CN106290190B - The method for measuring MPO halogenase activity - Google Patents
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Classifications
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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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
- G01N2021/3185—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry typically monochromatic or band-limited
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method of measurement MPO halogenase activity, the purpose of the present invention is isolated from HL-60 cell with ion chromatography with nature protein structure and enzymatic activity MPO, measurement MPO halogenase activity method is established, uses purifying MPO as calibration object, measures MPO halogenase activity unit in human serum/slurry.The MPO halogenase that the present invention measures is separated from HL-60 cell.The present invention establishes NaI-TMB(3,3 ', 5,5 '-tetramethylbenzidine, tetramethyl benzidine) method, directly MPO halogenase activity in measurement serum/slurry, for predicting cardiovascular and cerebrovascular disease possibility occurrence and disease prognosis.
Description
Technical field
The invention belongs to field of biotechnology, in particular to natural protease purification method.
Background technique
Myeloperoxidase (Myeloperoxidase, MPO) is also known as peroxidase, is thin by neutrophil leucocyte, monokaryon
The glycosylation of the macrophages secrete of born of the same parents and certain tissues and hemoprotein enzyme containing prosthetic heme group, are ferroheme peroxidating
One of object enzyme superfamily member.Human body granulocyte enters before circulation, in marrow close MPO at and be stored in azurophilic granule
Interior, environmental stimuli can lead to neutrophil accumulation, discharge MPO.In mature granulocyte (mainly neutrophil leucocyte and monokaryon
Cell) in, MPO is the most abundant glycoprotein of content, accounts for about peripheral blood polymorphonuclear neutrophils (PMNs) interior gross protein and contains
The 5% of amount, 95% MPO derives from PMNs in blood
Myeloperoxidase (MPO) is the dimer as made of 2 subunit polymerizations, and each subunit is again by a heavy chain
(α chain, 466 amino acid residues) and a light chain (β chain, 108 amino acid residues) are constituted.2 subunits at α chain by
1 disulfide bond is connected.Heavy chain has ferroporphyrin group, shows that MPO is iron dependence oxide enzyme.
MPO gene is located at chromosome 17q23 q24, contains 12 exons and 11 intrones, is about 14638 bp, adjusts
Control its gene expression is growth factor.For the mRNA of MPO in the expression highest of progranulocyte, followed by original grain is thin
Born of the same parents, naivety and monoblast；When cell differentiation to mature period, MPO gene expression dose declines rapidly.MPO is known
It is a relative molecular mass is that 84KDa precursor MPO albumen by post translational processing is cut into α and β two that gene is expressed first
Kind subunit, then the MPO molecule for being polymerized to maturation have enzymatic activity MPO in addition sugar chain, eventually forms glycosylation.MPO mono- important
It is characterized in that isoelectric point (pI) is very high, is 9.2.Hydrogen peroxide (H2O2) oxidation MPO after, become oxidized form MPO, have oxidizing ferment and
Halogenase activity, MPO enzymatic activity Optimal pH is 4.5~5.5, when pH >=10, or when≤2, loses MPO enzymatic activity.
The major function of MPO is that microorganism is killed in phagocyte, utilizes H2O2With chloride ion (Cl-) generate hypochlorous acid
Salt (HOCl), and the free radical with oxidability is formed, constitute MPO-H2O2Halogen system.It is many study found that MPO not only
Phagocytosis can be killed in intracellular microorganism, and releasably arrive extracellular, destruction target substances of multiple types, as tumour cell, blood are small
Plate, NK cell etc. generate and are adjusted to body the various aspects such as inflammatory reaction and play a role.However, under given conditions, MPO is urged
Change reaction and generate excessive oxidant (HOCl etc.), more than topical antioxidant defense reaction when, will lead to oxidisability group
Damage is knitted, such as intravascular cortical sites inflammatory reaction and the damage of blood vessel endothelium confluent monolayer cells and death.
Atherosclerosis is the important pathologic basis to form cardiovascular and cerebrovascular disease.Studies have shown that atherosclerosis is
A kind of chronic inflammatory reaction disease, and MPO is as inflammatory factor, the starting formed in atherosclerotic plaque, development, rupture and
It plays an important role during falling off etc..
The study found that MPO in blood, combining with vascular endothelial cell, invade profit to blood vessel endothelium hypobasal, pass through MPO enzyme oxygen
Change reaction, directly consumption endothelial cell generates signaling molecule NO, activation guanylic acid activating enzymes function is reduced, to cause intravascular
Skin diastolic dysfunction.
MPO is generated diffusible oxidant (HOCl) in vivo, promote oxidation LDL(oxLDL) generate, oxLDL be easier to by
Macrophage phagocytosis, is converted into foam cells, is deposited under endangium, so that atherosclerotic plaque be promoted to be formed；
HOCl can activate matrix metalloproteinase former (MMPs), make stromatin Polyose degradation, lead to atherosclerotic plaque fiber
Cap is thinning, and plaque surface corrosion, the formation of rupture and thrombus causes atherosclerotic plaque unstable.HOCl can induce blood
Endothelial cell generates P selectin and tissue factor expression, promotes platelet aggregation, so as to cause the formation of thrombus.
As it can be seen that early stage of the MPO to Dysfunction of vascular endothlial cells, formation and subsequent patch to atherosclerotic plaque
The formation whole process of rupture and thrombus has served key, eventually leads to coronarospasm, angina pectoris, or even cardiac muscle stalk
Dead generation.
The study found that MPO measures MPO enzymatic activity or concentration in blood as a kind of Inflammation Marker in blood；(1) energy
It can be used for the early diagnosis of acute coronary syndrome (CVD) as atherosclerotic plaque unstability early prediction index (1), comment
Estimate the CVD state of an illness and prognosis: (3) can be used to predict that the risk of cardiovascular and cerebrovascular disease to occur for healthy population.
In addition, the generation of blood of human body middle and high concentration MPO and a variety of diseases of the mankind, developing closely related, neuroinflamation disease
Sick aspect includes: multiple sclerosis, Alzheimer's disease, Parkinson's disease etc..Other inflammatory diseases are such as: chronic obstructive
Tuberculosis, cystic fibrosis, glomerular injury and rheumatoid arthritis etc..MPO enzymatic activity or concentration can be to these in measurement blood
The prevention of a little diseases, diagnosis and treatment have very great help.
In clinical examination, detects MPO concentration method in blood and be mainly enzyme-linked immunization (ELISA) and directly measure blood
Liquid MPO enzyme activity method, two measuring methods require purifying MPO as calibration object, this calibration object will have MPO nature albumen knot
Structure and enzymatic activity.It is that 84KDa precursor MPO albumen is cut by post translational processing that MPO gene gives expression to a molecular mass first
Two kinds of subunits of α and β are cut into, then are polymerized to mature MPO molecule, are further glycosylated, enzymatic activity saccharification MPO has been eventually formed.
Purifying MPO method is obtained first is that isolating leucocyte from blood of human body, then largely extract nature from these cells
MPO.This method disadvantage, second is that there are many infectants in blood, prepares MPO calibration object first is that blood sources are restricted
It is dangerous.Another method is MPO to be expressed in human body cell with gene engineering research, but MPO not can be carried out effective albumen after expression
Processing, not can be carried out correct glycosylation, and recombination MPO albumen has very weak enzymatic activity, should not be used as MPO calibration object.
Currently, having the kit of MPO in detection blood both at home and abroad, ELISA is mainly used, this method is simple, and it is easy to operate,
It is used widely.But the content of MPO, shows MPO enzymatic activity in blood, sometimes MPO indirectly in this method measurement blood
Content to MPO enzymatic activity be it is uncorrelated, as there is the crowd of MPO mortifier in MPO genetic mutation crowd and blood；In addition, preparation
It measures MPO ELISA kit and needs several MPO monoclonal antibodies, preparation cost is high, and the time is long；Meanwhile ELISA method step
It is more, need the disadvantages of time is longer, and accuracy is poor.And artery sclerosis activity situation and MPO halogenase activity correlation in blood
Maximum, artery sclerosis activity situation can most be represented by measuring MPO halogenase activity in blood.
Summary of the invention
The purpose of the present invention is isolated from HL-60 cell with nature protein structure and enzymatic activity with ion chromatography
MPO establishes measurement MPO halogenase activity method, uses purifying MPO as calibration object, measures MPO halogenation enzyme activity in human serum/slurry
Property unit.
The step of MPO halogenase that the present invention measures is separated from HL-60 cell is:
A, MPO is isolated from HL-60 cell: cell is collected by centrifugation in culture HL-60 cell, molten with phosphate buffer
Cell is solved, further ruptures cell with Dounce homogenization, cell deposition object, cell deposition are collected in centrifugation
Contain MPO in object；
B, 1% CTAB- phosphate buffer dissolves MPO in above-mentioned cell deposition object；
C, it prepares Q-Sepharose chromatographic column: balancing Q-Sepharose layers with 0.5% CTAB- phosphate buffer
Column is analysed, above-mentioned MPO lysate is by Q-Sepharose chromatographic column, on binding protein to Q-Sepharose chromatographic column, collects
Filtered solution, filtered solution contain the MPO of pI=9.2；
D, SP-Sepharose chromatographic column is prepared: with 0.2% CTAB- phosphate solution balance SP-Sepharose chromatography
Column, above-mentioned to pass through SP-Sepharose chromatographic column containing MPO filtered solution, the protein binding of pI >=8.0 is to SP-Sepharose layers
It analyses on column, with 600mM K2PO4Phosphate buffer elutes SP-Sepharose chromatographic column binding protein, and collection contains MPO's
Eluent；
E, it puts and states eluent into albumen bag filter, dialysed with 150mM NaCl- phosphate buffer；
F, it prepares ConA-Sepharose4B chromatographic column: washing ConA- with 100mM NaCl- phosphate solution
Sepharose4B, then mixed with protein liquid after dialysis, ConA-Sepharose4B combines saccharification MPO, with 500mM NaCl- phosphorus
Phthalate buffer washs ConA-Sepharose4B chromatographic column, with 600mM a-D-methylmannoside-phosphate-buffered
Liquid elutes ConA-Sepharose4B chromatographic column combination glycated protein, collects the eluent containing MPO；
G, it puts and states eluent into albumen bag filter, dialysed with 150mM NaCl- phosphate buffer, collect albumen
Liquid, packing, puts -80 °C and freezes.
The protein liquid that the present invention freezes measures MPO purity using argentation,
A, it freezes protein liquid centrifuge tube to be put into 37 ° of water-baths, flash melt is put into ice cube；
B, 10ul protein liquid adds 4ul 4X albumen sample adding liquid, and 100 °, 3 minutes, wherein sample liquid was 50 mM Tris, pH
6.8,2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol
blue；
C, plus in 10% SDS-PAGE gel pore of 10ul protein liquid sample liquid, electrophoresis is carried out；
D, after completing electrophoresis, SDS-PAGE gel is obtained, carries out Silver stain, steps are as follows:
1. plus 50% Methanol of 100ml at room temperature, slowly shakes into the container of PAGE gel, 15 minutes,
Remove Methanol liquid；
2. plus 5% Methanol of 100ml at room temperature, slowly shakes into PAGE gel container, 10 minutes, goes
Except Methanol liquid, it is washed with water PAGE gel 1 time；
3. plus in 10ul DTT to SDS-PAGE glue container, have 200ml water, at room temperature, slowly shake, 10 minutes, removal
DTT liquid is washed with water PAGE gel 1 time；
4. plus 0.1% AgNO of 100ml3It into PAGE gel container, at room temperature, slowly shakes, 15 minutes, removes
AgNO3Liquid is washed with water PAGE gel 2 times；
5. plus 3% Na of 100ml2CO30.05% Formaldehyde of & is into PAGE gel container, at room temperature instead
It answers, it is seen that until clear protein band；
6. plus Critic Acid is into PAGE gel container；Terminate reaction；
7. after washing with water SDS-PAGE glue, drying PAGE gel judges MPO purity.
Bradford method measurement purifying MPO concentration of the present invention；
1. taking 1ml concentration dye reagent and 4ml H2O is uniformly mixed, and obtains dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. plus 78ul PBS is into micro titer plate well, adds 2ul various concentration BSA titer, 2ul purifies MPO liquid, often
A sample adds 3 holes；
4. plus 80ul dilution dye reagent, into micro titer plate well, room temperature is placed 5 minutes；
5. putting microtiter plate into Biochemical Analyzer, wavelength 495nm is selected, measures OD value；
6. preparing BSA concentration standard curve, purifying MPO concentration is calculated.
The present invention measures purifying MPO halogenase activity unit:
1. plus 50ul working solution enters in micropore titer plate well；
2. plus 50ul purifying MPO is into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution is mixed into every hole, reacts 5min；
4. 20ul catalase is added into every hole, it is mixed, reacts 5min；
5. 30ul substrate solution is added into every hole, it is mixed；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min。
The present invention measures MPO halogenase activity unit in serum/slurry sample:
1. plus 50ul working solution enters in micropore titer plate well；
2. plus 50ul serum and MPO calibration object are into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution is mixed into every hole, reacts 5min；
4. 20ul Catalase is added into every hole, it is mixed, reacts 5min；
5. 30ul substrate solution is added into every hole, it is mixed；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min。
The present invention establishes NaI-TMB(3,3 ', 5,5 '-tetramethylbenzidine, tetramethyl benzidine) method, directly
MPO halogenase activity in serum/slurry is measured, for predicting cardiovascular and cerebrovascular disease possibility occurrence and disease prognosis.Benefit of the invention
Use H2O2After aoxidizing MPO, halogenation Cl-Further aoxidizing TauNH2 for HOCl, HOCl is TauNHCl, then uses I-Catalyst action,
Quick catalysis TauNHCl aoxidizes TMB, forms oxidized form TMB, is blue product, with 650nm wavelength, directly measurement blue product
OD value, this OD value represent MPO halogenase activity.
The method advantage be it is sensitive, it is quickly, quantitative.Important feature be in blood MPO be in oxidizing ferment and halogenase activity, but
Identical as MPO oxidase active there are many oxidizing ferment in blood, MPO oxidase active is not special；There was only MPO in blood is in halogenation
Enzymatic activity measures halogenase activity in blood and represents MPO halogenase activity, and cardiovascular and cerebrovascular disease is better anticipated in high specificity
Possibility occurrence.Simply, quickly, MPO halogenase activity unit method in quantitative detection human serum/slurry is used for the clinical assessment heart
A possibility that cranial vascular disease occurs.
Detailed description of the invention
Fig. 1 is BSA concentration curve；
Fig. 2 is HOCL concentration standard curve；
Fig. 3 is purifying MPO halogenase activity standard curve；
Fig. 4 is MPO calibration object halogenase activity unit (U) standard curve；
Fig. 5 is NaI-TMB method measurement MPO halogenase activity reaction principle figure；
Fig. 6 is ion chromatography separation MPO albumen.
Specific embodiment
The step of MPO halogenase that the present invention measures is separated from HL-60 cell is:
A, MPO is isolated from HL-60 cell: culture HL-60 cell is collected by centrifugation cell, uses phosphate buffer
(pH8.0) cell to be dissolved, further ruptures cell with Dounce homogenization, cell deposition object is collected in centrifugation,
Contain MPO in cell deposition object；
B, 1% CTAB(Cetyltrimethylammonium bromide) in-phosphate buffer (pH8.0) dissolution
State MPO in cell deposition object；
C, it prepares Q-Sepharose chromatographic column: balancing Q- with 0.5% CTAB- phosphate buffer (pH8.0)
Sepharose chromatographic column, above-mentioned MPO lysate arrive Q- by Q-Sepharose chromatographic column, binding protein (pI≤8.0)
On Sepharose chromatographic column, collect filtered solution (containing MPO, pI=9.2)；
D, it prepares SP-Sepharose chromatographic column: balancing SP- with 0.2% CTAB- phosphate solution (pH8.0)
Sepharose chromatographic column, it is above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, the albumen of pI >=8.0 (including
MPO, pI=9.2) it is integrated on SP-Sepharose chromatographic column, with 600mM K2PO4Phosphate buffer (pH9.5) elution
SP-Sepharose chromatographic column binding protein collects the eluent containing MPO；
E, it puts and states eluent into albumen bag filter, dialysed with 150mM NaCl- phosphate buffer (pH7.5)；
F, it prepares ConA-Sepharose4B chromatographic column: washing ConA- with 100mM NaCl- phosphate solution (pH7.5)
Sepharose4B, then mixed with protein liquid after dialysis, ConA-Sepharose4B combines saccharification MPO, with 500mM NaCl- phosphorus
Phthalate buffer (pH7.5) washs ConA-Sepharose4B chromatographic column, with 600mM a-D-methylmannoside-phosphorus
Phthalate buffer (pH7.5) elutes ConA-Sepharose4B chromatographic column combination glycated protein, collects the eluent containing MPO；
G, it puts and states eluent into albumen bag filter, dialysed with 150mM NaCl- phosphate buffer (pH7.5), received
Collect protein liquid, packing is put -80 °C and frozen.
The protein liquid that the present invention freezes measures MPO purity using argentation,
A, it freezes protein liquid centrifuge tube to be put into 37 ° of water-baths, flash melt is put into ice cube；
B, 10ul protein liquid adds 4ul 4X albumen sample adding liquid, and 100 °, 3 minutes, wherein sample liquid was 50 mM Tris, pH
6.8,2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol
blue；
C, plus in 10% SDS-PAGE gel pore of 10ul protein liquid sample liquid, electrophoresis is carried out；
D, after completing electrophoresis, SDS-PAGE gel is obtained, carries out Silver stain, steps are as follows:
1. plus 50% Methanol of 100ml at room temperature, slowly shakes into the container of PAGE gel, 15 minutes,
Remove Methanol liquid；
2. plus 5% Methanol of 100ml at room temperature, slowly shakes into PAGE gel container, 10 minutes, goes
Except Methanol liquid, it is washed with water PAGE gel 1 time；
3. plus in 10ul DTT to SDS-PAGE glue container, have 200ml water, at room temperature, slowly shake, 10 minutes, removal
DTT liquid is washed with water PAGE gel 1 time；
4. plus 0.1% AgNO of 100ml3It into PAGE gel container, at room temperature, slowly shakes, 15 minutes, removes
AgNO3Liquid is washed with water PAGE gel 2 times；
5. plus 3% Na of 100ml2CO30.05% Formaldehyde of & is into PAGE gel container, at room temperature instead
It answers, it is seen that until clear protein band；
6. plus Critic Acid is into PAGE gel container；Terminate reaction；
7. after washing with water SDS-PAGE glue, drying PAGE gel judges MPO purity.
Bradford method measurement purifying MPO concentration of the present invention；
1. take 1ml concentration dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) with
4ml H2O is uniformly mixed, and obtains dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. plus 78ul PBS is into micro titer plate well, adds 2ul various concentration BSA titer, 2ul purifies MPO liquid, often
A sample adds 3 holes；
4. plus 80ul dilution dye reagent, into micro titer plate well, room temperature is placed 5 minutes；
5. putting microtiter plate into Biochemical Analyzer, wavelength 495nm is selected, measures OD value；
6. preparing BSA concentration standard curve, purifying MPO concentration is calculated.
The present invention measures purifying MPO halogenase activity unit:
1. plus 50ul working solution (20mM NaPO4,pH6.5,200mM NaCl,10mM TauNH2) enter micropore titer plate well
In；
2. plus 50ul(0,50,100,200,400,800ng/ml) MPO is purified into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution (2mM) is mixed into every hole, reacts 5min；
4. 20ul catalase (Catalase buys Sigma) (20ug/ml) is added into every hole, it is mixed, reacts
5min；
5. 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) is added into every hole, mix
?；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min。
The present invention measures MPO halogenase activity unit in serum/slurry sample:
1. plus 50ul working solution (20mM NaPO4,pH6.5,100mM NaCl,5mM TauNH2) enter micropore titer plate well
In；
2. plus 50ul serum and MPO calibration object (0,50,100,200,400,600U) are into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution (2mM) is mixed into every hole, reacts 5min；
4. 20ul Catalase (20ug/ml) is added into every hole, it is mixed, reacts 5min；
5. 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) is added into every hole, mix
?；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min。
The present invention is mainly that neutrophil cell largely generates MPO directly from HL-60 cell extraction MPO, blood of human body,
And HL-60 cell is acute neutrophilic Leukemia Cell Lines, generates a large amount of maturation MPO.The growth of HL-60 cell is fast,
Culture medium requires low, energy mass propgation, and reduction is at low cost.It is important be mature MPO isoelectric point (pI) be 9.2, and be glycosylation
Albumen isolates MPO and provides basis for ion chromatography and glycosylation protein binding method.The present invention separates MPO experimentation and uses
Mild buffer does not change MPO protein structure；With Oxidizing and Reducing Agents buffer is free of, MPO enzymatic activity is not interfered, finally
It is purified into nature protein structure and enzymatic activity MPO, it can be as the calibration of MPO enzymatic activity and concentration in measurement blood preparation
Product.
The invention will be described in further detail below:
The first step of the present invention isolates MPO from HL-60 cell:
1. mass propgation HL-60 cell, is collected by centrifugation cell, cell is dissolved with phosphate buffer (pH8.0), then into
One step Dounce homogenization ruptures cell, and cell deposition object (containing MPO) is collected in centrifugation
2. 1% Cetyltrimethylammonium bromide(CTAB) in-phosphate buffer (pH8.0) dissolution
State MPO in cell deposition object
3. preparing Q-Sepharose chromatographic column, Q- is balanced with 0.5% CTAB- phosphate buffer (pH8.0)
Sepharose chromatographic column, above-mentioned MPO lysate arrive Q- by Q-Sepharose chromatographic column, binding protein (pI≤8.0)
On Sepharose chromatographic column, collect filtered solution (containing MPO, pI=9.2)
4. preparing SP-Sepharose chromatographic column, SP- is balanced with 0.2% CTAB- phosphate solution (pH8.0)
Sepharose chromatographic column, it is above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, the albumen of pI >=8.0 (including
MPO, pI=9.2) it is integrated on SP-Sepharose chromatographic column, with 600mM K2PO4Phosphate buffer (pH9.5) elution
SP-Sepharose chromatographic column binding protein is collected eluent (containing MPO)
Eluent is stated into albumen bag filter 5. putting, and is dialysed with 150mM NaCl- phosphate buffer (pH7.5)
6. preparing ConA-Sepharose4B chromatographic column, washed with 100mM NaCl- phosphate solution (pH7.5)
ConA-Sepharose4B, then mixed with protein liquid after dialysis, ConA-Sepharose4B combines saccharification MPO, uses 500mM
NaCl- phosphate buffer (pH7.5) washs ConA-Sepharose4B chromatographic column, with 600mM a-D-
Methylmannoside-phosphate buffer (pH7.5) elutes ConA-Sepharose4B chromatographic column combination glycated protein,
It collects eluent (containing MPO)
Eluent is stated into albumen bag filter 7. putting, and is dialysed with 150mM NaCl- phosphate buffer (pH7.5),
Protein liquid is collected, packing is put -80 °C and frozen
Second step measurement separation MPO purity of protein of the present invention and concentration:
1. the above-mentioned protein liquid that freezes carries out SDS-PAGE protein electrophoresis, then do argentation, measurement MPO purity reach 95% with
On
2. measuring MPO protein concentration with Bradford method
Third step of the present invention establishes measurement MPO halogenase activity method:
1.NaI-TMB method measures MPO halogenase activity principle: (see figure 5)
Reaction process is H2O2Aoxidize ferroporphyrin group (Fe3 in MPO albumen+), it is converted into oxidized form MPO(Fe4=O+),
This MPO(Fe4=O+) oxidation CL-, HOCl is generated, HOCl further aoxidizes TauNH2, form TauNHCl, I-Catalysis rapidly
TauNHCl aoxidizes TMB, forms oxidized form TMB, blue, there is very strong light absorption value, and with 650nm wavelength, directly measurement blue is produced
Object OD value.
2. establishing measurement purifying MPO halogenase activity NaI-TMB method, purifying MPO and Taurine (TauNH2)-phosphoric acid
Salt buffer mixing, uses H2O2MPO after oxidation converts Cl-Further aoxidizing TauNH2 for HOCl, HOCl is TauNHCl, is added
Catalase (Catalase) decomposes remaining H2O2, after adding NaI-TMB substrate solution, I-Quickly/catalysis TauNHCl oxygen
Change TMB, form oxidized form TMB, switch to blue, with 650nm wavelength, directly measurement blue product OD value, this OD value represents purifying
MPO halogenase activity.
3. establishing MPO halogenase activity unit (U), MPO halogenase activity unit (U) is set as in NaI-TMB of the present invention
Under the conditions of method measures MPO halogenase activity, MPO aoxidizes Cl within 1 minute-, generate equivalent HOCl (umol/ml/min, U).The present invention
It is standard items with pure HOCl solution, prepares 0,40,80,120,160,200umol HOCl solution standards, HOCl oxygen
Change TauNH2, TauNHCl is generated, in I-Under catalysis, TauNHCl aoxidizes MTB, forms blue product, with wavelength 650nm, measures OD
Value.Various concentration HOCl standard items OD value is determined, HOCl standard concentration and OD value affinity criterions curve is drawn out, calculates
The slope of curve (K): such as K=(HOCl 120nmo -80umol)/(120umol OD value -80umolOD value).It further measures pure
Change MPO halogenase activity unit (U).
4. measurement purifying MPO halogenase activity unit (U), 20ul purify MPO(0,50,100,200,400,800ng/
Ml it) is reacted with zymolyte (TauNH2, NaI, TMB), generation oxidized form TMB, reaction blue, the measurement differential responses time (0,
5,10,15min), 650nm wavelength OD value calculates MPO halogenase activity unit: such as 200ng/ml MPO halogen according to the following formula
Change unit of enzyme activity (umol/ml/min, U)=K (200ng/ml MPO OD5min-200ng/ml MPO OD0min)/5min/
0.05ml。
5. using purifying MPO as calibration object, measure for detecting MPO halogenase activity unit, root in serum/slurry samples
According to previous investigation, within the scope of 20-400ng/ml, the present invention selects 0 to 1000ng/ml pure MPO concentration in human blood
Change MPO albumen as calibration object, is converted into corresponding MPO halogenase activity unit (U).With NaI-TMB method measurement MPO calibration object and
In serum/slurry after MPO halogenase activity, MPO calibration object halogenase activity unit (U) standard curve is prepared, for calculating inspection
Survey MPO halogenase activity unit (U) in serum/slurry samples.
Experimentation of the present invention:
One, myeloperoxidase (MPO) is separated from HL-60 cell
1. contain 18% FBS with RPMI-1640() HL-60 cell is cultivated in 150mm Tissue Culture Dish
2. HL-60 cell is collected into 250ml centrifuge tube, 4 °C, centrifugation, 3000rpm, 10 minutes
3. removing supernatant, add 150ml PBS into centrifuge tube, is mixed HL-60 cell, 4 °C, is centrifuged, 3000rpm, 10
Minute, supernatant is removed, HL-60 cell is collected
4. adding 30ml Buffer A (10mM NaPO4, pH8.0,1.0mM MgCl2, 1.0mM CaCl2,0.5mM
PMSF, 10ug/ml leupetine, 10ug/ml Aprotinin) to having in HL-60 cell centrifuge tube, pressure-vaccum is mixed,
It is placed in ice cube 5 minutes
5. rupturing cell with Dounce homogenization
6. centrifugation, 20000g 10 minutes, removes supernatant under the conditions of 4 °C
7. adding in 30ml Buffer A to HL-60 cell precipitate, add Cetyltrimethylammonium bromide
(CTAB, buy Sigma) into BufferA- cell precipitation mixed liquor, concentration to 1%；Add NaCl concentration to 100mM, in 4 °C of items
Under part, shake 2 hours
8. centrifugation, 20000g 30 minutes, collects supernatant, into new 50ml centrifuge tube under the conditions of 4 °C
9. Q-sepharose chromatographic column (purchase GE Healthcare Life Sciences) is prepared, with Buffer B
(20mM NaPO4, pH8.0,100mM NaCl, 0.5% CTAB) and balance Q-Sepharose chromatographic column.
10. above-mentioned 30ml supernatant, is added in Q-Sepharose chromatographic column, by Q-Sepharose chromatographic column, receive
Collect filtered solution, be then added in Q-Sepharose chromatographic column, after quadruplication, collects filtered solution
11. preparing SP-Sepharose chromatographic column (purchase GE Healthcare Life Sciences), Buffer is used
C(50mM K2PO4, pH8.0,100mM NaCl, 0.2% CTAB) and balance SP-Sepharose chromatographic column.
12. above-mentioned filtered solution collects filtered solution, is then added to SP-Sepharose by SP-Sepharose chromatographic column
In chromatographic column, quadruplication
13. adding in 100ml Buffer C to SP-Sepharose chromatographic column, Buffer C slow transits through SP-
Sepharose chromatographic column
14. adding 10ml eluent (0.6M K2PO respectively4, pH9.5,200mM NaCl, 0.2% CTAB) and arrive SP-
In Sepharose chromatographic column, eluent slow transits through SP-Sepharose chromatographic column, collects 10ml eluent
15. an eluent is transferred in bag filter (Spectra/Pro), dialyzate (20mM NaPO is placed into4,
PH7.5,100mM NaCl, 5% Glycerol, 1mM DTT, 1mM EDTA, 1mM PMSF) in, under the conditions of 4-8 °C,
Slowly agitation, 6 hours
16. protein liquid of dialysing in bag filter is moved on in centrifuge tube, under the conditions of 4 °C, centrifugation, 20000g, 10 minutes
17. take 1.5ml ConA-Sepharose4B(to buy GE Healthcare Life Sciences) it is put into 15ml
In centrifuge tube
18. adding 10ml cleaning solution (20mM NaPO4, pH7.5, 100mM NaCl, 5% Glycerol,0.01%
NP-40,1mM EDTA, 1mM DTT, 1mM PMSF, 10ug/ml leupetine, 10ug/ml Aprotinin) it arrives
In ConA Sepharose4B centrifuge tube
19. centrifugation, 3000g 2 minutes, are sucked out supernatant, wash repeatedly 2 times
20. adding 15ml dialysis protein liquid into ConA-Sepharose4B centrifuge tube
21. under the conditions of 4 °C, centrifuge tube is slowly shaken, 2.0 hours
22. under the conditions of 4 °C, ConA-Sepharose4B to Poly-prep column (0.8 X after transfer reaction
4cm Pharmacia Biotech, 731-1550), form ConA-Sepharose4B chromatographic column
23. adding 20ml cleaning solution (20mM NaPO4, pH7.5, 500mM NaCl, 5% Glycerol,0.01% NP-
40,1mM EDTA, 1mM DTT, 1mM PMSF, 10ug/ml leupetine, 10ug/ml Aprotinin) arrive ConA-
In Sepharose 4B chromatographic column, cleaning solution slow transits through ConA-Sepharose 4B- chromatographic column
24. adding 2.0ml eluent (20mM NaPO4,pH7.5 150mM NaCl, 600mM a-D-
Methylmannoside) into ConA-Sepharose 4B- chromatographic column, eluent slow transits through chromatographic column, collects eluent
25. an eluent is transferred in bag filter (Spectra/Pro), bag filter is put in dialyzate (20mM
NaHPO4, pH7.5,150mM NaCl, 10% Glycerol, 1mM DTT, 1mM EDTA, 1mM PMSF) in, at 4 °C
Under the conditions of, it slowly stirs, overnight
26. protein liquid in bag filter is moved on in microcentrifugal tube, under the conditions of 4 °C, centrifugation, 13000rpm, 10 points
Clock,
Protein liquid is dispensed into different microcentrifugal tubes, -80 °C of long-term preservations.
Two, MPO purity of protein and concentration are isolated in measurement
(1) argentation measures MPO purity
1. freezing protein liquid centrifuge tube to be put into 37 °C of water-baths, flash melt is put into ice cube,
2.10ul protein liquid adds 4ul 4X albumen sample adding liquid (50 mM Tris, pH 6.8,2% SDS, 10%
Glycerol, 1% β-mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol blue), 100 °C 3
Minute
3. adding in 10% SDS-PAGE gel pore of 10ul protein liquid sample liquid, electrophoresis is carried out
4. after completing electrophoresis, obtaining SDS-PAGE gel, Silver stain is carried out, steps are as follows:
A. plus 50% Methanol of 100ml is into the container of PAGE gel, at room temperature, slowly shakes, 15 points
Clock,
Remove Methanol liquid
B. plus 5% Methanol of 100ml is into PAGE gel container, at room temperature, slowly shakes, 10 minutes, goes
Except Methanol liquid, it is washed with water PAGE gel 1 time
C. plus in 10ul DTT to SDS-PAGE glue container, there is 200ml water, at room temperature, slowly shake, 10 minutes, removal
DTT liquid is washed with water PAGE gel 1 time
Plus 0.1% AgNO of 100ml d.3It into PAGE gel container, at room temperature, slowly shakes, 15 minutes, goes
Except AgNO3Liquid is washed with water PAGE gel 2 times
Plus 3% Na of 100ml e.2CO30.05% Formaldehyde of & is into PAGE gel container, at room temperature instead
It answers, it is seen that until clear protein band
F. plus Critic Acid is into PAGE gel container, terminates reaction
After washing with water SDS-PAGE glue, drying PAGE gel judges MPO purity.
(2) Bradford method measurement purifying MPO concentration
1. take 1ml concentration dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) with
4ml H2O
It is uniformly mixed, obtains dilution dye reagent
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml
3. adding 78ul PBS into micro titer plate well, adding 2ul various concentration BSA titer, 2ul purifies MPO liquid,
Each sample adds 3 holes
4. adding 80ul dilution dye reagent into micro titer plate well, room temperature is placed 5 minutes
5. putting microtiter plate into Biochemical Analyzer, wavelength 495nm is selected, measures OD value
6. preparing BSA concentration standard curve, purifying MPO concentration is calculated
Purifying MPO OD average value is 1.212, MPO concentration=1.6498 x, 1.212-0.6059=1.393mg/ml
。
Three, establish HOCl concentration standard curve
1. add 50ul HOCl(to buy NovaBay Pharmaceuticals, Inc), concentration 0,40,80,120,
160,200uM and 40ul PBS is into microwell plate corresponding aperture
2. adding 50ul working solution (20mM NaPO4,pH6.5,200mM NaCl,10mM Taurine(TauNH2) enter it is each
Kong Zhong reacts 5min
3. 30ul substrate solution (2mM TMB, 150um NaI, 400mM Acetate, 10% DMF) is added into every hole,
It is mixed, reacts 5min
4. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures OD value
5. drawing out HOCl concentration and OD value affinity criterions curve, the slope of curve (K) is calculated: such as K=(HOCl
120uM-80uM)/(OD120uM-OD180uM)=120-80/0.638667-0.3906=161
。
Four, measurement purifying MPO halogenase activity unit (U)
1. adding 50ul working solution (20mM NaPO4,pH6.5,200mM NaCl,10mM TauNH2) enter micropore titer plate well
In
2. add 50ul(0,50,100,200,400,800ng/ml) MPO is purified into corresponding microtiter plate hole
3. adding 20ul H again2O2Solution (2mM) is mixed into every hole, reacts 5min
4. 20ul catalase (Catalase buys Sigma) (20ug/ml) is added into every hole, it is mixed, reacts
5min
5. 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) is added into every hole,
It is mixed
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value
8. calculating OD value=OD5min–OD0min
MPO concentration and OD value affinity criterions curve are drawn out, MPO halogenase activity unit (U) is calculated.Such as 200ng/
MlMPO halogenase activity unit (U)=K(200ng/ml OD5min – 200ng/ml OD0min)/5min/0.05ml=161
(0.321667-0.135667)/5/0.05=120uM/ml/min
。
Five, measure MPO halogenase activity unit (U) in serum/slurry sample
1. adding 50ul working solution (20mM NaPO4,pH6.5,100mM NaCl,5mM TauNH2) enter in micropore titer plate well
2. adding 50ul serum and MPO calibration object (0,50,100,200,400,600U) into corresponding microtiter plate hole
2. adding 20ul H again2O2Solution (2mM) is mixed into every hole, reacts 5min
3. 20ul Catalase (20ug/ml) is added into every hole, it is mixed, reacts 5min
4. 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) is added into every hole,
It is mixed
5. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value
6. after sustained response 5min, with 650nm wavelength, measuring 5min OD value
7. calculating OD value=OD5min–OD0min
8. MPO calibration object halogenase activity unit (U) and OD value affinity criterions curve are drawn out, with this standard curve meter
/ slurry MPO halogenase activity unit (U) is calculated in serum
Citing: 1 MPO(U of serum)=910.38 X, 0.208-45.686=143.67 U
。
Claims (3)
1. a kind of method for measuring MPO halogenase activity, it is characterised in that: the MPO halogenase of measurement is separated from HL-60 cell
The step of be:
A, MPO is isolated from HL-60 cell: cell is collected by centrifugation in culture HL-60 cell, is dissolved with phosphate buffer thin
Born of the same parents further rupture cell with Dounce homogenization, are centrifuged, collection cell deposition object, in cell deposition object
Containing MPO；
B, 1% CTAB- phosphate buffer dissolves MPO in above-mentioned cell deposition object；
C, it prepares Q-Sepharose chromatographic column: balancing Q-Sepharose chromatographic column with 0.5% CTAB- phosphate buffer,
The above-mentioned cell deposition object containing MPO through the dissolution of 1% CTAB- phosphate buffer passes through Q-Sepharose chromatographic column, in conjunction with
On albumen to Q-Sepharose chromatographic column, filtered solution is collected, filtered solution contains the MPO of pI=9.2；
D, it prepares SP-Sepharose chromatographic column: balancing SP-Sepharose chromatographic column with 0.2% CTAB- phosphate solution,
The above-mentioned MPO filtered solution containing pI=9.2 passes through SP-Sepharose chromatographic column, the protein binding of pI >=8.0 to SP-
On Sepharose chromatographic column, with 600mM K2PO4Phosphate buffer elutes SP-Sepharose chromatographic column binding protein,
Collect the eluent containing MPO；
E, the eluent A of above-mentioned steps d acquisition is put into albumen bag filter, is dialysed with 150mM NaCl- phosphate buffer；
F, it prepares ConA-Sepharose4B chromatographic column: washing ConA- with 100mM NaCl- phosphate solution
Sepharose4B, then mixed with protein liquid after dialysis, ConA-Sepharose4B combines saccharification MPO, with 500mM NaCl- phosphorus
Phthalate buffer washs ConA-Sepharose4B chromatographic column, with 600mM a-D-methylmannoside-phosphate-buffered
Liquid elutes ConA-Sepharose4B chromatographic column combination glycated protein, collects the eluent containing MPO；
G, the eluent B of above-mentioned steps f acquisition is put into albumen bag filter, is dialysed with 150mM NaCl- phosphate buffer,
Protein liquid is collected, packing is put -80 °C and frozen；
Wherein measurement purifies MPO halogenase activity unit:
1. plus 50ul working solution enters in micropore titer plate well；Wherein working solution is by NaPO4,pH6.5, NaCl, TauNH2Group
At；
2. plus 50ul purifying MPO is into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution is mixed into every hole, reacts 5min；
4. 20ul catalase is added into every hole, it is mixed, reacts 5min；
5. 30ulNaI-TMB substrate solution is added into every hole, it is mixed；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min；
Wherein measure MPO halogenase activity unit in serum/slurry sample:
1. plus 50ul working solution enters in micropore titer plate well；Wherein working solution is by NaPO4,pH6.5, NaCl, TauNH2Group
At；
2. plus 50ul serum and MPO calibration object are into corresponding microtiter plate hole；
3. adding 20ul H again2O2Solution is mixed into every hole, reacts 5min；
4. 20ul Catalase is added into every hole, it is mixed, reacts 5min；
5. 30ulNaI-TMB substrate solution is added into every hole, it is mixed；
6. putting microwell plate into Biochemical Analyzer, 650nm wavelength is selected, measures 0min OD value；
7. after sustained response 5min, with 650nm wavelength, measuring 5min OD value；
8. calculating OD value=OD5min–OD0min。
2. measuring the method for MPO halogenase activity according to claim 1, it is characterised in that: the protein liquid frozen is using silver
Dye method measures MPO purity,
A, it freezes protein liquid centrifuge tube to be put into 37 ° of water-baths, flash melt is put into ice cube；
B, 10ul protein liquid adds 4ul 4X albumen sample adding liquid, and 100 °, 3 minutes, wherein sample liquid was 50 mM Tris, pH 6.8,
2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol blue；
C, plus in 10% SDS-PAGE gel pore of 10ul protein liquid sample liquid, electrophoresis is carried out；
D, after completing electrophoresis, SDS-PAGE gel is obtained, carries out Silver stain, steps are as follows:
1. plus 50% Methanol of 100ml at room temperature, slowly shakes into the container of PAGE gel, 15 minutes, removes
Methanol liquid；
2. plus 5% Methanol of 100ml at room temperature, slowly shakes into PAGE gel container, 10 minutes, removes
Methanol liquid is washed with water PAGE gel 1 time；
3. plus in 10ul DTT to SDS-PAGE glue container, have 200ml water, at room temperature, slowly shake, 10 minutes, remove DTT
Liquid is washed with water PAGE gel 1 time；
4. plus 0.1% AgNO of 100ml3It into PAGE gel container, at room temperature, slowly shakes, 15 minutes, removes AgNO3
Liquid is washed with water PAGE gel 2 times；
5. plus 3% Na of 100ml2CO30.05% Formaldehyde of & reacts at room temperature into PAGE gel container,
Until seeing clear protein band；
6. plus Critic Acid is into PAGE gel container；Terminate reaction；
7. after washing with water SDS-PAGE glue, drying PAGE gel judges MPO purity.
3. measuring the method for MPO halogenase activity according to claim 1, it is characterised in that: the measurement purifying of Bradford method
MPO concentration；
1. taking 1ml concentration dye reagent and 4ml H2O is uniformly mixed, and obtains dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. plus 78ul PBS adds 2ul various concentration BSA titer, 2ul purifying MPO liquid, each sample into micro titer plate well
This adds 3 holes；
4. plus 80ul dilution dye reagent, into micro titer plate well, room temperature is placed 5 minutes；
5. putting microtiter plate into Biochemical Analyzer, wavelength 495nm is selected, measures OD value；
6. preparing BSA concentration standard curve, purifying MPO concentration is calculated.
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| CN104950111A (en) * | 2015-05-22 | 2015-09-30 | 北京协和洛克生物技术有限责任公司 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
| CN105606595A (en) * | 2016-01-12 | 2016-05-25 | 杭州检康生物技术有限公司 | Chemiluminescence immunoassay method for quantitative detection of myeloperoxidase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104950111A (en) * | 2015-05-22 | 2015-09-30 | 北京协和洛克生物技术有限责任公司 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
| CN105606595A (en) * | 2016-01-12 | 2016-05-25 | 杭州检康生物技术有限公司 | Chemiluminescence immunoassay method for quantitative detection of myeloperoxidase |
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