CN109406655A - A kind of method and its detection kit for identifying Huppert's disease biomarker - Google Patents
A kind of method and its detection kit for identifying Huppert's disease biomarker Download PDFInfo
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- G—PHYSICS
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Abstract
The present invention provides the methods and its detection kit of a kind of biomarker for identifying Huppert's disease.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, 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 can distinguish normal person and multiple myeloma patients, 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 have very important meaning for the relationship between research free serum glucose and mannose and Huppert's disease, the novel Huppert's disease clinical detection marker of searching with mannose and glucose free in fast quantification multiple myeloma patients serum.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a method of the biomarker of identification Huppert's disease
And its detection kit.
Background technique
Huppert's disease is a kind of plasma-cell malignancy, its main feature is that marrow mesoplasmatocyte clonal expansion, and point
Secrete monoclonal immunoglobulin or light chain.Huppert's disease onset low, early stage non-evident sympton, diverse clinical manifestations,
Seriously jeopardize body health.The multiple systems of whole body, patient Shou Zhen department can be appeared in by the clinical manifestation of Huppert's disease
It is more miscellaneous, it often will cause mistaken diagnosis and fail to pinpoint a disease in diagnosis, eventually lead to state of an illness delay.It is living that the diagnosis of Huppert's disease generally requires progress marrow
Inspection, imageological examination and blood test etc., and diagnosed in conjunction with clinical manifestation.These diagnostic methods can bring certain
Drawback.Therefore, a kind of convenience is found, the detection method of hurtless measure is very necessary.Blood test is easy to operate, noninvasive because of its
The characteristics of hurting is as ideal detection method, so screening suitable blood biomarker is Huppert's disease clinic
The key of diagnosis.
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
The object of the present invention is to provide for identifying biomarker and its application of Huppert's disease, the present invention is adopted
The high performance liquid chromatography derived from PMP before column (HPLC) detects the monosaccharide in multiple myeloma patients serum, more particularly to
The detection of free mannose and glucose in serum.Technical solution of the present invention can be used for detecting multiple myeloma patients or
The ratio of normal human serum free glucose and mannose, can be used for identifying multiple myeloma patients.
To achieve the purpose of the present invention, the present invention is achieved by the following technical programs:
For identifying that the biomarker of Huppert's disease, the biomarker are that serum is derivative by PMP before column
The obtained ratio of free glucose and mannose of high performance liquid chromatography.
For identifying the quantitative analysis method of the biomarker of Huppert's disease, 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
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 Huppert's disease.
Biomarker is preparing the purposes in the kit for detecting Huppert's disease, it is characterised in that: described
It include the titer that glucose is 62.72 with mannose ratio in kit.
A kind of kit for identifying Huppert's disease, which is characterized in that the kit contains biomarker, i.e. serum
By the ratio of free glucose and mannose that high performance liquid chromatography derived from PMP before column obtains.
It include the titer that glucose is 62.72 with mannose ratio in the kit.
Identify that the kit of Huppert's disease is preparing the purposes in the kit for detecting Huppert's disease.
Compared with prior art, high performance liquid chromatography derived from PMP before column is used for Huppert's disease trouble by the present invention
The detection of person's 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 statistics of the ratio (G/M ratio) to free serum mannose, glucose and glucose and mannose total amount
Data analysis, establishes the relationship between Huppert's disease and three.It can quickly be distinguished using technical solution of the present invention
Normal person and multiple myeloma patients.
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 multiple myeloma patients and the free mannose of normal human serum, glucose and glucose and mannose are total
Measure the scatter plot of ratio；
Fig. 4 is the ROC song of multiple myeloma patients free serum mannose, glucose and glucose and mannose ratio
Line；
Fig. 5 is that normal person and multiple myeloma patients free serum mannose, glucose and glucose and mannose are total
The 3 dimensional drawing of the ratio building of amount.
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；
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% |
| 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 (G1c) 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:
| 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 0.0005mg/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 pH5.5 |
| 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 185 normal human sera samples and 185 Huppert's diseases made a definite diagnosis respectively
10 μ L of patient serum 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 mixed
It is even；
(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. multiple myeloma patients free serum monosaccharide and G/M ratio and normal person
| Mannose | Glucose | G/M ratio | |
| Huppert's disease | ↑*** | ↑*** | ↓*** |
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 in 2. multiple myeloma patients of table and normal human serum
As a result
| Mannose | Glucose | G/M ratio | |
| Normal person | 69.05±30.26 | 5583±2116 | 82.59±15.58 |
| Huppert's disease | 82.75±42.19 | 6450±2654 | 58.58±18.17 |
In addition, to free mannose, glucose and the glucose and mannose for having significant difference in table 1 with normal person
Ratio (G/M) has carried out ROC curve analysis, finds the higher finger of area under the curve (AUC), sensitivity and specificity with expectation
Mark, to determine best screening positive critical value (cutoff value), AUC illustrates that diagnosis effect is better closer to 1.Testing result
See Fig. 4, Fig. 4 result is summarized to table 3.
Ratio (the G/ of free mannose, glucose and glucose and mannose in 3. multiple myeloma patients serum of table
M cutoff value (μm ol/L) and its sensitivity (%) and specificity (%))
| AUC | Cutoff value | Sensitivity | Specificity | |
| Mannose | 0.6237 | 60.10 | 70.18 | 49.19 |
| Glucose | 0.6272 | 5123 | 67.03 | 57.30 |
| G/M | 0.8460 | 62.72 | 63.78 | 92.43 |
From Fig. 3, table 1, table 2 is as can be seen that multiple myeloma patients free serum glucose and mannose ratio (G/M)
It significantly reduces.In addition, table 3 is shown, the specificity of the ratio (G/M) of glucose and mannose and sensitivity are higher, therefore can
Multiple myeloma patients are detected so that the ratio (G/M) with dissociate glucose and mannose is biomarker.
Embodiment 5
It is used to detect the application of Huppert's disease using biomarker of the present invention, specifically includes following step
It is rapid:
(1) it creates alkaline environment: 10 μ L of blood serum sample to be detected being taken to add 30 μ L ultrapure waters in 1.5mL EP pipe, be added
40 μ L 0.3mol/L sodium hydroxides are vortexed and mix；
(2) PMP is derivative: 60 μ L 0.5mol/L PMP are added in each sample, are vortexed and mix, 70 DEG C of baking oven reactions after centrifugation
1h；
(3) acid adding neutralization reaction: the sample in baking oven is taken out, and places cooling, and 40 μ L 0.3mol/L are added in each sample
Hydrochloric acid is vortexed and mixes；
(4) extract: 500 μ L chloroforms are added in every pipe, are vortexed, and centrifugation removes lower layer's chloroform, retain supernatant, weight
It is 3 times multiple；
(5) sample 13000r/min is centrifuged 10min, takes 80 μ L supernatants in the brown loading that 150 μ L interpolation pipes are housed
In bottle, lid is covered tightly, is centrifuged, is ready to use in high-efficient liquid phase analysis.High performance liquid chromatography is 1260 efficient liquid phase system of Agilent,
Agilent Poroshell EC-C18 chromatographic column (4.6mm × 100mm, 2.7 μm)；
Chromatographic condition:
Detection wavelength: 254nm, bandwidth 4nm；
Reference wavelength: 350nm, bandwidth 100nm；
Column temperature: 37 DEG C；
Flow velocity: 1mL/min；
Sampling volume: 20 μ L；
Mobile phase: 0.1mol/L ammonium acetate-acetic acid buffer solution, acetonitrile；
Change of gradient:
(6) draw the standard curve of mannose and glucose: precision weighs mannose and proper amount of glucose, adds deionized water
It is configured to each 0.5mg/mL, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005mg/mL containing the above monosaccharide,
The hybrid standard product solution of 0.0025mg/mL, 0.001mg/mL, 0.0005mg/mL, matching while using；Take 40 μ L mixing monosaccharide marks
Quasi- product are managed in 1.5mL EP, and 40 μ L 0.3mol/L sodium hydroxides are added, and are vortexed and are mixed；Remaining step with (2), (3), (4),
(5)。
(7) standard curve in the chromatogram for obtaining step (5) and step (5) compares simultaneously analytical calculation, determines sweet dew
The ratio of sugar, the concentration of glucose and glucose and mannose total amount.And it is made between three by 9 software of Origin
Can 3 dimensional drawing distinguish normal person and multiple myeloma patients more intuitively to observe triple combination, see figure
5。
If the ratio (G/M) of free glucose and mannose in test serum sample determines to be measured less than 62.72
Blood serum sample is multiple myeloma patients.
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 multiple myeloma patients, be suitble to clinically use
In the diagnosis of multiple myeloma patients.
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 Huppert's disease, it is characterised in that: the biomarker is that serum passes through column
The ratio of free glucose and mannose that high performance liquid chromatography derived from preceding 1- phenyl -5- methylpyrazole quinoline ketone (PMP) obtains.
2. it is according to claim 1 for identifying the quantitative analysis method of the biomarker of Huppert's disease, it is special
Sign is: 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；Sample in baking oven is taken out, cooling is placed, hydrochloric acid is added in each sample, is vortexed and mixes；
(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. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of Huppert's disease, it is special
Sign is: 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.
4. it is according to claim 3 for identifying the quantitative analysis method of the biomarker of Huppert's disease, it is special
Sign is: 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. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of Huppert's disease, it is special
Sign is: the concentration of hydrochloric acid is 0.3mol/L in the step (3).
6. it is according to claim 2 for identifying the quantitative analysis method of the biomarker of Huppert's disease, it is special
Sign is: 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 Huppert's disease is in multiple marrow described in claim 2-6
Application in tumor disease, it is characterised in that the blood serum sample is the serum of multiple myeloma patients.
8. a kind of kit for identifying Huppert's disease, which is characterized in that the kit contains biology described in claim 1
The ratio of free glucose and mannose that marker, i.e. serum are obtained by high performance liquid chromatography derived from PMP before column.
9. the kit of identification Huppert's disease according to claim 8, which is characterized in that include in the kit
Glucose and mannose total amount are than the titer for 62.72.
10. the described in any item reagent consumptive materials of claim 2-9 are in preparation for mannose in multiple myeloma patients serum
With the purposes in the kit of glucose quantitation.
11. a kind of kit for identifying Huppert's disease, it is characterised in that pass through the mannose and glucose quantitation in serum
Identify Huppert's disease.
12. the kit of the described in any item identification Huppert's diseases of claim 8-11 detects Huppert's disease in vitro
Purposes.
13. purposes according to claim 12, it is characterised in that identified by mannose in serum and glucose quantitation
Huppert's disease.
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Non-Patent Citations (5)
| Title |
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| 丁克祥: "《简明临床生化参考值手册》", 31 December 1987 * |
| 中国科协学会学术部: "《网络药理学一中药现代化的新思路与新方法》", 30 November 2014 * |
| 吴梧桐等: "《生物化学》", 31 August 2015 * |
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