CN109490436A

CN109490436A - A kind of method and its detection kit for identifying coronary heart disease biomarker

Info

Publication number: CN109490436A
Application number: CN201811323315.4A
Authority: CN
Inventors: 张丽娟; 徐颖婕; 于玲范
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: 2019-03-19

Abstract

The present invention provides the methods and its detection kit of a kind of biomarker of identification coronary atherosclerotic heart disease (referred to as, coronary heart disease, CAD).The biomarker is the free mannose and glucose obtained in serum by high performance liquid chromatography derived from PMP before column.Detection method is high performance liquid chromatography derived from 1- phenyl -5- methylpyrazole quinoline ketone (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 CAD patient, 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 CAD, searching novel C AD clinical detection marker with mannose and glucose free in fast quantification CAD patients serum.

Description

A kind of method and its detection kit for identifying coronary heart disease 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 CAD Kit.

Background technique

Coronary heart disease has become the biggest threat for endangering population health in global range, and leads to the main original of people's death One of because.In recent years, although developed country's death rate as caused by coronary heart disease is declined, in developing countries such as China In, the disease incidence and lethality of coronary heart disease are still growing steadily, and seriously endanger compatriots' health.Most of early coronary disease patients without Manifest symptom, once it is to improve to be preced with that morbidity, which may develop as myocardial infarction, the acute illness such as sudden death, therefore reasonable early screening, Cardiaopath's cure rate, the key for improving patients ' life quality.Electrocardiogram is relied primarily on to the diagnosis of coronary heart disease at present, it is coronal dynamic Arteries and veins radiography and intravascular imaging technique, the image technologies such as coronary artery CT.In addition to this to blood lipid, the hematology of the indexs such as blood glucose Check to be also to assess whether that there are one of the important means of coronary heart disease, cardiac troponin and c reactive protein are that laboratory is common Major blood clinical diagnosis index.But these physiochemical indices only serve certain booster action to the diagnosis of coronary heart disease, still need to To carry out further patient diagnosed's state of an illness by results such as image, pathologic findings.And the patient made a definite diagnosis by means such as image technologies, Its coronary artery position has often had already appeared in various degree narrow, affects the early prevention and treatment of coronary artery disease.Because of this person Still need preferably to facilitate coronary disease disease early diagnosis and serum biological indicator that prognosis determines.

Reductive monosaccharide seems to be only limitted to mannose and grape in human serum, wherein glucose (Glc) content highest, just Normal range is 3.9~6.1mM, and mannose (Man) content is about the 1% of glucose content.In eukaryocyte, by 14 monosaccharide groups At oligosaccharides (9 mannoses, 3 glucose and 2 N-acetyl-glucosamines) be transferred in endoplasmic reticulum it is most of newly synthesized In the N- sugar chain connection site of protein.Studies have reported that abnormal protein glycosylation has with diseases such as metabolic disease, cancers It closes.Therefore, the content for measuring free monosaccharide in human serum will be for the various complex diseases including systemic loupus erythematosus Diagnosis provides novel molecular horizontal information.

The content of mannose is about the 1% of glucose content.It is considered as by the glucose isomerase in cell that it is most of Change.And some researches show that the free mannose flowed out in cell is the mannose that dissociates in mammalian recently Main source.The free mannose in this two parts source maintains the stabilization of mannose in serum.D-MANNOSE is glycoprotein, carefully Required monosaccharide in the structures such as cellular surface glycoconjugate and glycosylphosphatidylinositol-anchored proteins.Wherein, mannose is various The important component of N- sugar chain in polysaccharide compound, glucose or sweet dew of the mannose residue in blood in N- glycan Sugar.Free mannose is normal plasma composition.Currently, the measuring method of serum mannose mainly have enzyme process, gas-liquid chromatography, High resolution liquid chromatography, gas-liquid chromatography-mass spectrography and capillary electrophoresis etc..But there is certain deficiency in these methods Place.For example, when using enzyme process, gas-liquid chromatography and high resolution liquid chromatography, for avoid high concentration glucose influence needs It removes it, causes pretreatment process comparatively laborious；Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for routine purpose；Institute Need serum sample amount big.

Summary of the invention

The object of the present invention is to provide for identifying biomarker and its application of CAD, the present invention uses PMP before column Dissociate in monosaccharide more particularly to serum in derivative high performance liquid chromatography (HPLC) detection CAD patients serum mannose and The detection of glucose.Technical solution of the present invention can be used for detecting CAD patient or the free mannose of normal human serum and grape Sugared concentration can be used for identifying CAD patient.

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

For identifying that the biomarker of CAD, the biomarker are serum by efficient liquid phase derived from PMP before column The free mannose and glucose that chromatography obtains.

For identifying the quantitative analysis method of the biomarker of CAD, 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 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 CAD.

Biomarker is preparing the purposes in the kit for detecting CAD, it is characterised in that: wraps in the kit Include the titer for the glucose that concentration is 64.71 μm of ol/L mannoses and concentration is 5149 μm of ol/L.

A kind of kit for identifying CAD, which is characterized in that the kit contains biomarker, i.e., serum is by before column The free mannose and glucose that high performance liquid chromatography derived from 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 CAD is preparing the purposes in the kit for detecting CAD.

Compared with prior art, high performance liquid chromatography derived from PMP before column is used for CAD patients serum and dissociates by the present invention The detection of 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 CAD and three.Using technical solution of the present invention can quickly distinguish normal person and CAD patient.

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 CAD patient and the free mannose of normal human serum, glucose and glucose and the ratio of mannose concentration dissipate Point diagram；

Fig. 4 is the ROC curve of CAD patients serum free mannose and glucose；

Fig. 5 is the ratio structure of normal person and CAD patients serum free mannose, glucose and glucose and mannose concentration The 3 dimensional drawing built.

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 pH5.5
			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；

Change of gradient:

Time	Acetonitrile	0.1mol/L ammonium acetate pH5.5
			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 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:

Mobile phase variable gradient E:

Figure it is seen that only Man is eluted out under the conditions of gradient A；Under the conditions of gradient B, although three Can be separated and, but the peak of solvent PMP and Man from it is relatively close；And under the conditions of gradient C, solvent peak and the peak Man weight It closes；Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient D item Under part, appearance is symmetrical, and separating degree is good.Abscissa is time (min), and ordinate is DAD signal strength.1,2 and 3 is successively The chromatographic peak of Man, Rha and Glc.

Embodiment 3

(1) precision weighs mannose and proper amount of glucose, and deionized water is added to be configured to each 0.5mg/mL containing the above monosaccharide, 0.25mg/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 242 normal human sera samples and 360 CAD patients serum's samples made a definite diagnosis respectively 10 μ L of 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.CAD patients serum free monosaccharide and G/M ratio and normal person

	Glucose	Mannose	G/M ratio
				Coronary heart disease	↑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 table 2.CAD patient and normal human serum

	Glucose	Mannose	G/M ratio
				Normal person	4625±645.9	53.17±10.61	89.09±14.31
Coronary heart disease	4964±2380	76.94±57.04	56.12±22.18

In addition, there is the mannose of significant difference and G/M ratio to carry out ROC curve analysis with normal person in table 1, with It is expected that the higher index of area under the curve (AUC), sensitivity and specificity is found, to determine best screening positive critical value (cutoff value), AUC illustrate that diagnosis effect is better closer to 1.Testing result is shown in Fig. 4, and Fig. 4 result is summarized to table 3.

The cutoff value (μm ol/L) and its sensitivity (%) of mannose and G/M ratio and spy in table 3.CAD patients serum Anisotropic (%)

	AUC	Cutoff value	Sensitivity	Specificity
					Mannose	0.6095	72.17	40.22	95.9
G/M ratio	0.9198	71.98	81.67	89.23

From Fig. 3, table 1, as can be seen that CAD patients serum dissociates, mannose and G/M ratio significantly rise table 2.In addition, table 3 The specificity of display, mannose and G/M ratio is higher, therefore can be using free mannose and G/M ratio as biomarker To detect CAD patient.

Embodiment 5

It is used to detect the application of CAD 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 CAD patient more intuitively to observe triple combination, see Fig. 5.

If the free mannose concentration in test serum sample is greater than 64.71 μm of ol/L and free concentration of glucose is less than 71.98 μm of ol/L then determine test serum sample for CAD patient.

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 CAD patient, It is suitble to clinically be used for the diagnosis of CAD patient.

The above examples are only used to illustrate the technical scheme of the present invention, rather than is limited；Although referring to aforementioned reality Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.

Claims

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

2. according to claim 1 for identifying the quantitative analysis method of the biomarker of CAD, 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 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 CAD, 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 CAD, 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 CAD, 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 CAD, 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 CAD in CAD disease is identified described in claim 2-6, It is characterized in that the blood serum sample is the serum of CAD patient.

8. a kind of kit for identifying CAD, which is characterized in that the kit contains biomarker described in claim 1, i.e., The free mannose and glucose that serum is obtained by high performance liquid chromatography derived from PMP before column.

9. the kit of identification CAD 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 fixed for mannose in CAD patients serum and glucose in preparation Purposes in the kit of amount.

11. a kind of kit for identifying CAD, it is characterised in that pass through the mannose and glucose quantitation identification CAD in serum.

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

13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation CAD。