator determined afterwards.

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 gastritis sufferer's serum dissociate it is sweet Dew sugar and glucose.

Summary of the invention

Before for identifying that biomarker and its application of gastritis, the present invention use column High performance liquid chromatography derived from PMP (HPLC) detects the sweet dew that dissociates in monosaccharide more particularly to serum in gastritis sufferer's serum The detection of sugar and glucose.Technical solution of the present invention can be used for detecting gastritis sufferer or normal human serum dissociate mannose and Concentration of glucose can be used for identifying gastritis sufferer.

To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:

For identifying that the biomarker of gastritis, the biomarker are serum by efficient liquid derived from PMP before column The ratio (G/M concentration ratio) of free mannose and glucose that phase chromatography obtains and mannose concentration.

For identifying the quantitative analysis method of the biomarker of gastritis, it is characterised in that: this method is derivative for PMP before column 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 gastritis.

Biomarker is preparing the purposes in the kit for detecting gastritis, it is characterised in that: in the kit It is 59.06 μm of ol/L mannoses and G/M concentration than the titer for 65.00 including concentration.

A kind of kit for identifying gastritis, which is characterized in that the kit contains biomarker, i.e., serum is by before column The free mannose and G/M concentration ratio that high performance liquid chromatography derived from PMP obtains.

In the kit include concentration be 59.06 μm of ol/L mannoses and G/M concentration than the titer for 65.00.

Identify that the kit of gastritis is preparing the purposes in the kit for detecting gastritis.

Compared with prior art, high performance liquid chromatography derived from PMP before column is used for the trip of gastritis sufferer's serum by the present invention Detection from 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 statistical data analysis to free serum mannose and G/M concentration ratio, establish between gastritis and three Relationship.Normal person and gastritis sufferer 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 gastritis sufferer and the free glucose of normal human serum, mannose and G/M concentration compare scatter plot；

Fig. 4 is the ROC curve of gastritis sufferer's free serum mannose and G/M concentration ratio；

Fig. 5 is the 3 D stereo of the free glucose of normal person's stomach function regulating inflammation patients serum, 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:

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；

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；

(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；

(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 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；

(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.

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 variable gradient:

(7) standard curve of mannose and glucose is drawn.

Embodiment 4

(1) it creates alkaline environment: taking 237 normal human sera samples and 237 gastritis sufferer's 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. gastritis sufferer's free serum monosaccharide and G/M ratio and normal person

	Mannose	Glucose	G/M ratio
				Gastritis	↑***	↓*	↓***

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. gastritis sufferer of table and normal human serum

	Mannose	Glucose	G/M ratio
				Normal person	46.85±23.40	6264±2251	83.82±16.59
Gastritis	59.59±27.22	5739±2226	64.46±25.38

In addition, ROC curve analysis has been carried out to the mannose and G/M concentration ratio that have extremely significant difference in table 1 with normal person, The higher index of area under the curve (AUC), sensitivity and specificity is found with expectation, 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. gastritis sufferer's serum of table the cutoff value (μm ol/L) and its sensitivity (%) of mannose and G/M concentration ratio and Specific (%)

	AUC	Cutoff value	Sensitivity	Specificity
					Mannose	0.6659	59.06	48.95	83.12
G/M	0.7711	65.00	60.34	88.61

From Fig. 3, table 1, table 2 is as can be seen that gastritis sufferer's free serum mannose concentration significantly rises with G/M concentration than aobvious Write decline.In addition, table 3 is shown, the specificity of mannose and G/M concentration ratio is higher, therefore can be with free mannose and G/M Concentration ratio is biomarker to detect gastritis sufferer.

Embodiment 5

It is used to detect the application of gastritis 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；

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 gastritis sufferer more intuitively to observe triple combination, see Fig. 5.

If the free mannose concentration in test serum sample is greater than 59.06 μm of ol/L and G/M concentration ratios and is less than 65.00, then determine test serum sample for gastritis sufferer.

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 gastritis sufferer can be distinguished by calculating the mannose to dissociate in serum and G/M concentration ratio, be suitble to clinically suffer from for gastritis The diagnosis of person.

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

1. for identifying the biomarker of gastritis, it is characterised in that: the biomarker is serum by 1- phenyl-before column The ratio of free mannose and glucose that high performance liquid chromatography derived from 5- methylpyrazole quinoline ketone (PMP) obtains and mannose concentration It is worth (G/M concentration ratio).

2. according to claim 1 for identifying the quantitative analysis method of the biomarker of gastritis, it is characterised in that: should 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；

(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；

(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 the dense of mannose Degree and G/M concentration ratio.

3. according to claim 2 for identifying the quantitative analysis method of the biomarker of gastritis, it is characterised in that: institute Stating 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；

4. according to claim 3 for identifying the quantitative analysis method of the biomarker of gastritis, it is characterised in that: institute State the change of gradient of mobile phase in 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 gastritis, it is characterised in that: institute The concentration for stating hydrochloric acid in step (3) is 0.3mol/L.

6. according to claim 2 for identifying the quantitative analysis method of the biomarker of gastritis, it is characterised in that: institute Stating organic solvent in step (4) is at least one of chloroform, methylene chloride, ethyl acetate and n-hexane.

7. application of the quantitative analysis method of the biomarker of identification gastritis in gastritis disease described in claim 2-6, It is characterized in that the blood serum sample is the serum of gastritis sufferer.

8. a kind of kit for identifying gastritis, which is characterized in that the kit contains biomarker described in claim 1, That is the free mannose and G/M concentration ratio that are obtained by high performance liquid chromatography derived from PMP before column of serum.

9. the kit of identification gastritis according to claim 8, which is characterized in that be including concentration in the kit 59.06 μm of ol/L mannoses and G/M concentration are than the titer for 65.00.

10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and G/M concentration in gastritis sufferer's serum Than the purposes in quantitative kit.

11. a kind of kit for identifying gastritis, it is characterised in that by mannose in serum and G/M concentration than Quantitative measurement stomach It is scorching.

12. the purposes that the kit of the described in any item identification gastritis of claim 8-11 detects gastritis in vitro.

13. purposes according to claim 12, it is characterised in that pass through the mannose and the quantitative mirror of G/M concentration ratio in serum Determine gastritis.