CN106290190A - The method measuring MPO halogenase activity - Google Patents
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- CN106290190A CN106290190A CN201610636169.5A CN201610636169A CN106290190A CN 106290190 A CN106290190 A CN 106290190A CN 201610636169 A CN201610636169 A CN 201610636169A CN 106290190 A CN106290190 A CN 106290190A
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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
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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
- G01N1/30—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
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- 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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- 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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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of method measuring MPO halogenase activity, it is an object of the invention to isolate from HL 60 cell with ion chromatography that there is nature protein structure and enzymatic activity MPO, set up and measure MPO halogenase activity method, with purification MPO as calibration object, measure MPO halogenase activity unit in human serum/slurry.The MPO chlB4 that the present invention measures separates from HL 60 cell.The present invention sets up NaI TMB(3,3 ', 5,5 ' tetramethylbenzidine, tetramethyl benzidine) method, directly measure MPO halogenase activity in serum/slurry, be used for predicting cardiovascular and cerebrovascular disease possibility occurrence and disease prognosis.
Description
Technical field
The invention belongs to biological technical field, particularly to natural protease purification method.
Background technology
Myeloperoxidase (MPO) (Myeloperoxidase, MPO), also known as peroxidase, is thin by neutrophilic granulocyte, monokaryon
The glycosylation of the macrophages secrete of born of the same parents and some tissue the hemoprotein enzyme containing prosthetic heme group, be haemachrome peroxidating
One of thing enzyme superfamily member.Before human body granulocyte enters circulation, in bone marrow, close MPO become and be stored in azurophilic granule
In, environmental stimuli may result in neutrophil accumulation, discharges MPO.At ripe granulocyte (mainly neutrophilic granulocyte and monokaryon
Cell) in, MPO is the glycoprotein that content is the abundantest, accounts for peripheral blood M7 (PMNs) interior gross protein and contains
The 5% of amount, in blood, the MPO of 95% derives from PMNs
Myeloperoxidase (MPO) (MPO) is by the dimer of 2 subunit polymerization, 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) constituted.2 subunits at α chain by 1
Individual disulfide bond is connected.Heavy chain has ferroporphyrin group, shows that MPO is ferrum dependency oxide enzyme.
MPO gene is positioned at chromosome 17q23 q24, containing 12 exons and 11 introns, is about 14638 bp, adjusts
Control its gene expression is somatomedin.The mRNA of MPO is the highest at the expression of promyelocyte, next to that original grain is thin
Born of the same parents, naivety and monoblast；When cell breaks up mature period, and MPO gene expression dose declines rapidly.Know MPO
Gene first express be a relative molecular mass be 84KDa precursor MPO albumen, through post translational processing, cut into α and β two
Planting subunit, repolymerization is ripe MPO molecule, adds sugar chain, eventually forms glycosylation, has enzymatic activity MPO.MPO mono-is important
Feature is that isoelectric point, IP (pI) is the highest, is 9.2.Hydrogen peroxide (H2O2) oxidation MPO after, become oxidized form MPO, have oxidase 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 killing microorganisms in phagocyte, utilizes H2O2With chloride ion (Cl-) produce hypochlorous acid
Salt (HOCl), and form the free radical with oxidability, constitute MPO H2O2Halogen system.Many researchs find, MPO is not only
Can kill and swallow in intracellular microorganism, and releasably arrive extracellular, destroy target substances of multiple types, as little in tumor cell, blood
Plate, NK cell etc., play a role to many-sides such as body generation and regulation inflammatory reactions.But, under given conditions, MPO urges
Change reaction and generate the oxidant (HOCl etc.) of excess, when exceeding the defense reaction of topical antioxidant, may result in oxidisability group
Knit damage, as Ink vessel transfusing cortical sites inflammatory reaction and blood vessel endothelium confluent monolayer cells damage and dead.
Atherosclerosis is to form the important pathologic basis of cardiovascular and cerebrovascular disease.Research shows, atherosclerosis is
A kind of chronic inflammatory reaction disease, and MPO is as inflammatory factor, atherosclerotic plaque formed initiate, develop, rupture and
Play an important role during coming off etc..
Research finds, MPO in blood, combining with vascular endothelial cell, invades profit and arrives blood vessel endothelium hypobasal, by MPO enzyme oxygen
Change reaction, directly consume endotheliocyte and produce signaling molecule NO, reduce and activate guanyl activating enzymes function, thus cause Ink vessel transfusing
Skin diastolic dysfunction.
MPO produces diffusible oxidant (HOCl) in vivo, promotes oxidation LDL(oxLDL) produce, oxLDL be more easy to by
Macrophage phagocytic, is converted into foam cell, is deposited under tunica intima, thus promotes that atheromatous plaque is formed；
HOCl can activate matrix metalloproteinase former (MMPs), makes stromatin Polyose degradation, causes atheromatous plaque fiber
Cap is thinning, and plaque surface is corroded, and ruptures the formation with thrombosis, causes atheromatous plaque unstable.HOCl can induce blood
Endothelial cell produces P and selects element and tissue factor expression, promotes platelet aggregation, thus causes the formation of thrombosis.
Visible, the MPO early stage to Dysfunction of vascular endothlial cells, to the formation of atherosclerotic plaque, and speckle subsequently
The whole process with the formation of thrombosis that ruptures has played key effect, ultimately results in coronary vasospasm, angina pectoris, even cardiac muscle stalk
Dead generation.
Research finds, in blood, MPO is as a kind of Inflammation Marker, measures MPO enzymatic activity or concentration in blood；(1) energy
The early diagnosis of acute coronary syndrome (CVD) can be used for as atherosclerotic plaque unstability early prediction index (1), comment
Estimate the CVD state of an illness and prognosis: (3) can be used for predicting the danger of healthy population generation cardiovascular and cerebrovascular disease.
It addition, the generation of blood of human body middle and high concentration MPO disease multiple with the mankind, develop closely related, neuroinflamation disease
Sick aspect includes: multiple sclerosis, Alzheimer's disease, Parkinson's disease etc..Other inflammatory diseases is such as: chronic obstructive
Pneumonopathy, cystic fibrosis, glomerular injury and rheumatoid arthritis etc..In mensuration blood, MPO enzymatic activity or concentration can be to these
The prevention of a little diseases, diagnosis, and treatment have very great help.
In Clinical Laboratory, in detection blood, MPO concentration method is mainly euzymelinked immunosorbent assay (ELISA) (ELISA) and directly measures blood
Liquid MPO enzyme activity method, two assay methods are required for purification MPO as calibration object, and this calibration object MPO to be had nature albumen is tied
Structure and enzymatic activity.First MPO gene gives expression to a molecular mass is 84KDa precursor MPO albumen, through post translational processing, cuts
Being slit into two kinds of subunits of α and β, repolymerization is ripe MPO molecule, and further glycosylation has eventually formed enzymatic activity saccharifying MPO.
Obtaining purification MPO method one is to isolate leukocyte from blood of human body, then extracts nature from these cells in a large number
MPO.This method shortcoming, one is that blood sources is restricted, and two is to have the multi-infection factor in blood, prepares MPO calibration object
Dangerous.Other method is to express MPO in human body cell with gene engineering research, but after expressing, MPO can not carry out effective albumen
Processing, it is impossible to carry out correct glycosylation, restructuring MPO albumen has the most weak enzymatic activity, should not be as MPO calibration object.
At present, having detected the test kit of MPO in blood, mainly used ELISA, this method is simple, easily operates,
It is used widely.But this method measures the content of MPO in blood, indirectly shows MPO enzymatic activity in blood, sometimes MPO
Content is uncorrelated with MPO enzymatic activity, as there being the crowd of MPO mortifier in MPO genovariation crowd and blood；It addition, preparation
Measuring MPO ELISA test kit and need several MPO monoclonal antibodies, preparation cost is high, and the time is long；Meanwhile, ELISA method step
Many, require time for the shortcomings such as longer, poor accuracy.And arteriosclerosis activity situation and MPO halogenase activity dependency in blood
Maximum, measures in blood MPO halogenase activity and can represent arteriosclerosis activity situation.
Summary of the invention
It is an object of the invention to isolate from HL-60 cell with ion chromatography that there is nature protein structure and enzymatic activity
MPO, sets up and measures MPO halogenase activity method, with purification MPO as calibration object, measures MPO chlB4 in human serum/slurry and lives
Property unit.
The step that the MPO chlB4 that the present invention measures separates from HL-60 cell is:
A, from HL-60 cell, isolate MPO: cultivate HL-60 cell, centrifugal collecting cell, dissolve with phosphate buffer thin
Born of the same parents, further rupture cell with Dounce homogenization, centrifugal, collect cell deposition thing, in cell deposition thing
Containing MPO；
B, 1% CTAB-phosphate buffer dissolve MPO in above-mentioned cell deposition thing；
C, preparation Q-Sepharose chromatographic column: balance Q-Sepharose chromatographic column with 0.5% CTAB-phosphate buffer,
Above-mentioned MPO lysate passes through Q-Sepharose chromatographic column, on associated proteins to Q-Sepharose chromatographic column, collects and filters
Liquid, filtered solution contains the MPO of pI=9.2；
D, preparation SP-Sepharose chromatographic column: balance SP-Sepharose chromatographic column with 0.2% CTAB-phosphate solution,
Above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, pI >=8.0 protein binding is to SP-Sepharose chromatographic column
On, use 600mM K2PO4-phosphate buffer eluting SP-Sepharose chromatographic column associated proteins, collects the eluting containing MPO
Liquid；
E, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer；
F, preparation ConA-Sepharose4B chromatographic column: wash ConA-with 100mM NaCl-phosphate solution
Sepharose4B, then mix with protein liquid after dialysis, ConA-Sepharose4B combines saccharifying MPO, with 500mM NaCl-phosphorus
Phthalate buffer washing ConA-Sepharose4B chromatographic column, by 600mM a-D-methylmannoside-phosphate-buffered
Liquid eluting ConA-Sepharose4B chromatographic column combines glycated protein, collects the eluent containing MPO；
G, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer, collect protein liquid, point
Dress, puts-80 ° of C frozen.
The protein liquid employing argentation mensuration MPO purity that the present invention is frozen,
A, frozen protein liquid centrifuge tube are put in 37 ° of water-baths, and flash melt is put in 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, add in 10ul protein liquid sample liquid 10% SDS-PAGE gel pore, carry out electrophoresis；
D, complete electrophoresis after, obtain SDS-PAGE gel, carry out Silver stain, step is as follows:
1. add 100ml 50% Methanol in the container of PAGE gel, under room temperature, slowly shake, 15 minutes, remove
Methanol liquid；
2. add 100ml 5% Methanol in PAGE gel container, under room temperature, slowly shake, 10 minutes, remove
Methanol liquid, washes PAGE gel with water 1 time；
3. add in 10ul DTT to SDS-PAGE glue container, have 200ml water, under room temperature, slowly shake, 10 minutes, remove DTT
Liquid, washes PAGE gel with water 1 time；
4. 100ml 0.1% AgNO is added3In PAGE gel container, under room temperature, slowly shake, 15 minutes, remove
AgNO3Liquid, washes PAGE gel with water 2 times；
5. 100ml 3% Na is added2CO3& 0.05% Formaldehyde, in PAGE gel container, reacts under room temperature,
Till seeing clear protein band；
6. Critic Acid is added in PAGE gel container；Terminate reaction；
7. after cleaning SDS-PAGE glue with water, drying PAGE gel, it is judged that MPO purity.
Bradford method of the present invention measures purification MPO concentration；
1. take 1ml and concentrate dye reagent and 4ml H2O mix homogeneously, it is thus achieved that dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. add 78ul PBS in micro titer plate well, add 2ul variable concentrations BSA titer, 2ul purification MPO liquid, each sample
Originally 3 holes are added；
4. 80ul dilution dye reagent is added in micro titer plate well, room temperature, place 5 minutes；
5. put microtitration plate in biochemistry analyzer, select wavelength 495nm, measure OD value；
6. prepare BSA concentration standard curve, calculate purification MPO concentration.
The present invention measures purification MPO halogenase activity unit:
1. add 50ul working solution to enter in micropore titer plate well；
2. 50ul purification MPO is added in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution, in every hole, is mixed, and reacts 5min；
4. add 20ul catalase in every hole, be mixed, react 5min；
5. add 30ul substrate solution in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
The present invention measures MPO halogenase activity unit in serum/slurry specimen:
1. add 50ul working solution to enter in micropore titer plate well；
2. 50ul serum and MPO calibration object are added in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution, in every hole, is mixed, and reacts 5min；
4. add 20ul Catalase in every hole, be mixed, react 5min；
5. add 30ul substrate solution in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
The present invention sets up NaI-TMB(3,3 ', 5,5 '-tetramethylbenzidine, tetramethyl benzidine) method, directly
Measure MPO halogenase activity in serum/slurry, be used for predicting cardiovascular and cerebrovascular disease possibility occurrence and disease prognosis.Present invention profit
Use H2O2After oxidation MPO, halogenation Cl-Aoxidizing TauNH2 further for HOCl, HOCl is TauNHCl, then uses I-Catalyst action,
Quick catalysis TauNHCl aoxidizes TMB, forms oxidized form TMB, for blue product, uses 650nm wavelength, directly measure blue product
OD value, this OD value represents MPO halogenase activity.
The method advantage is sensitive, quickly, quantitatively.Key character is that in blood, MPO is oxidase and halogenase activity, but
Having multiple oxidase identical with MPO oxidase active in blood, MPO oxidase active is the most special；In blood, only MPO is halogenation
Enzymatic activity, measures halogenase activity in blood and represents MPO halogenase activity, high specificity, cardiovascular and cerebrovascular disease is better anticipated
Possibility occurrence.Simply, quickly, MPO halogenase activity unit method in detection by quantitative human serum/slurry, for the clinical assessment heart
The probability that cerebrovascular disease occurs.
Accompanying drawing explanation
Fig. 1 is BSA concentration curve；
Fig. 2 is HOCL concentration standard curve；
Fig. 3 is purification MPO halogenase activity standard curve；
Fig. 4 is MPO calibration object halogenase activity unit (U) standard curve；
Fig. 5 is that NaI-TMB method measures MPO halogenase activity reaction principle figure；
Fig. 6 is that ion chromatography separates MPO albumen.
Detailed description of the invention
The step that the MPO chlB4 that the present invention measures separates from HL-60 cell is:
A, from HL-60 cell, isolate MPO: cultivate HL-60 cell, centrifugal collecting cell, use phosphate buffer
(pH8.0) dissolve cell, further rupture cell with Dounce homogenization, centrifugal, collect cell deposition thing,
Containing MPO in cell deposition thing；
B, 1% CTAB(Cetyltrimethylammonium bromide)-phosphate buffer (pH8.0) dissolve above-mentioned carefully
MPO in born of the same parents' deposit；
C, preparation Q-Sepharose chromatographic column: with 0.5% CTAB-phosphate buffer (pH8.0) balance Q-Sepharose
Chromatographic column, above-mentioned MPO lysate passes through Q-Sepharose chromatographic column, and associated proteins (pI≤8.0) arrives Q-Sepharose layer
On analysis post, collect filtered solution (containing MPO, pI=9.2)；
D, preparation SP-Sepharose chromatographic column: with 0.2% CTAB-phosphate solution (pH8.0) balance SP-Sepharose
Chromatographic column, above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, pI >=8.0 albumen (including MPO, pI=9.2) is tied
Close on SP-Sepharose chromatographic column, use 600mM K2PO4-phosphate buffer (pH9.5) eluting SP-Sepharose layer
Analysis post associated proteins, collects the eluent containing MPO；
E, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer (pH7.5)；
F, preparation ConA-Sepharose4B chromatographic column: with 100mM NaCl-phosphate solution (pH7.5) washing ConA-
Sepharose4B, then mix with protein liquid after dialysis, ConA-Sepharose4B combines saccharifying MPO, with 500mM NaCl-phosphorus
Phthalate buffer (pH7.5) washing ConA-Sepharose4B chromatographic column, with 600mM a-D-methylmannoside-phosphorus
Phthalate buffer (pH7.5) eluting ConA-Sepharose4B chromatographic column combines glycated protein, collects the eluent containing MPO；
G, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer (pH7.5), collect egg
White liquor, subpackage, put-80 ° of C frozen.
The protein liquid employing argentation mensuration MPO purity that the present invention is frozen,
A, frozen protein liquid centrifuge tube are put in 37 ° of water-baths, and flash melt is put in 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, add in 10ul protein liquid sample liquid 10% SDS-PAGE gel pore, carry out electrophoresis；
D, complete electrophoresis after, obtain SDS-PAGE gel, carry out Silver stain, step is as follows:
1. add 100ml 50% Methanol in the container of PAGE gel, under room temperature, slowly shake, 15 minutes, remove
Methanol liquid；
2. add 100ml 5% Methanol in PAGE gel container, under room temperature, slowly shake, 10 minutes, remove
Methanol liquid, washes PAGE gel with water 1 time；
3. add in 10ul DTT to SDS-PAGE glue container, have 200ml water, under room temperature, slowly shake, 10 minutes, remove DTT
Liquid, washes PAGE gel with water 1 time；
4. 100ml 0.1% AgNO is added3In PAGE gel container, under room temperature, slowly shake, 15 minutes, remove
AgNO3Liquid, washes PAGE gel with water 2 times；
5. 100ml 3% Na is added2CO3& 0.05% Formaldehyde, in PAGE gel container, reacts under room temperature,
Till seeing clear protein band；
6. Critic Acid is added in PAGE gel container；Terminate reaction；
7. after cleaning SDS-PAGE glue with water, drying PAGE gel, it is judged that MPO purity.
Bradford method of the present invention measures purification MPO concentration；
1. take 1ml and concentrate dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) and 4ml
H2O mix homogeneously, it is thus achieved that dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. add 78ul PBS in micro titer plate well, add 2ul variable concentrations BSA titer, 2ul purification MPO liquid, each sample
Originally 3 holes are added；
4. 80ul dilution dye reagent is added in micro titer plate well, room temperature, place 5 minutes；
5. put microtitration plate in biochemistry analyzer, select wavelength 495nm, measure OD value；
6. prepare BSA concentration standard curve, calculate purification MPO concentration.
The present invention measures purification MPO halogenase activity unit:
1. 50ul working solution (20mM NaPO is added4,pH6.5,200mM NaCl,10mM TauNH2) enter in micropore titer plate well；
2. 50ul(0,50,100,200,400,800ng/ml is added) purification MPO is in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution (2mM), in every hole, is mixed, and reacts 5min；
4. add 20ul catalase (Catalase, buy Sigma) (20ug/ml) in every hole, be mixed, reaction
5min；
5. add 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
The present invention measures MPO halogenase activity unit in serum/slurry specimen:
1. 50ul working solution (20mM NaPO is added4,pH6.5,100mM NaCl,5mM TauNH2) enter in micropore titer plate well；
2. 50ul serum and MPO calibration object (0,50,100,200,400,600U) are added in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution (2mM), in every hole, is mixed, and reacts 5min；
4. add 20ul Catalase (20ug/ml) in every hole, be mixed, react 5min；
5. add 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
The present invention is directly from HL-60 cell extraction MPO, and in blood of human body, mainly neutrophil cell produces MPO in a large number,
And HL-60 cell is acute neutrophilic Leukemia Cell Lines, produce a large amount of ripe MPO.The growth of HL-60 cell is fast,
Culture medium requires low, energy mass propgation, reduces low cost.Important be ripe MPO isoelectric point, IP (pI) be 9.2, and be glycosylation
Albumen, isolates MPO for ion chromatography and glycosylation protein binding method and provides basis.The present invention separates MPO experimentation and uses
Gentle buffer, does not change MPO protein structure；With without Oxidizing and Reducing Agents buffer, do not disturb MPO enzymatic activity, finally
It is purified into and there is nature protein structure and enzymatic activity MPO, can be as measuring MPO enzymatic activity and the calibration of concentration in blood preparation
Product.
Hereinafter the present invention is described in further detail:
The first step of the present invention isolates MPO from HL-60 cell:
1. mass propgation HL-60 cell, centrifugal collecting cell, dissolves cell with phosphate buffer (pH8.0), further
Cell is ruptured with Dounce homogenization, centrifugal, collect cell deposition thing (containing MPO)
2. 1% Cetyltrimethylammonium bromide(CTAB)-phosphate buffer (pH8.0) dissolve above-mentioned carefully
MPO in born of the same parents' deposit
3. preparation Q-Sepharose chromatographic column, with 0.5% CTAB-phosphate buffer (pH8.0) balance Q-Sepharose
Chromatographic column, above-mentioned MPO lysate passes through Q-Sepharose chromatographic column, and associated proteins (pI≤8.0) arrives Q-Sepharose layer
On analysis post, collect filtered solution (containing MPO, pI=9.2)
4. preparation SP-Sepharose chromatographic column, with 0.2% CTAB-phosphate solution (pH8.0) balance SP-Sepharose
Chromatographic column, above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, pI >=8.0 albumen (including MPO, pI=9.2) is tied
Close on SP-Sepharose chromatographic column, use 600mM K2PO4-phosphate buffer (pH9.5) eluting SP-Sepharose layer
Analysis post associated proteins, collects eluent (containing MPO)
5. put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer (pH7.5)
6. preparation ConA-Sepharose4B chromatographic column, washs ConA-with 100mM NaCl-phosphate solution (pH7.5)
Sepharose4B, then mix with protein liquid after dialysis, ConA-Sepharose4B combines saccharifying MPO, with 500mM NaCl-phosphorus
Phthalate buffer (pH7.5) washing ConA-Sepharose4B chromatographic column, with 600mM a-D-methylmannoside-phosphorus
Phthalate buffer (pH7.5) eluting ConA-Sepharose4B chromatographic column combines glycated protein, collects eluent (containing MPO)
7. put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer (pH7.5), collect
Protein liquid, subpackage, put-80 ° of C frozen
Second step of the present invention measures and separates MPO purity of protein and concentration:
The most above-mentioned frozen protein liquid carries out SDS-PAGE protein electrophoresis, then does argentation, measures MPO purity and reaches more than 95%
2. measure MPO protein concentration by Bradford method
The present invention the 3rd step foundation mensuration MPO halogenase activity method:
1.NaI-TMB method mensuration MPO halogenase activity principle: (see figure 5)
Course of reaction is H2O2Ferroporphyrin group (Fe3 in oxidation MPO albumen+), it is converted into oxidized form MPO(Fe4=O+), this
MPO(Fe4=O+) oxidation CL-, produce HOCl, HOCl and aoxidize TauNH further2, form TauNHCl, I-It is catalyzed rapidly TauNHCl
Oxidation TMB, forms oxidized form TMB, in blueness, has very strong light absorption value, uses 650nm wavelength, directly measures blue product OD
Value.
2. set up and measure purification MPO halogenase activity NaI-TMB method, purification MPO Yu Taurine (TauNH2)-phosphoric acid
Salt buffer mixes, and uses H2O2MPO after oxidation, converts Cl-Further aoxidizing TauNH2 for HOCl, HOCl is TauNHCl, adds
Catalase (Catalase) decomposes residue H2O2, after adding NaI-TMB substrate solution, I-Quickly/catalysis TauNHCl oxygen
Changing TMB, form oxidized form TMB, transfer blueness to, use 650nm wavelength, directly measure blue product OD value, this OD value represents purification
MPO halogenase activity.
3. setting up MPO halogenase activity unit (U), setting MPO halogenase activity unit (U) is at NaI-TMB of the present invention
Under the conditions of method measures MPO halogenase activity, within 1 minute, MPO aoxidizes Cl-, produce equivalent HOCl (umol/ml/min, U).The present invention
It is standard substance with pure HOCl solution, prepares 0,40,80,120,160,200umol HOCl solution standard, HOCl oxygen
Change TauNH2, generate TauNHCl, at I-Under catalysis, TauNHCl aoxidizes MTB, forms blue product, uses wavelength 650nm, measures OD
Value.Determine variable concentrations HOCl standard substance OD value, draw out HOCl standard concentration and OD value affinity criterions curve, calculate
The slope of curve (K): such as K=(HOCl 120nmo-80umol)/(120umol OD value-80umolOD value).Measure pure further
Change MPO halogenase activity unit (U).
4. mensuration purification MPO halogenase activity unit (U), 20ul purification MPO(0,50,100,200,400,800ng/
Ml) react with zymolyte (TauNH2, NaI, TMB), produce oxidized form TMB, react in blueness, the mensuration differential responses time (0,
5,10,15min), 650nm wavelength OD value, calculate MPO halogenase activity unit according to below equation: such as 200ng/ml MPO halogen
Change unit of enzyme activity (umol/ml/min, U)=K (200ng/ml MPO OD5min-200ng/ml MPO OD0min)/5min/
0.05ml。
5., with purification MPO as calibration object, measure and be used for detecting MPO halogenase activity unit, root in serum/slurry samples
According to previous investigation, in human blood, MPO concentration is in the range of 20-400ng/ml, and the present invention selects 0 to 1000ng/ml pure
Change MPO albumen, as calibration object, is converted into corresponding MPO halogenase activity unit (U).With NaI-TMB method measure MPO calibration object and
In serum/slurry after MPO halogenase activity, prepare MPO calibration object halogenase activity unit (U) standard curve, be used for calculating inspection
Survey MPO halogenase activity unit (U) in serum/slurry samples.
Experimentation of the present invention:
One, from HL-60 cell, myeloperoxidase (MPO) (MPO) is separated
1. with RPMI-1640(containing 18% FBS) cultivate HL-60 cell in 150mm Tissue Culture Dish
2. collect HL-60 cell in 250ml centrifuge tube, 4 ° of C, centrifugal, 3000rpm, 10 minutes
3. removing supernatant, add 150ml PBS in centrifuge tube, be mixed HL-60 cell, 4 ° of C, centrifugal, 3000rpm, 10 points
Clock, removes supernatant, collects HL-60 cell
4. add 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 centrifugation pipe, pressure-vaccum, it is mixed,
It is placed in ice cube 5 minutes
5. rupture cell with Dounce homogenization
6. under the conditions of 4 ° of C, centrifugal, 20000g, 10 minutes, removes supernatant
7. add in 30ml Buffer A to HL-60 cell precipitate, add Cetyltrimethylammonium bromide
(CTAB buys Sigma) is precipitated in mixed liquor to BufferA-cell, concentration to 1%；Add NaCl concentration to 100mM, at 4 ° of C bars
Under part, shake 2 hours
8. under the conditions of 4 ° of C, centrifugal, 20000g, 30 minutes, collects supernatant, in new 50ml centrifuge tube
9. preparation Q-sepharose chromatographic column (buying GE Healthcare Life Sciences), with Buffer B (20mM
NaPO4, pH8.0,100mM NaCl, 0.5% CTAB) and balance Q-Sepharose chromatographic column.
The most above-mentioned 30ml supernatant, is added in Q-Sepharose chromatographic column, by Q-Sepharose chromatographic column, receives
Collection filtered solution, is then added in Q-Sepharose chromatographic column, after quadruplication, collects filtered solution
11. preparation SP-Sepharose chromatographic columns (buying GE Healthcare Life Sciences), use Buffer C
(50mM K2PO4, pH8.0,100mM NaCl, 0.2% CTAB) and balance SP-Sepharose chromatographic column.
12. above-mentioned filtered solutions, by SP-Sepharose chromatographic column, collect filtered solution, are then added to SP-Sepharose
In chromatographic column, quadruplication
13. add in 100ml Buffer C to SP-Sepharose chromatographic column, and Buffer C slow transits through SP-Sepharose layer
Analysis post
14. add 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. eluents are transferred in bag filter (Spectra/Pro), place into dialysis solution (20mM NaPO4, pH7.5,
100mM NaCl, 5% Glycerol, 1mM DTT, 1mM EDTA, 1mM PMSF) in, under the conditions of 4-8 ° of C, slowly stir
Dynamic, 6 hours
Protein liquid of dialysing in bag filter is moved on in centrifuge tube by 16., under the conditions of 4 ° of C, centrifugal, 20000g, 10 minutes
17. take 1.5ml ConA-Sepharose4B(buy GE Healthcare Life Sciences) be put into 15ml be centrifuged
Guan Zhong
18. add 10ml cleaning mixture (20mM NaPO4, pH7.5, 100mM NaCl, 5% Glycerol,0.01% NP-40,
1mM EDTA, 1mM DTT, 1mM PMSF, 10ug/ml leupetine, 10ug/ml Aprotinin) arrive ConA
In Sepharose4B centrifuge tube
19. are centrifuged, 3000g, 2 minutes, sucking-off supernatant, repeated washing 2 times
20. add 15ml dialysis protein liquid in ConA-Sepharose4B centrifuge tube
21. under the conditions of 4 ° of C, slowly shake centrifuge tube, 2.0 hours
22. under the conditions of 4 ° of C, ConA-Sepharose4B to Poly-prep column (0.8 X 4cm after transfer reaction
Pharmacia Biotech, 731-1550), form ConA-Sepharose4B chromatographic column
23. add 20ml cleaning mixture (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 mixture slow transits through ConA-Sepharose 4B-chromatographic column
24. add 2.0ml eluent (20mM NaPO4, pH7.5 150mM NaCl, 600mM a-D-methylmannoside)
In ConA-Sepharose 4B-chromatographic column, eluent slow transits through chromatographic column, collects eluent
25. eluents are transferred in bag filter (Spectra/Pro), put bag filter at dialysis solution (20mM NaHPO4,
PH7.5,150mM NaCl, 10% Glycerol, 1mM DTT, 1mM EDTA, 1mM PMSF) in, under the conditions of 4 ° of C, slow
Slow agitation, overnight
26. by during in bag filter, protein liquid moves on to microcentrifugal tube, under the conditions of 4 ° of C, centrifugal, 13000rpm, 10 minutes,
Subpackage protein liquid is in different microcentrifugal tubes, and-80 ° of C preserve for a long time.
Two, mensuration isolates MPO purity of protein and concentration
(1) argentation measures MPO purity
The most frozen protein liquid centrifuge tube is put in 37 ° of C water-baths, and flash melt is put in 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 ° of C 3 minutes
3. add in 10ul protein liquid sample liquid 10% SDS-PAGE gel pore, carry out electrophoresis
4. after completing electrophoresis, obtaining SDS-PAGE gel, carry out Silver stain, step is as follows:
A. add 100ml 50% Methanol in the container of PAGE gel, under room temperature, slowly shake, 15 minutes,
Remove Methanol liquid
B. add 100ml 5% Methanol in PAGE gel container, under room temperature, slowly shake, 10 minutes, remove
Methanol liquid, washes PAGE gel with water 1 time
C. add in 10ul DTT to SDS-PAGE glue container, have 200ml water, under room temperature, slowly shake, 10 minutes, remove DTT
Liquid, washes PAGE gel with water 1 time
D. 100ml 0.1% AgNO is added3In PAGE gel container, under room temperature, slowly shake, 15 minutes, remove
AgNO3Liquid, washes PAGE gel with water 2 times
E. 100ml 3% Na is added2CO3& 0.05% Formaldehyde, in PAGE gel container, reacts under room temperature,
Till seeing clear protein band
F. add Critic Acid in PAGE gel container, terminate reaction
After cleaning SDS-PAGE glue with water, drying PAGE gel, it is judged that MPO purity.
(2) Bradford method measures purification MPO concentration
1. take 1ml and concentrate dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) and 4ml
H2O mix homogeneously, it is thus achieved that dilution dye reagent
2. preparation BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml
3. add 78ul PBS in micro titer plate well, add 2ul variable concentrations BSA titer, 2ul purification MPO liquid, each sample
Originally 3 holes are added
4. add 80ul dilution dye reagent in micro titer plate well, room temperature, place 5 minutes
5. put microtitration plate in biochemistry analyzer, select wavelength 495nm, measure OD value
6. preparation BSA concentration standard curve, calculating purification MPO concentration purification MPO OD meansigma methods is 1.212, MPO concentration=
1.6498 x 1.212 – 0.6059=1.393mg/ml
。
Three. set up HOCl concentration standard curve
1. add 50ul HOCl(and buy NovaBay Pharmaceuticals, Inc), concentration is 0,40,80,120,160,
200uM and 40ul PBS is in microwell plate respective aperture
2. add 50ul working solution (20mM NaPO4,pH6.5,200mM NaCl,10mM Taurine(TauNH2) enter each hole
In, react 5min
3. add 30ul substrate solution (2mM TMB, 150um NaI, 400mM Acetate, 10% DMF) in every hole, be mixed,
Reaction 5min
4. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure OD value
5. draw out HOCl concentration and OD value affinity criterions curve, calculate the slope of curve (K): such as K=(HOCl 120uM-
80uM)/(OD120uM-OD180uM)=120-80/0.638667-0.3906=161
。
Four. measure purification MPO halogenase activity unit (U)
1. add 50ul working solution (20mM NaPO4,pH6.5,200mM NaCl,10mM TauNH2) enter in micropore titer plate well
2. adding 50ul(0,50,100,200,400,800ng/ml) purification MPO is in corresponding microtiter plate hole
Add 20ul H the most again2O2Solution (2mM), in every hole, is mixed, and reacts 5min
4. add 20ul catalase (Catalase, buy Sigma) (20ug/ml) in every hole, be mixed, react 5min
5. add 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) in every hole, be mixed
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value
8. calculate OD value=OD5min–OD0min
Draw out MPO concentration and OD value affinity criterions curve, calculate MPO halogenase activity unit (U).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 specimen
1. add 50ul working solution (20mM NaPO4,pH6.5,100mM NaCl,5mM TauNH2) enter in micropore titer plate well
2. add 50ul serum and MPO calibration object (0,50,100,200,400,600U) in corresponding microtiter plate hole
Add 20ul H the most again2O2Solution (2mM), in every hole, is mixed, and reacts 5min
3. add 20ul Catalase (20ug/ml) in every hole, be mixed, react 5min
4. add 30ul substrate solution (2mM TMB, 100um NaI, 400mM Acetate, 10% DMF) in every hole, be mixed
5. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value
6. after sustained response 5min, use 650nm wavelength, measure 5min OD value
7. calculate OD value=OD5min–OD0min
8. draw out MPO calibration object halogenase activity unit (U) and OD value affinity criterions curve, calculate with this standard curve
In serum/slurry MPO halogenase activity unit (U)
Citing: serum 1 MPO(U)=910.38 X 0.208 45.686=143.67 U
。
Claims (5)
1. the method measuring MPO halogenase activity, it is characterised in that: the MPO chlB4 of mensuration separates from HL-60 cell
Step be:
A, from HL-60 cell, isolate MPO: cultivate HL-60 cell, centrifugal collecting cell, dissolve with phosphate buffer thin
Born of the same parents, further rupture cell with Dounce homogenization, centrifugal, collect cell deposition thing, in cell deposition thing
Containing MPO；
B, 1% CTAB-phosphate buffer dissolve MPO in above-mentioned cell deposition thing；
C, preparation Q-Sepharose chromatographic column: balance Q-Sepharose chromatographic column with 0.5% CTAB-phosphate buffer,
Above-mentioned MPO lysate passes through Q-Sepharose chromatographic column, on associated proteins to Q-Sepharose chromatographic column, collects and filters
Liquid, filtered solution contains the MPO of pI=9.2；
D, preparation SP-Sepharose chromatographic column: balance SP-Sepharose chromatographic column with 0.2% CTAB-phosphate solution,
Above-mentioned containing MPO filtered solution by SP-Sepharose chromatographic column, pI >=8.0 protein binding is to SP-Sepharose chromatographic column
On, use 600mM K2PO4-phosphate buffer eluting SP-Sepharose chromatographic column associated proteins, collects the eluting containing MPO
Liquid；
E, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer；
F, preparation ConA-Sepharose4B chromatographic column: wash ConA-with 100mM NaCl-phosphate solution
Sepharose4B, then mix with protein liquid after dialysis, ConA-Sepharose4B combines saccharifying MPO, with 500mM NaCl-phosphorus
Phthalate buffer washing ConA-Sepharose4B chromatographic column, by 600mM a-D-methylmannoside-phosphate-buffered
Liquid eluting ConA-Sepharose4B chromatographic column combines glycated protein, collects the eluent containing MPO；
G, put and state eluent in albumen bag filter, dialyse with 150mM NaCl-phosphate buffer, collect protein liquid, point
Dress, puts-80 ° of C frozen.
The method measuring MPO halogenase activity the most according to claim 1, it is characterised in that: frozen protein liquid uses silver
Dye method measures MPO purity,
A, frozen protein liquid centrifuge tube are put in 37 ° of water-baths, and flash melt is put in 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, add in 10ul protein liquid sample liquid 10% SDS-PAGE gel pore, carry out electrophoresis；
D, complete electrophoresis after, obtain SDS-PAGE gel, carry out Silver stain, step is as follows:
1. add 100ml 50% Methanol in the container of PAGE gel, under room temperature, slowly shake, 15 minutes, remove
Methanol liquid；
2. add 100ml 5% Methanol in PAGE gel container, under room temperature, slowly shake, 10 minutes, remove
Methanol liquid, washes PAGE gel with water 1 time；
3. add in 10ul DTT to SDS-PAGE glue container, have 200ml water, under room temperature, slowly shake, 10 minutes, remove DTT
Liquid, washes PAGE gel with water 1 time；
4. 100ml 0.1% AgNO is added3In PAGE gel container, under room temperature, slowly shake, 15 minutes, remove AgNO3
Liquid, washes PAGE gel with water 2 times；
5. 100ml 3% Na is added2CO3& 0.05% Formaldehyde, in PAGE gel container, reacts under room temperature,
Till seeing clear protein band；
6. Critic Acid is added in PAGE gel container；Terminate reaction；
7. after cleaning SDS-PAGE glue with water, drying PAGE gel, it is judged that MPO purity.
The method measuring MPO halogenase activity the most according to claim 1, it is characterised in that: Bradford method measures purification
MPO concentration；
1. take 1ml and concentrate dye reagent and 4ml H2O mix homogeneously, it is thus achieved that dilution dye reagent；
2. preparing BSA titer, concentration is respectively 0,0.25,0.5,1.0,1.5,2.0mg/ml；
3. add 78ul PBS in micro titer plate well, add 2ul variable concentrations BSA titer, 2ul purification MPO liquid, each sample
Originally 3 holes are added；
4. 80ul dilution dye reagent is added in micro titer plate well, room temperature, place 5 minutes；
5. put microtitration plate in biochemistry analyzer, select wavelength 495nm, measure OD value；
6. prepare BSA concentration standard curve, calculate purification MPO concentration.
The method measuring MPO halogenase activity the most according to claim 1, it is characterised in that: measure purification MPO chlB4 and live
Property unit:
1. add 50ul working solution to enter in micropore titer plate well；
2. 50ul purification MPO is added in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution, in every hole, is mixed, and reacts 5min；
4. add 20ul catalase in every hole, be mixed, react 5min；
5. add 30ul substrate solution in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
The method measuring MPO halogenase activity the most according to claim 1, it is characterised in that: measure in serum/slurry specimen
MPO halogenase activity unit:
1. add 50ul working solution to enter in micropore titer plate well；
2. 50ul serum and MPO calibration object are added in corresponding microtiter plate hole；
Add 20ul H the most again2O2Solution, in every hole, is mixed, and reacts 5min；
4. add 20ul Catalase in every hole, be mixed, react 5min；
5. add 30ul substrate solution in every hole, be mixed；
6. put microwell plate in biochemistry analyzer, select 650nm wavelength, measure 0min OD value；
7. after sustained response 5min, use 650nm wavelength, measure 5min OD value；
8. OD value=OD is calculated5min–OD0min。
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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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