CN108929895A - Hypoxanthine and xanthine concentration analysis enzymatic compositions as the purine metabolism object for diagnosing tumor - Google Patents
Hypoxanthine and xanthine concentration analysis enzymatic compositions as the purine metabolism object for diagnosing tumor Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
- C12Y117/03—Oxidoreductases acting on CH or CH2 groups (1.17) with oxygen as acceptor (1.17.3)
- C12Y117/03002—Xanthine oxidase (1.17.3.2)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/9029—Oxidoreductases (1.) acting on -CH2- groups (1.17)
Abstract
The present invention relates to a kind of purine metabolism object Concentration Testing enzymatic compositions for diagnosing tumor, in more detail, it is related to a kind of enzymatic compositions, the enzymatic compositions be used for detect be present in urine as the hypoxanthine (Hypoxanthine) of purine metabolism object (Purine met abolite) and the concentration of xanthine (Xanthine), and including xanthine oxidase (Xanthine Oxidase), 1- methoxyl group -5- toluphenazine Methylsulfate salt (1-Methoxy-5-meth ylphenazinium methyl sulfate, MePMS), EZ-cytox (WST-8) and buffer solution of potassium phosphate.
Description
Technical field
The present invention relates to a kind of to be used to detect the hypoxanthine and xanthine as purine metabolism object for diagnosing tumor
The enzymatic compositions of (hereinafter referred to as purine metabolism object) concentration are related to a kind of enzymatic compositions in more detail, and the enzymatic compositions are used for
Detect the concentration for the purine metabolism object being present in urine, including xanthine oxidase (Xanthine Oxidase), 1- methoxy
Base -5- toluphenazine Methylsulfate salt (1-Methoxy-5-methylphenazinium methyl sulfate, MePMS),
EZ-cytox (WST-8) and potassium phosphate (Potassium Phosphate) buffer solution.
Background technique
According to the related data of statistics Room publication in 2014, through investigating, the domestic all cancer patients of South Korea have 217,057,
And because death toll is 76,855 caused by cancer, the death rate holds pride of place in each major causes of death.
Existing diagnosing tumor has invasion and is accompanied by pain, it is difficult to avoid and be difficult to deagnostic test.Because described in
The diagnostic method that reason, invasion minimum and the early stage to tumour and prognosis are predicted becomes diagnostic method of new generation.
According to nearest research, in terms of the energetic supersession of cancer cell and nucleic acid synthesis, the correlation of purine metabolism object is illustrated
Property.It is well known that different from normal cell, cancer cell rapidly synthetic DNA and carries out cellular replication, for this purpose, largely consuming core
The substances such as acid, protein, amino acid, glucose.
Composition substance of the purine base as nucleic acid, is the aromatic organic compounds with hexagon ring.The life of purine base
Object synthesis is formed by number of ways, is divided into de novo synthesis (De novo pathway) and salvage route
(Salvage pathway).For with regard to the de novo synthesis the case where, by with PRPP (phosphoribosylpyrophosphate), amino acid,
The reaction of ATP (atriphos), carbon dioxide etc. generates, and salvage route passes through PRPP and hypoxanthine
(hypoxanthine) be implemented in combination with.
According to the paper delivered recently, increase in the usage amount of cancer glutamine, this is reported as in mitochondria
(mitochondria) de novo synthesis is generated in ATP synthesis, is increased so that the ATP of cancer cell is synthesized.And recognize
For the unlimited division of the feature as cancer cell, to need a large amount of nucleic acid, generating de novo synthesis and salvage route
Active reaction, but the usage amount in cancer cell glutamine increases, and compared with the biosynthesis of de novo synthesis, mends
That rescues route of synthesis reuses rate increase, and accordingly, the concentration of purine metabolism object reduces in urine.
Advanced technical literature
【Patent document】
(patent document 0001) KR published patent the 2017-0021349th
Summary of the invention
For the purine metabolism analyte detection enzymatic compositions for diagnosing tumor of the invention, pass through the knot of case study
Fruit, it is known that, when compared with ordinary people, with regard to tumour, there is a situation where the purine metabolism objects for patient, being present in urine
Concentration is lower, and the purine metabolism object can be used as tumour and diagnosis biomarker occurs.
Accordingly, an object of the invention is a kind of enzymatic compositions are provided, to be present in fast in the urine of subject
Purine metabolin reacts with enzymatic compositions according to the present invention, by carrying out to the color change occurred in the reaction process
Absorbance measurement calculates the concentration of purine metabolism object in urine, whether so as to diagnosing tumour generation.
It is further an object that providing a kind of purine measured using the enzymatic compositions for diagnosing tumor
The method of the concentration of metabolism body.
In addition, the present invention provides a kind of purine metabolism analyte detection enzymatic compositions for diagnosing tumor, it is used to detect
It is present in the concentration of the purine metabolism object in urine, including xanthine oxidase (Xanthine Oxidase), 1- methoxyl group -5-
Toluphenazine Methylsulfate salt (1-Methoxy-5-methylphenazinium methyl sulfate, MePMS), EZ-
Cytox (WST-8) and buffer solution of potassium phosphate.
In addition, the side that the present invention is measured as the concentration to the purine metabolism object in the urine for being present in subject
Method, its purpose is to provide a kind of method for measurement of concentration of purine metabolism object for diagnosing tumor, for the fast of diagnosing tumor
The method for measurement of concentration of purine metabolin is characterized in that, using the enzymatic compositions, xanthine oxidase aoxidizes hypoxanthine
The hydrogen ion generated during turning to uric acid (Uric acid) for xanthine, by xanthine oxidase makes the EZ-cytox's
Thaumatropy Wei formazan (Formazan) form, in the conversion process, color changes, by the color to variation into
Row absorbance measurement, to calculate the concentration of purine metabolism object in urine, and then whether be diagnosed to be tumour and occur.
For enzymatic compositions according to the present invention, the purine metabolism object being present in urine is detected with the method for non-invasion
Concentration, so as to whether early diagnosing out in-vivo tumour (pernicious or benign) generation.Accordingly, treatment can be predicted
Direction and degree so not only can effectively be treated, but also can reduce the oncotherapy for the writing that may be paid
Take.
In addition, not equipping (nuclear magnetic resonance using used at present for enzymatic compositions according to the present invention
(NMR：Nuclear Magnetic Resonance), red, orange, green, blue, yellow (ROGBY), mass spectrograph etc.), it is possible to not experimenter's
Whether being more easily diagnosed to be tumour generation in the case where a series of process such as the pretreatment of deviation or urine sample.
Detailed description of the invention
Fig. 1 is the chart of confirmation analysis performance.
Fig. 2 is the chart for confirming detection limit, determination limit and rectilinear propagation.
Fig. 3 is the chart for confirming clinical performance (cancer of pancreas).
Fig. 4 is the chart for confirming clinical performance (colorectal cancer).
Fig. 5 is to be related to the photo of the form of kit.
Fig. 6 is xanthine oxidase expression polar plot (xanthine oxidase expression vector map).
Specific embodiment
Hereinafter, being illustrated in more detail to the present invention.
The present invention relates to a kind of purine metabolism analyte detection enzymatic compositions for diagnosing tumor, are used for detection and are present in
The concentration of purine metabolism object in urine, and including xanthine oxidase (Xanthine Oxidase), 1- methoxyl group -5- first
Base phenazine methosulfate salt (1-Methoxy-5-methylphenazinium methyl sulfate, MePMS), EZ-cytox
(WST-8) and buffer solution of potassium phosphate.
The buffer solution of potassium phosphate can extraly include NP-40 (Nonidet P 40：Nonidet P40).
The EZ-cytox is cell Proliferation/toxicity detection using water-soluble tetrazolium salts (tetrazolium salt)
Kit.
The enzymatic compositions can be used for the diagnosis of cancer of pancreas, colorectal cancer etc., but be not limited to this.
The present invention is specifically explained, the hypoxanthine and xanthine as purine metabolism object are because of xanthine oxidase
And it is oxidized to xanthine, uric acid (reference picture 1) respectively.In the process, as following reaction formula 1, two oxygen are generated
With two hydrogen.
【Picture 1】
【Reaction equation 1】
R-H+H2O+2O2->ROH+2O2-+2H+
The hydrogen ion generated in the reaction process makes EZ-cytox (WST-8, water soluble
tetrazolium salt：Water-soluble tetrazolium salts) thaumatropy Wei formazan (Formazan) form (reference picture 2).
【Picture 2】
In this process, the color of originally EZ-cytox (WST-8) shows light orange, but is converted to formazan form
Darkorange is showed simultaneously.In addition, enable that the hydrogen ion and EZ-cytox react is 1- methoxyl group -5- methyl
Phenazine methosulfate salt (1-Methoxy-5-methylphenazinium methyl sulfate, MePMS), the 1- methoxy
Base -5- toluphenazine Methylsulfate salt plays the role of induced color (reference picture 3).
【Picture 3】
Generated hydrionic quantity and structure are sent out during the hypoxanthine and xanthine oxidase turn to uric acid
The quantity for having given birth to the EZ-cytox formazan (Formazan) of conversion is identical, in other words, the concentration phase of hypoxanthine and xanthine
Together.
Accordingly, the color showed is more rendered as darkorange, then means that conversion formazan structure is more, it is meant that
A large amount of hydrogen ions are generated in oxidation process, it means that the concentration of internal purine metabolism object is higher, so such result master
It appears in the ordinary people there is no tumour.
On the contrary, the color showed is more rendered as light orange, then mean that conversion formazan structure is fewer, this is because
A small amount of hydrogen ion is generated in oxidation process, in other words, the concentration of internal hypoxanthine and xanthine is low, so such
As a result it mainly appears in tumorigenic patient.
Accordingly, the reaction is by being measured to measure indirectly to the concentration for converting formazan by hydrogen ion
The method of the concentration of purine metabolism object leads to the accuracy of reaction in urine because of many kinds of substance and pH, but at this
These problems are overcome by the optimization of response composite in invention.
Purine metabolism object method for measurement of concentration according to the present invention for diagnosing tumor, as to being present in subject's
The method that the concentration of purine metabolism object in urine is measured, which is characterized in that utilize the enzymatic compositions, xanthine oxidation
Hypoxanthine is oxidized to xanthine, xanthine oxidase is turned to generated hydrogen ion during uric acid (Uric acid) by enzyme
So that thaumatropy Wei formazan (Formazan) form of the EZ-cytox, and color becomes in the conversion process
Change, by carrying out absorbance measurement to the color of variation, to calculate the concentration of hypoxanthine and xanthine in urine, and then examines
Whether disconnected tumour occurs.
For the case where light orange is presented in the color, absorbance can be digitized as 0.654 in 450nm ±
0.0616(Mean±SEM：Average ± standard error), the case where darkorange is presented in the color, absorbance can be in 450nm
It is digitized as 2.02 ± 0.175 (Mean ± SEM).
In an implementation form of the invention, the measuring method can also be additionally using the clinical letter by subject
Breath, such as clinical pathology inspection, medical imaging check the result that the same method obtains.
Hereinafter, more specifically being illustrated by embodiment to the present invention.It will be understood by those skilled in the art that aobvious and easy
See, the embodiment is only used for that the present invention will be described, and the scope of the present invention is not limited to the embodiment.
Production Example 1：The manufacture of xanthine oxidase
Xanthine oxidase (xanthine oxidase) is in ox xanthine dehydrogenase (Bos taurus xanthine
dehydrogenase：XDH), ensure the part oxidizing ferment (oxidase) in registration number (Accession No.) NM_173972.2
Gene, carry out protein expression and expression in Escherichia coli and use (referring to Fig. 6).
(1) xanthine oxidase (Xanthine oxidase) gene ensures step
Using registration number (Accession No.) NM_173972.2's as NCBI gene pool (NCBI gene bank)
Based on ox xanthine dehydrogenase (Bos taurus xanthine dehydrogenase), in IDT (Integrated
Device Technoligy, INC., USA) recombinant bacterial strain Escherichia coli (E.coli：Escherichia coli) synthesis
For the coding of optimization.The gene of synthesis is 5 ' Not I, 3 ' NdeI, and be inserted into pUCIDT-kan carrier.
Xanthine oxidase (Xanthine oxidase) carries the gene ensured by the method insertion pET24a
Body (vector), 6x His are made as being attached to the fused protein form of c-terminus, convert in BL21 (DE3) Escherichia coli,
To produce protein (referring to Fig. 6).The process is as follows.
The information of primer pair for the reaction such as following table 1.
【Table 1】
(2) Escherichia coli incubation step is converted
A. xanthine oxidase (xanthine oxidase) gene is inserted in the Nde I of pET24a carrier, Xho I limitation
Enzyme site (site), to complete the recombinant DNA of production 6x His fused protein.
B. recombinant DNA produced is converted in BL21 (DE3) Escherichia coli.
C. in 3L LB (Luria broth：Miller is modified) in culture solution, the Escherichia coli are trained at 16 DEG C, 200rpm
It supports to OD600~0.7, is handled with 0.1M IPTG, so that inducible protein matter produces.
(3) Escherichia coli destruction step
A. after being precipitated with center of circle seperator to the Escherichia coli cultivated, supernatant is outwelled, is made with 200ml PBS
The Escherichia coli settling flux precipitated, then makes its precipitating with center of circle seperator, to carry out cleaning process.
B. by 40ml-0.5M sodium chloride (NaCl), 5mM imidazole radicals (imidazole), 20mM tri- (methylol) aminomethane
(Tris-HCl), pH 7.9 is put into the Escherichia coli of precipitating, and makes its settling flux.
C. in ultrasonic cell-break machine, the time is crushed in energy 38% maximum (Energy 38%max), total cell
(total cell breakingtime) 10 minutes (2 seconds sonicateds (sonic treatment)/pause (pause) in 4 seconds)
Under the conditions of Escherichia coli are crushed.Bottle equipped with Escherichia coli sample keeps the shape for being put into ice during clasmatosis
State.
D. the Escherichia coli being broken carry out the center of circle separation under the conditions of 13000rpm, 30 minutes, 4 DEG C.
E. after the separation of the center of circle, only supernatant is built up in conical tube (conical tube).
(4) histidine tag (his the tag)-fusion protein of Ni-NTA agarose resin (Agarose resin) is utilized
Matter purifying
It a. will include the supernatant and Ni-NTA agarose resin of the water soluble protein obtained from previous step
(Agarose resin) is mixed, and histidine tag (his tag)-fusion is induced while shaking 30 minutes under the conditions of 4 DEG C
Protein and Ni-NTA agarose resin (Agarose resin) be combined with each other.
B. the supernatant for including water soluble protein and resin (resin) mixed liquor are devoted into chromatographic column
(column), chromatographic column cork is opened, separates liquid with gravity mode.
C. make cleaning solution (Washing buffer) (0.5M sodium chloride (NaCl), 60mM imidazole radicals (imidazole),
20mM tri- (methylol) aminomethane (Tris-HCl), pH 7.9) 400ml passes through, to remove impurity and remove fused protein
Protein in addition.
D. eluent (Elution buffer) (0.25M sodium chloride (NaCl), 500mM imidazole radicals is put into
(imidazole), 20mM tri- (methylol) aminomethane (Tris-HCl), pH 7.9) 3ml, places ten minutes later, recycles albumen
Matter.
E. it is repeated twice d process.
(5) protein purification of FPLC- volume exclusion (size exclusion) method is utilized
A. the protein recycled is devoted and Xiu Podaikesi (superdex) S200 (GE healthcare) is installed
The FPLC of chromatographic column, and protein is separated according to size.
B. in classification (fraction) sample obtained, with polyacrylamide gel electrophoresis (polyacrylamide
Gel electrophoresis) method confirmation belongs to the classification (fraction) of histidine tag (his tag) fused protein,
Finally ensure desired histidine tag (his tag) fused protein.
C. 280 value of Abs is measured, from value obtained conversion protein concentration, is recorded in following table 2.
【Table 2】
| It distinguishes | Protein concentration (OD280nm) | Enzymatic activity |
| 1mg xanthine oxidase/ml | 2.42 | 2.7μU |
Xanthine oxidase DNA sequence dna (Xanthine oxidase DNA sequence) and xanthine oxidase egg
White matter sequence (Xanthine oxidase protein sequence) attached respectively in the form of sequence catalogue 3 and 4.
In addition, being used for 1- methoxyl group -5- toluphenazine Methylsulfate salt (1-Methoxy-5- of the invention
Methylphenazinium methyl sulfate, MePMS), EZ-cytox (WST-8), buffer solution of potassium phosphate and NP-
Following table 3 is recorded at 40 purchase.
【Table 3】
| Name of product | Company | Catalog number (Cat.No.) |
| EZ-cytox | Greatly-laboratory services Co., Ltd | EZ-3000 |
| 1- methoxyl group PMs | Colleague's molecular engineering Co., Ltd | M003 |
| Potassium dihydrogen phosphate | Moral Chinese yam product | 432 |
| NP-40,70%in H2O | Sigma Aldrich | NP-40S |
Embodiment 1：The manufacture of enzymatic compositions
It is such as recorded in the shown of following table 4, in the xanthine that enzymatic activity (enzyme activity) is 7.5 μ unit/ μ l
Oxidizing ferment (xanthine oxidase)It is respectively put into W-response capacity10%EZ-Cytox and 0.5%
MePMS mixed solution (Mix solution)The 50mM kaliumphosphate buffer pH value 7.5 of 0.1%NP-40 mixes molten
Liquid (potassium Phosphate buffer pH 7.5Mix solution)To manufacture enzyme reaction composition
(Reaction mixture) is total
【Table 4】
Embodiment 2：Urine sample collection
The urine collection of normal person and tumor patient be it is identical, proceed as follows.
After get up in the morning, in the urine that collection first is returned, the initial urine outflowed from the urethra is not collected, but from
Sample is collected in intermediate urine.
Experimental example 1：The concentration mensuration of purine metabolism object
In order to measure the concentration for the purine metabolism object being present in urine, so that collected normal person and tumor patient
Urine is eachEnzyme reaction composition phase reaction with the embodiment 1 is (respectively in total).For reactant, normal
Light is covered under warm (25 DEG C) and reacts it 5 minutes, and absorbance is measured in 450nm using microplate reader (ELISA reader),
To calculate the concentration in urine.Its result is recorded in following table 5.
【Table 5】
As shown in table 5 above, for the dense of purine metabolism object calculated and being reacted with enzyme reaction composition
Degree, when the urine of normal person is compared with the urine of tumor patient, confirms, and darkorange is presented in the urine of normal person, with this
On the contrary, light orange is presented in the urine of tumor patient.
It follows that the concentration of purine metabolism object reduces in the urine of tumor patient.
Experimental example 2：Analyze confirming performance
In order to confirm the analysis performance of enzymatic compositions of the invention, pass through linearity (linearity), detection limit
(Limit Of Detect, LOD), determination limit (Limit Of Quantitation, LOQ), the coefficient of variation
The project of (Coefficient of Variation, CV) etc. verifies (validation) to implement analytic approach.This process is made
Whether pass through the process that the concentration dependent of purine metabolism object is successfully reacted for verifying enzymatic compositions, carries out following letter
Slightly explanation.
Linearity is to carry out enzyme reaction by the concentration dependent of purine metabolism object and carried out by linear regression analysis
The method of verifying.Detection limit and determination limit are tested the detectability of the purine metabolism object of enzymatic compositions of the invention
The method of card.The coefficient of variation is that the process of deviation is observed after linearity experiment three times is repeated.
In order to verify analysis performance, 0,2,4,8 are set using the concentration of the calibration curve for drawing purine metabolism object,
The range of 16,32 μM of concentration.The regression straight line of purine metabolism object is y=0.0013x-0.0001, coefficient R2
(coefficient of correlation) is 0.9987, is shown good linearity (Linearity) (Fig. 1), and
And show LOD (32 μM), LOQ (0,2,4,8,16,32 μM), CV (2.8%) (Fig. 2).
Experimental example 3：Clinical performance confirmation
Using enzymatic compositions of the invention, to 33 normal persons, 48 Pancreas cancer patients, 32 colorectal cancer patients into
Go experiment, for the people for participating in clinical test, age-based, gender, the type of cancer, the root of cancer, pathology opinion, image doctor
It learns opinion, attending physician's opinion etc. to classify, and is compared (Soul E Shan with the diagnosis of enzymatic compositions through the invention
Hospital's research is clinical, IRB-20160815).
Its result is as shown in Figure 3,4, it is thus identified that, it is compared with normal people, purine metabolism object concentration is in cancer of pancreas, colorectum
Cancer patient reduces with it.In addition, being recorded in following table 6 for the content of each ROC curve shown by Fig. 3,4.
【Table 6】
| Cancer type | Conspicuousness | AUC | Specificity | Susceptibility | Likelihood ratio |
| Cancer of pancreas | < 0.0001 | 0.9272 | 90.32% | 78.72% | 8.13 |
| Colorectal cancer | < 0.0001 | 0.9366 | 90.32% | 82.76% | 8.55 |
More than, specific part of the invention is described in detail, has one in the technical field of the invention
As knowledge technical staff for, it is obvious that specific describe is only preferred to realize example, model of the invention
It encloses and is not limited to this.It, then can be with the content if there is the technical staff of general knowledge in the technical field of the invention
Based on, a variety of applications and deformation are carried out within the scope of the present invention.
Accordingly, the range of essence of the invention is defined by attached claims and his equivalent.
Claims (5)
1. a kind of purine metabolism object Concentration Testing enzymatic compositions for diagnosing tumor are used for detection and are present in urine
As the hypoxanthine of purine metabolism object and the concentration of xanthine, and including xanthine oxidase, 1- methoxyl group -5- methyl pheno
Piperazine Methylsulfate salt, EZ-cytox (WST-8) and buffer solution of potassium phosphate.
2. the purine metabolism object Concentration Testing enzymatic compositions according to claim 1 for diagnosing tumor, feature exist
In,
The buffer solution of potassium phosphate extraly includes NP-40.
3. the purine metabolism object Concentration Testing enzymatic compositions according to claim 1 for diagnosing tumor, feature exist
In,
Absorbance measurement is carried out to the color showed when the composition is reacted with urine, to calculate purine generation in urine
Whether thanking to the concentration of object, and then be diagnosed to be tumour generation.
4. a kind of method for measurement of concentration of the purine metabolism object for diagnosing tumor is present in the urine of subject as measurement
Purine metabolism object concentration method, which is characterized in that
Using enzymatic compositions according to claim 1, hypoxanthine is oxidized to xanthine, by xanthine oxidase by xanthine oxidase
The thaumatropy Wei formazan form that generated hydrogen ion during uric acid makes the EZ-cytox is turned to, in the conversion
Color changes in the process, carries out absorbance measurement to the color of variation, so that the concentration of purine metabolism object in urine is calculated,
And then whether diagnosing tumour generation.
5. the method for measurement of concentration of the purine metabolism object according to claim 4 for diagnosing tumor, which is characterized in that
The color of the variation more shows light orange, and generated hydrogen ion number is fewer, to mean that first can not be converted into
Za form, this is because the internal hypoxanthine of subject and the concentration of xanthine are low, so being diagnosed to be generation tumour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020170064733A KR101899426B1 (en) | 2017-05-25 | 2017-05-25 | Enzyme Composition for analysing purine metabolite concentration such as hypoxanthine and xanthine for tumor diagnosis |
| KR10-2017-0064733 | 2017-05-25 |
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| CN108929895A true CN108929895A (en) | 2018-12-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113670911A (en) * | 2021-08-30 | 2021-11-19 | 武汉纺织大学 | Method for determining total number of viable bacteria based on WST-8 chromogenic reaction |
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