CN109406656A - A kind of method and its detection kit for identifying psoriasis biomarker - Google Patents

A kind of method and its detection kit for identifying psoriasis biomarker Download PDF

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Publication number
CN109406656A
CN109406656A CN201811323458.5A CN201811323458A CN109406656A CN 109406656 A CN109406656 A CN 109406656A CN 201811323458 A CN201811323458 A CN 201811323458A CN 109406656 A CN109406656 A CN 109406656A
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Prior art keywords
psoriasis
mannose
phase
glucose
biomarker
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Inventor
张丽娟
杨丹丹
王君
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Affiliated Hospital of University of Qingdao
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Affiliated Hospital of University of Qingdao
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • G01N2030/067Preparation 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 psoriasis.The biomarker is the ratio of the free glucose and mannose 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 psoriatic, 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 glucose and mannose and psoriasis, the novel psoriasis clinical detection marker of searching with mannose and glucose free in fast quantification Sera in Patients with Psoriasis Vulgaris.

Description

A kind of method and its detection kit for identifying psoriasis biomarker
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 psoriasis Test agent box.
Background technique
Psoriasis is commonly called as " psoriasis ", be it is a kind of it is common it is chronic can recurrent inflammatory dermatoses, illness rate exists There is notable difference in different crowd all over the world.The course of disease of psoriasis is long, obstinate, easy to recur, and the body and mind for seriously affecting patient is strong Health and quality of life.The diagnostic method of psoriasis mainly includes skin lesion observation, dermoscopy and imageological examination etc. at present.Skin lesion is seen It is more to examine complex steps, observation item, and mistaken diagnosis easily occurs.Imageological examination includes skin CT, X-ray film etc., but iconography is examined Radiation has been looked into, adverse effect can be generated to human body.Therefore, find a kind of convenience, simple and direct detection method is very necessary, and Optimal early detection is exactly blood analysis easy to operate, that wound is minimum.
Reductive monosaccharide seems to be only limitted to mannose and glucose in serum.Glucose contains in human serum free monosaccharide Highest is measured, the range of normal person is 3.90~6.16mmol/L.Currently, hospital laboratory can be directly by surveying biochemical indicator inspection Concentration of glucose is surveyed, mannose concentration temporarily cannot be directly detected.The concentration range of free mannose in health adult's blood plasma For 20~80 μm of ol/L.The content of mannose is about the 1% of glucose content.It is considered as by the grape in cell that it is most of Sugared isomerization.And recently some researches show that, the free mannose flowed out in cell be dissociate in mammalian it is sweet Reveal the main source of sugar.The free mannose in this two parts source maintains the stabilization of mannose in serum.D-MANNOSE is sugar Required monosaccharide in the structures such as albumen, cell surface glycoconjugates and glycosylphosphatidylinositol-anchored proteins.Wherein, mannose The important component of N- sugar chain in various polysaccharide compounds, glucose of the mannose residue in N- glycan in the blood or Mannose.Free mannose is normal plasma composition.Currently, the measuring method of serum mannose mainly has enzyme process, gas-liquid color Spectrometry, high resolution liquid chromatography, gas-liquid chromatography-mass spectrography and capillary electrophoresis etc..But these methods exist it is certain not Foot place.For example, when using enzyme process, gas-liquid chromatography and high resolution liquid chromatography, for avoid high concentration glucose influence It needs to remove it, causes pretreatment process comparatively laborious;Gas-liquid chromatography-mass spectrography instrument price is expensive, is not suitable for routine mesh 's;Required serum sample amount is big.
Summary of the invention
Before for identifying that biomarker and its application of psoriasis, the present invention use column Dissociate in monosaccharide more particularly to serum in high performance liquid chromatography derived from PMP (HPLC) detection Sera in Patients with Psoriasis Vulgaris sweet The detection of dew sugar and glucose.Technical solution of the present invention can be used for detecting psoriatic or the free grape of normal human serum The ratio of sugar and mannose, can be used for identifying psoriatic.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of psoriasis, the biomarker are serum by efficient derived from PMP before column The ratio of free glucose and mannose that liquid chromatogram obtains.
For identifying the quantitative analysis method of the biomarker of psoriasis, 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 The ratio of sugar, the concentration of glucose and glucose and mannose.
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 psoriasis.
Biomarker is preparing the purposes in the kit for detecting psoriasis, it is characterised in that: the kit In include titer that glucose and mannose ratio are 65.85.
A kind of kit for identifying psoriasis, which is characterized in that the kit contains biomarker, i.e. serum passes through column The ratio of free glucose and mannose that high performance liquid chromatography derived from preceding PMP obtains.
It include the titer that glucose is 65.85 with mannose ratio in the kit.
Identify that the kit of psoriasis is preparing the purposes in the kit for detecting psoriasis.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for Sera in Patients with Psoriasis Vulgaris by the present invention The detection of free monosaccharide, advantageous effects are:
1, pre-treatment is simple.
2, analysis time is short.
3, required amount of serum is few, simply needs to be less than the blood volume of 1mL every time.
4, instrument price is reasonable, meets conventional use.
5, operating procedure is easy to learn.
6, detection is accurate.
The present invention uses hplc simultaneous determination free serum mannose and glucose derived from PMP before column, Simplify operation, and amount of serum is few, and the measurement for having proven to the method mannose concentration will not be by high concentration glucose Influence.Pass through the statistics of the ratio (G/M ratio) to free serum mannose, glucose and glucose and mannose total amount Data analysis, establishes the relationship between psoriasis and three.Normal person can be quickly distinguished using technical solution of the present invention And psoriatic.
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 psoriatic and the free mannose of normal human serum, glucose and glucose and mannose total amount ratio Scatter plot;
Fig. 4 is the ROC curve of Sera in Patients with Psoriasis Vulgaris free glucose and mannose ratio;
Fig. 5 is the ratio of normal person and Sera in Patients with Psoriasis Vulgaris free mannose, glucose and glucose and mannose total amount It is worth the 3 dimensional drawing of building.
Specific embodiment
Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.
All blood samples involved in embodiment are provided by affiliated hospital, University Of Qingdao, Qingdao City.
Technical solution of the present invention the following steps are included:
Embodiment 1
(1) precision weighs mannose (Man), gucosamine (GlcN), galactosamine (GalN), glucuronic acid (GlcUA), glucose (Glc), galactolipin (Gal), xylose (Xyl), fucose (Fuc) in right amount, add deionized water to prepare two parts Containing the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using;
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes;
(3) PMP is derivative: 60 μ L 0.5mol/L 1- phenyl -5- methylpyrazole quinoline ketone (PMP) are added in each sample, are vortexed It mixes, 70 DEG C of baking ovens react 1h after centrifugation;
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes;
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple;
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, two parts of samples carry out high-efficient liquid phase analysis, efficient liquid phase using different chromatographic column A and B respectively Chromatography is 1260 efficient liquid phase system of Agilent, and gained chromatogram is shown in Fig. 1.
Chromatographic column A:Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm);
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm) chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm;
Reference wavelength: 350nm, bandwidth 100nm;
Column temperature: 37 DEG C;
Flow velocity: 1mL/min;
Sampling volume: 20 μ L;
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile;
Change of gradient:
Time Acetonitrile 0.1mol/L ammonium acetate pH 5.5
0min 15% 85%
10min 22% 78%
15min 24% 76%
15.1min 15% 85%
20min 15% 85%
Chromatographic column B:Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm);
Agilent ZORBAX XDB-C18 chromatographic column (4.6mm × 150mm, 5 μm) chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm;
Reference wavelength: 350nm, bandwidth 100nm;
Column temperature: 37 DEG C;
Flow velocity: 1mL/min;
Sampling volume: 20 μ L;
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile;
Change of gradient:
Time Acetonitrile 0.1mol/L ammonium acetate pH 5.5
0min 15% 85%
40min 22% 78%
40.1min 15% 85%
55min 15% 85%
It will be seen from figure 1 that the Agilent ZORBAX XDB-C18 chromatogram column analysis time is long, chromatographic peak separating degree is poor, The Agilent Poroshell EC-C18 chromatogram column analysis time is the 1/3 of ZORBAX XDB-C18 chromatographic column, and chromatographic peak is good. Abscissa is time (min), and ordinate is DAD signal strength.1,2,3,4,5,6,7,8 successively Man, GlcN, GalN, GlcUA, The chromatographic peak of Glc, Gal, Xyl, Fuc.
Embodiment 2
(1) precision weighs mannose (Man), rhamnose (Rha) and glucose (Glc) in right amount, and deionized water is added to prepare phase Same 5 parts contain the above monosaccharide 0.1mg/mL hybrid standard product solution, matching while using;
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes;
(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h;
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes;
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple;
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, 5 parts of samples are used for high-efficient liquid phase analysis, gained chromatography under different mobile phase variable gradients respectively Figure, is shown in Fig. 2.
High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm);
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm;
Reference wavelength: 350nm, bandwidth 100nm;
Column temperature: 37 DEG C;
Flow velocity: 1mL/min;
Sampling volume: 20 μ L;
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile;
Mobile phase variable gradient A:
Time Acetonitrile 0.1mol/L ammonium acetate pH5.5
0min 15% 85%
20min 15% 85%
Mobile phase variable gradient B:
Time Acetonitrile 0.1mol/L ammonium acetate pH5.5
0min 20% 80%
20min 20% 80%
Mobile phase variable gradient C:
Time Acetonitrile 0.1mol/L ammonium acetate pH5.5
0min 22% 78%
20min 22% 78%
Mobile phase variable gradient D:
Time Acetonitrile 0.1mol/L ammonium acetate pH5.5
0min 15% 85%
10min 22% 78%
15min 15% 85%
20min 15% 85%
Mobile phase variable gradient E:
Figure it is seen that only Man is eluted out under the conditions of gradient A;Under the conditions of gradient B, although three Can be separated and, but the peak of solvent PMP and Man from it is relatively close;And under the conditions of gradient C, solvent peak and the peak Man weight It closes;Under the conditions of gradient D, the overlong time that the peak Glc is eluted out leads to cannot to be formed point and high peak.Only in gradient E item Under part, appearance is symmetrical, and separating degree is good.Abscissa is time (min), and ordinate is DAD signal strength.1,2 and 3 is successively The chromatographic peak of Man, Rha and Glc.
Embodiment 3
(1) precision weighs mannose and proper amount of glucose, and deionized water is added to be configured to each 0.5mg/mL containing the above monosaccharide, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL, 0.0025mg/mL, 0.001mg/mL, The hybrid standard product solution of 0.0005mg/mL, matching while using;
(2) it creates alkaline environment: taking 40 μ L mixing monosaccharide standard to manage in 1.5mL EP, 40 μ L 0.3mol/L hydrogen are added Sodium oxide molybdena is vortexed and mixes;
(3) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation 1h;
(4) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample Hydrochloric acid is vortexed and mixes;
(5) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight It is 3 times multiple;
(6) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.
High performance liquid chromatography is 1260 efficient liquid phase system of Agilent, Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm);
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm;
Reference wavelength: 350nm, bandwidth 100nm;
Column temperature: 37 DEG C;
Flow velocity: 1mL/min;
Sampling volume: 20 μ L;
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile;
Mobile phase variable gradient:
Time Acetonitrile 0.1mol/L ammonium acetate pH 5.5
0min 15% 85%
10min 22% 78%
15min 24% 76%
15.1min 15% 85%
20min 15% 85%
(7) standard curve of mannose and glucose is drawn.
Embodiment 4
(1) it creates alkaline environment: taking 99 normal human sera samples and 99 Sera in Patients with Psoriasis Vulgaris 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 total The ratio of amount.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. Sera in Patients with Psoriasis Vulgaris free monosaccharide and G/M ratio and normal person
Mannose Glucose G/M ratio
Psoriasis ↑NS ↑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 total amount ratio result in 2. psoriatic of table and normal human serum
Mannose Glucose G/M ratio
Normal person 62.66±23.13 5174±1853 83.49±15.31
Psoriasis 64.92±25.36 5196±1644 60.91±19.03
In addition, having carried out ROC to the ratio (G/M) of the glucose and mannose that have significant difference in table 1 with normal person Tracing analysis finds the higher index of area under the curve (AUC), sensitivity and specificity 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 total It ties to table 3.
The cutoff value (μm ol/L) and its spirit of the ratio (G/M) of glucose and mannose in 3. Sera in Patients with Psoriasis Vulgaris of table Sensitivity (%) and specificity (%)
AUC Cutoff value Sensitivity Specificity
G/M 0.8274 65.85 67.68 88.89
From Fig. 3, table 1, as can be seen that Sera in Patients with Psoriasis Vulgaris dissociates, glucose and mannose ratio (G/M) significantly drop table 2 It is low.In addition, table 3 is shown, the specificity of the ratio (G/M) of glucose and mannose and sensitivity are higher, therefore can be with trip Psoriatic is detected from the ratio (G/M) of glucose and mannose is biomarker.
Embodiment 5
It is used to detect the application of psoriasis 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:
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%
(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 total amount.And it is made between three by 9 software of Origin Can 3 dimensional drawing distinguish normal person and psoriatic more intuitively to observe triple combination, see Fig. 5.
Determine if the ratio (G/M) of free glucose and mannose in test serum sample is less than 65.85 to be measured Blood serum sample is psoriatic.
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 dissociate in serum glucose and mannose can distinguish normal person and psoriatic, be suitble to clinically for silver bits The diagnosis of 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 psoriasis, it is characterised in that: the biomarker is serum by 1- benzene before column The ratio of free glucose and mannose that high performance liquid chromatography derived from base -5- methylpyrazole quinoline ketone (PMP) obtains.
2. according to claim 1 for identifying the quantitative analysis method of the biomarker of psoriasis, 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 of sugar and the ratio of glucose and mannose total amount.
3. according to claim 2 for identifying the quantitative analysis method of the biomarker of psoriasis, 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 psoriasis, 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 psoriasis, 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 psoriasis, 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 psoriasis answering in psoriasis disease described in claim 2-6 With, it is characterised in that the blood serum sample is the serum of psoriatic.
8. a kind of kit for identifying psoriasis, which is characterized in that the kit contains biomarker described in claim 1 The ratio of free glucose and mannose that object, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification psoriasis according to claim 8, which is characterized in that include glucose in the kit With mannose total amount than the titer for 65.85.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose and grape in Sera in Patients with Psoriasis Vulgaris Purposes in the quantitative kit of sugar.
11. a kind of kit for identifying psoriasis, it is characterised in that pass through the mannose and glucose quantitation identification silver in serum Bits disease.
12. the purposes that the kit of the described in any item identification psoriasis of claim 8-11 detects psoriasis in vitro.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation Psoriasis.
CN201811323458.5A 2018-11-01 2018-11-01 A kind of method and its detection kit for identifying psoriasis biomarker Pending CN109406656A (en)

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