CN109187819A - A kind of method and its detection kit for identifying malignant tumor of lung biomarker - Google Patents

Abstract

The present invention provides for identifying biomarker and its application of malignant tumor of lung.The biomarker is the free glucose and mannose that patients serum obtains by high performance liquid chromatography (HPLC) derived from 1- phenyl -5- methylpyrazole quinoline ketone (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 carries out the analysis of HPLC instrument and can distinguish normally to enter and patient, 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 quickly analyze the mannose and glucose to dissociate in serum, and mannose, glucose sugar concentration and the two peak area can be calculated, there is very important meaning for the marker of relationship, searching novel lung malignancy disease clinical detection between research free serum mannose and glucose and disease.

Description

A kind of method and its detection kit for identifying malignant tumor of lung biomarker

Technical field

The invention belongs to pharmaceutical technology fields, and in particular to for identifying biomarker and its detection of malignant tumor of lung Using.

Background technique

Malignant tumor of lung (also referred to as lung cancer) is that morbidity and mortality growth is most fast, to population health and life threat One of maximum malignant tumour.It is characterized in that uncontrolled cell growth in lung tissue, mainly includes Small Cell Lung Cancer (SCLC ,~15%) and non-small cell lung cancer (NSCLC ,~85%).NSCLC, which can be further divided into histology, is divided into gland cancer (AC ,~40%), squamous cell carcinoma (SC ,~25%), maxicell lung cancer (LCC ,~15%) and other (~5%) rare classes Type, including body of gland tumour, class cancer and undifferentiated carcinoma.The most common symptom is cough (including hemoptysis), and weight loss is short of breath And pectoralgia.In most cases, the early stage of lung cancer is mostly asymptomatic, becomes difficult early diagnosis.The detection of lung cancer at present and Diagnosis includes physics and biochemical method.Physical method includes X-ray, and (positron emission is disconnected by CT (computed tomography) and PET Layer scanning)/CT, it can show that pulmonary abnormalities are grown.There is also some problems for these methods: 1. early diagnosis lacks sensibility, It can not be detected because infantile tumour is typically too small；2. some Visible lumps detected in lung due to these methods are even It is not tumour, therefore false positive rate is very high；3. leading to the high risk of additional cancer by the radiation that these methods generate.Due to biochemistry Detection method has the lower advantages such as invasive, inexpensive, easy to operate, clinically also uses several thin by detection cancer Born of the same parents or lung neoplasm are discharged into the biomarker in serum/plasma to detect the biochemical method of lung cancer.Although in diagnosing and treating Aspect obtains certain progress, past over 40 years integrally 5 years survival rates of lung cancer only rise 4% (rising to 16% from 12%). Survival rate due to receiving the patient of tumorectomy is higher more than 80%, and scientists prediction can not be cured in entrance cancer to be turned Early detection and diagnosis before the shifting stage will greatly reduce the death rate of lung lung cancer.Establish reliable lung cancer early diagnosis screening Method is still theoretical and in practice.How the Susceptible population of malignant tumor of lung is accurately identified so as to early intervention, or can The progress etc. for screening effect or predictive disease that suitable biomarker is treated with predictive disease has become hot issue.

Reductive monosaccharide seems to be only limitted to mannose and grape in serum.The content of glucose in human serum free monosaccharide Highest, the range of normal Check-up crowd are 3.90~6.16mmol/L.Currently, hospital laboratory can directly be referred to by surveying biochemistry Mark detection concentration of glucose, temporarily cannot directly detect mannose concentration.The concentration of free mannose in health adult's blood plasma Range is 20~80 μm of ol/L.The content of mannose is about the 1% of glucose content.It is considered as by cell that it is most of Glucose isomerization.And some researches show that the free mannose flowed out in cell is in mammalian recently The main source of free mannose.The free mannose in this two parts source maintains the stabilization of mannose in serum.D- sweet dew Sugar is glycoprotein, required monosaccharide in the structures such as cell surface glycoconjugates and glycosylphosphatidylinositol-anchored proteins.Wherein, Mannose is the important component of N- sugar chain in various polysaccharide compounds, Portugal of the mannose residue in N- glycan in blood Grape sugar or mannose.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 these methods have one Fixed shortcoming.For example, when using enzyme process, gas-liquid chromatography and high resolution liquid chromatography, for the glucose for avoiding high concentration Influence needs remove it, cause pretreatment process comparatively laborious；Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for Conventional purpose；Required serum sample amount is big.It is derivative currently without 1- phenyl -5- methylpyrazole quinoline ketone (PMP) before document report column High performance liquid chromatography simultaneously detect in malignant tumor of lung patients serum dissociate mannose and glucose.

Therefore, establish in the serum of efficiently and accurately dissociate mannose detection method, for research free serum monosaccharide with The marker of relationship, searching Disease Clinical detection between disease has very important meaning.

The PMP derivatization method of monosaccharide is current more common monosaccharide detection method.This method including the following steps: (1) alkaline condition is created for PMP, (2) are added PMP, are derived at 70 DEG C, and (3) adjust the pH value of solution after deriving, will be remaining PMP is extracted.103969371 B of patent of invention CN discloses that " a kind of blood degrades to obtain and detect the method for monosaccharide in cancer Application in disease detection ".Polysaccharide therein and glycoprotein are degraded to by the patent of invention first by blood serum sample by acid degradation Monosaccharide component；Then the 8 kinds of monosaccharide obtained after PMP is derivative and efficient liquid phase is to degradation detect.Before degradation Serum, sample component and property have occurred and that great variety.Although the PMP of monosaccharide spreads out, method is wider in every field application It is general, but there is no inspection of the report by this method applied to dissociate in the serum of malignant tumor of lung patient mannose and glucose at present It surveys.

Summary of the invention

The object of the present invention is to provide for identifying biomarker and its application of malignant tumor of lung, the present invention is used Monosaccharide more particularly to serum before column in high performance liquid chromatography derived from PMP (HPLC) detection malignant tumor of lung patients serum In dissociate mannose and glucose detection.Technical solution of the present invention can be used for detecting malignant tumor of lung patient or regular Inspection crowd's free serum glucose and mannose concentration and to the two calculated by peak area ratio, can be used for identifying malignant tumor of lung 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 malignant tumor of lung, the biomarker are that patients serum spreads out by PMP before column The free mannose and glucose that raw high performance liquid chromatography obtains.

For identifying the quantitative analysis method of the biomarker of malignant tumor of lung, it is characterised in that: this method is before column High performance liquid chromatography derived from PMP, 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 (4.6 mm × 100mm, 2.7 μm), the chromatographic condition in the step (5) are as follows:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: A phase is acetonitrile, and B phase is 0.1mol/L ammonium acetate-acetic acid buffer solution that pH is 5.5.

The change of gradient of mobile phase in the step (5) are as follows:

0min, A phase 15%, B phase 85%；

10min, A phase 22%, B phase 78%；

15min, A phase 24%, B phase 76%；

15.1min, A phase 15%, B phase 85%；

20min, A phase 15%, B phase 85%.

The concentration of hydrochloric acid is 0.3mol/L in the step (3).

Organic solvent is at least one in chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4) Kind.

Biomarker is preparing the purposes in the kit for detecting malignant tumor of lung.

Biomarker is preparing the purposes in the kit for detecting malignant tumor of lung, it is characterised in that: the examination It include the titer for the glucose that concentration is 78.7 μm of ol/L mannoses and concentration is 5865 μm of ol/L in agent box.

A kind of kit for identifying malignant tumor of lung, which is characterized in that the kit contains biomarker, i.e. serum passes through Cross the free mannose and glucose that high performance liquid chromatography derived from PMP obtains before column.

It include the mark for the glucose that concentration is 78.7 μm of ol/L mannoses and concentration is 5865 μm of ol/L in the kit Quasi- liquid.

Identify that the kit of malignant tumor of lung is preparing the purposes in the kit for detecting malignant tumor of lung.

Compared with prior art, high performance liquid chromatography derived from PMP before column is used for malignant tumor of lung patient by the present invention The detection of free serum 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 ratio (G/M ratio) to free serum glucose, mannose concentration and glucose and mannose peak area Statistical data analysis, establish the relationship with malignant tumor of lung.Using technical solution of the present invention, by detection serum Free mannose and glucose calculates the ratio of mannose and concentration of glucose and glucose and mannose peak area, can Quickly to distinguish normal population and malignant tumor of lung 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 malignant tumor of lung patient and normal Check-up crowd free serum concentration of glucose, mannose concentration and grape The scatter plot of sugar and mannose peak area ratio

Fig. 4 is malignant tumor of lung patient and normal Check-up crowd glucose and mannose peak area ratio, as serum sugar The ROC curve figure of biomarker

Fig. 5 is malignant tumor of lung patient and normal Check-up crowd free serum concentration of glucose, mannose concentration and grape The 3 dimensional drawing of sugar and the building of mannose peak area ratio

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.

Embodiment 1

Technical solution of the present invention the following steps are included:

(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 PMP (1- phenyl -5- methylpyrazole quinoline ketone) is added in each sample, is vortexed and mixes, centrifugation 70 DEG C of baking ovens react 1h afterwards；

(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, two parts of samples carry out high-efficient liquid phase analysis, efficient liquid phase using different chromatographic column A and B respectively Chromatography is 1260 efficient liquid phase system of Agilent, and gained chromatogram is shown in Fig. 1.

Chromatographic column A:Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；

Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm) chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	Acetonitrile	0.1mol/L ammonium acetate pH 5.5
			0min	15%	85%
10min	22%	78%
			15min	24%	76%
15.1min	15%	85%
			20min	15%	85%

Chromatographic column B:Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm)；

Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm) chromatographic condition:

Detection wavelength: 254nm, bandwidth 4nm；

Reference wavelength: 350nm, bandwidth 100nm；

Column temperature: 37 DEG C；

Flow velocity: 1mL/min；

Sampling volume: 20 μ L；

Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	Acetonitrile	0.1mol/L ammonium acetate pH 5.5
			0min	15%	85%
40min	22%	78%
			40.1min	15%	85%
55min	15%	85%

It will be seen from figure 1 that the Agilent ZORBAX XDB-C18 chromatogram column analysis time is long, chromatographic peak separating degree is poor, The Agilent Poroshell EC-C18 chromatogram column analysis time is the 1/3 of ZORBAX XDB-C18 chromatographic column, and chromatographic peak is good. Abscissa is time (min), and ordinate is DAD signal strength.1,2,3,4,5,6,7,8 successively Man, GlcN, GalN, The chromatographic peak of GlcUA, Glc, Gal, Xyl, Fuc.

Embodiment 2

(1) precision weighs mannose (Man), rhamnose (Rha) and glucose (Glc) in right amount, and deionized water is added to prepare phase Same 5 parts contain the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using；

(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes；

(3) PMP is derivative: 60 μ L0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h；

(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 × 100 mm, 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 0.0005 mg/mL, matching while using；

(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes；

(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h；

(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes；

(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple；

(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.

High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100 mm, 2.7 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:

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

Embodiment 4

(1) it creates alkaline environment: taking 95 malignant tumor of lung patients made a definite diagnosis and 95 age-sexes matched respectively Normal 10 μ L of Check-up crowd blood serum sample adds 30 μ L ultrapure waters in 1.5mL EP pipe, and 40 μ L 0.3mol/L sodium hydroxides are added, It is vortexed and mixes；

(2) remaining step is the same as embodiment 3, analytical calculation glucose, the concentration of mannose and glucose and mannose peak The ratio of area.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. malignant tumor of lung patients serum free monosaccharide and G/M peak area ratio and normal Check-up crowd

	Glucose	Mannose	G/M ratio
				Malignant tumor of lung	-NS	↑NS	↓***

Annotation: there was no significant difference compared with normal Check-up crowd for " NS " representative, " * * * p < 0.001 ", " * * p < 0.01 " " * p < 0.05 ", which is respectively represented, has significant difference compared with normal Check-up crowd, and " ↑ " and " ↓ " respectively represents and normal physical examination Compared to rising or falling, "-" is indicated with normal Check-up crowd without significant change crowd.

2 kinds of free monosaccharide concentration (μm ol/L) and its peak face in 2. malignant tumor of lung patient of table and normal Check-up crowd serum Product ratio result

	Glucose	Mannose	G/M ratio
				Normal Check-up crowd	5609±2133	71.69±33.51	80.93±16.86
Malignant tumor of lung	5895±2218	78.70±34.57	53.17±16.35

In addition, to thering is the mannose of significant difference and glucose peaks area ratio to carry out ROC song 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.

Table 3

	AUC	Cutoff value	Sensitivity	Specificity
					G/M ratio	0.8916	65.76	85.26%	84.21%

From Fig. 3, table 1, table 2 can be seen that the malignant tumor of lung patient compared with the matched normal Check-up crowd of age-sex There was no significant difference for free serum glucose and mannose concentration, and glucose is remarkably decreased with mannose peak area ratio.In addition, Table 3 shows that the specificity of mannose and glucose peaks area ratio and sensitivity are higher, illustrate free serum glucose with Mannose peak area ratio and malignant tumor of lung disease have relationship, and G/M ratio can distinguish normal person and malignant tumor of lung patient. Therefore, malignant tumor of lung patient can be detected as biomarker using glucose free clearly and mannose peak area ratio.

Embodiment 5

It is used to detect the application of malignant tumor of lung 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 PMP are added in each sample, are vortexed and mix, and 70 DEG C of baking ovens react 1h after centrifugation；

(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 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: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；

Change of gradient:

Time	A (acetonitrile)	B (0.1mol/L ammonium acetate pH 5.5)
			0min	15%	85%
10min	22%	78%
			15min	24%	76%
15.1min	15%	85%
			20min	15%	85%

(6) draw the standard curve of glucose and mannose: precision weighs glucose and appropriate mannose, 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 is with (2), (3)), (4), (5)。

(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares simultaneously analytical calculation, determines grape The ratio of sugar, the concentration of mannose and glucose and mannose peak area.And it is made between three by 9 software of Origin 3 dimensional drawing, normal person and patients with lung cancer can be distinguished more intuitively to observe triple combination, see Fig. 5.

ROC curve is made by Prism software, to determine a possibility that G/M ratio is as marker, as a result, it has been found that quick Perception is 85.2%, and specificity is 84.2%, sensibility and specificity with higher.

If the free glucose and mannose peak area ratio in test serum sample determine to be measured less than 53.17 Blood serum sample is malignant tumor of lung 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.Institute of the present invention The method stated has very the marker of relationship, searching Disease Clinical detection between research free serum monosaccharide and lung cancer Important meaning will be suitble to clinically be used for the diagnosis of malignant tumor of lung 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 (13)

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

2. according to claim 1 for identifying the biomarker of malignant tumor of lung, it is characterised in that: this method is column High performance liquid chromatography derived from preceding PMP, 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 glucose and mannose is prepared；

(7) standard curve in the chromatogram for obtaining step (5) and step (6) compares and analytical calculation, determines glucose, sweet Reveal the concentration of sugar and the ratio of glucose and mannose peak area.

3. according to claim 1 for identifying the biomarker of malignant tumor of lung, it is characterised in that: the efficient liquid Phase chromatography be 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 biomarker of malignant tumor of lung, it is characterised in that: the step (5) change of gradient of mobile phase in 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 biomarker of malignant tumor of lung, it is characterised in that: the step (3) concentration of hydrochloric acid is 0.3mol/L in.

6. according to claim 2 for identifying the quantitative analysis method of malignant tumor of lung biomarker, feature exists In: organic solvent is at least one of chloroform, methylene chloride, ethyl acetate and n-hexane in the step (4).

7. the quantitative analysis method of identification malignant tumor of lung biomarker is in malignant tumor of lung disease described in claim 2-6 In application, it is characterised in that the blood serum sample be malignant tumor of lung patient serum.

8. a kind of kit for identifying malignant tumor of lung, which is characterized in that the kit contains biology mark described in claim 1 The free mannose and glucose that note object, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.

9. the kit of identification malignant tumor of lung according to claim 8, which is characterized in that include dense in the kit The titer for the glucose that degree is 78.7 μm of ol/L mannoses and concentration is 5865 μm of ol/L.

10. the described in any item reagent consumptive materials of claim 2-9 preparation for mannose in malignant tumor of lung patients serum and Purposes in the kit of glucose quantitation.

11. a kind of kit for identifying malignant tumor of lung, it is characterised in that pass through the mannose and glucose quantitation mirror in serum Determine malignant tumor of lung.

12. the use that the kit of the described in any item identification malignants tumor of lung of claim 8-11 detects malignant tumor of lung in vitro On the way.

13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation Malignant tumor of lung.