CN106841631A - Cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine kit and preparation method - Google Patents
Cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine kit and preparation method Download PDFInfo
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- CN106841631A CN106841631A CN201611165645.6A CN201611165645A CN106841631A CN 106841631 A CN106841631 A CN 106841631A CN 201611165645 A CN201611165645 A CN 201611165645A CN 106841631 A CN106841631 A CN 106841631A
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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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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- General Health & Medical Sciences (AREA)
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Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine agent box and preparation method, it is provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+Anti- cardiac muscle troponin I, anti-N ends Natriuretic Peptide, three kinds of monoclonal antibody microballoon coupled complexes of anti-D dimer, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide that fluorescent microsphere is marked respectively3+, it is stable under ground state, the fluorescence of wavelength 615nm is launched under the excitation source effect of 337nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, and the cell strain of monoclonal antibody for epitopes such as 26 different cardiac muscle troponin Is, N ends Natriuretic Peptide, D dimers is derived from respectively.Have the advantages that easy to operate, reaction is quick, sensitivity is high, high specificity.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically one kind can be quick and precisely
While to three cardiovascular and cerebrovasculars such as blood plasma and whole blood sample Myocardial Troponin I, NT-proBNP, D dimer
Disease index of correlation carries out the three-in-one measure kit and preparation method of quantitative analysis.
Background technology：
Cardiac muscle troponin I (Cardiac Troponin I, cTnI) can be used as myocardial injury markers.Due to it highly
Cardiac-specific, high susceptibility and window phase more long for myocardial damage, not only as judge myocardial damage, especially
It is diagnosing acute heart infarction " goldstandard ".And it is most suitable in myocardial damage risk to have turned into judgement coronary syndrome patient
Mark.The rising of troponin can also support that clinician makes antithrombotic, antiplatelet early as strong evidence
Aggegation and the decision of property of participation treatment.N-terminal Natriuretic Peptide (NT-proBNP) is that the one kind secreted by the heart, brain contains 32 ammonia
The polypeptide hormone of base acid, when ventricle tension force increases, heart overload promotes it to secrete, the row's of rising sodium, diuresis, expansion blood in body
The physiological action of pipe.Its elevated concentrations and heart failure (acute heart failure AHF chronic heart failure CHF) order of severity are consistent.Doubtful heart failure
(HF) when, the detection of first-selected N-terminal Natriuretic Peptide (NT-proBNP) is answered.N-terminal Natriuretic Peptide (NT-proBNP) is negative
There is predictive value very high, the presence of heart failure can be excluded.In expiratory dyspnea patient, N-terminal Natriuretic Peptide (NT-proBNP) is
One will occur the relatively strong indication factor of (HF), can effectively differentiate chronic obstructive expiratory dyspnea and cardiac dyspnea.
Especially obvious superiority can be shown in terms of Hazard degree assessment after screening left chamber function bad (LVD) and myocardial infarction.
DDi is that fibrinolysin decomposes fibrinous product.Only fiber egg is produced when fibrinogen is decomposed by fibrin ferment first
White polymer, then decomposing fibrin polymer by fibrinolysin again could generate DDi.DDi mainly reflects fibre
Fibrillarin dissolves function.As long as body Ink vessel transfusing has thrombosis and the Fibrinolysis activity of activation, DDi will be raised.
Myocardial infarction, cerebral infarction, pulmonary embolism, venous thronbosis, operation, tumour, disseminated intravascular coagulation, infection and necrosis
Deng DDi being caused to raise.Especially to the elderly and inpatient, because suffer from the disease such as bacteremia easily cause disturbance of blood coagulation and
DDi is caused to raise.Fiber DDi is DVT (DVT), pulmonary embolism (PE), disseminated intravascular coagulation
(DIC) key index
CTnI/NT-proBNP/D-Dimer joint-detections have certain clinical valency for the comprehensive descision of heart failure heart infarction
Value, is an advantage over the new emergency treatment three of cTnI/CK-MB/MYO combinations.
Immuno analytical method is detected such as using the immune response between trace antigen and corresponding antibody with high specificity
It is living in the organisms such as hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element
Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit is utilized
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be belonging to one kind of labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but
It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA uses isotope marks, has pole to human body
Big harm is simultaneously made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme is unstable in itself, pushes away
Wide application is restricted.The beginning of the eighties, people begin one's study and replace fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establish new ultramicron time-resolved fluoroimmunoassay point
Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology uses multidisciplinary advanced technology, collection
The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtains significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic for tracer replaces fluorescence
Matter, enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell,
Question response system is (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and target cell pairing effect cell
Lethal effect etc.) occur after, with TrFIA detectors determine product in fluorescence intensity.According to product fluorescence intensity and phase
To the ratio of fluorescence intensity, the concentration of analyte in reaction system is judged, so as to reach quantitative analysis.In common fluoremetry
In, due to containing various fluorescent components in test sample, background fluorescence (causes from the colloidal solid and solvent molecule in sample
Scattering light and the non-specific fluorescence that sends of Proteins in Serum and other compounds) intensity is big, interference is strong, as fluorescence
The bottleneck that analytic approach is promoted on a large scale.Why TrFIA can turn into after the new sensitive preparation method of the latter of EIA, RIA, main
To depend on wavelength resolution and time-delay technique and the dissociation-increasing used in the unique fluorescence feature of lanthanide series, detection
Strong technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, has in 17, be usually used in TrFIA mainly have europium (Eu),
Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela
Compound fluorescence decay time is long, is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60-
900 μ s, conventional Eu3+Fluorescence decay time is 714 μ s, and the fluorescence decay time of fluorogen only has in common fluorescent immunoassay
1-100 μ s, the fluorescence decay time of some protein is only 1-10 μ s in sample, therefore utilizes TIME RESOLVED TECHNIQUE, postpones one
Measured after fixing time, just can obtain Eu3+Specific fluorescence signal.Simultaneously because decay time is long, Eu3+Label is in measurement
Between in can be excited repeatedly, ground state is transitted to by excitation state quickly after exciting every time, just there is fluorescence to send, then again can be weighed
Newly excite, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series is glimmering
The maximum of light spectrum is characterized in that the Stokes displacements between exciting light and launching light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；While Eu3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, can adjust instrument to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after
And pass through time delay and wavelength resolution, and strong specificity fluorescent and background fluorescence are distinguished into open (therefore referred to as time resolution), make interference
Reach almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher
The fluorescent marker of sensitivity, the simplicity of operation and stabilization ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of utilization fluorescence immune chromatography it is sensitive
Property, combined with fluorescent immunochromatographiassays assays instrument realize sensitivity it is high, fast and simple, can simultaneously accurate quantitative analysis detection myocardium myo calcium
Protein I, NT-proBNP, DDi are three-in-one to determine agent box and preparation method.
The present invention can be reached by following measures：
A kind of three-in-one measure kit of cardiac muscle troponin I/NT-proBNP/D dimer, is provided with test paper
Card, it is characterised in that the test card is sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and suction
Before anti-cardiac muscle troponin I, anti-N- akrencephalon natriuretic peptide that rare-earth fluorescent microballoon is marked respectively are adsorbed with water cushion, wherein pad
Body, three kinds of monoclonal antibodies of anti-D dimer-microballoon coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon,
Rare-earth fluorescent microballoon containing one or more in rare earth lanthanide, in the excitation source of 300-400nm make by the stabilization under ground state
Launch the fluorescence that wave-length coverage is 550-650nm under；The monoclonal antibody is the monoclonal antibody for mixing after purification, point
The list for epitopes such as 2-6 different cardiac muscle troponin I, NT-proBNP, D dimers is not derived from
Clonal antibody cell line.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 120-200nm；The rare-earth fluorescent microballoon
Preferably comprise one or more rare earth lanthanides；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 on pad
The monoclonal cell cell line of individual different epitopes.
Pad of the present invention is obtained using following steps：Glass fibre membrane is soaked in 150mM Tris-HCL treatment
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 small
When, it is standby.Glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, is connect with Bio-Jet Quanti300 are non-
Anti- cardiac muscle troponin I, anti-NT-proBNP and anti-D dimerization that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon
Three kinds of coupled complexes of body antibody are sprayed onto on glass fibre membrane after mixing, and 37 DEG C of drying are obtained after 1 hour.
Before the cardiac muscle troponin I/N- akrencephalon natriuretic peptides of the rare-earth fluorescent microballoon mark in the present invention on pad
Three kinds of monoclonal antibodies are obtained using following steps in the three-in-one measure agent box of body/D dimer：
Step 1：The acquisition of cell strain of monoclonal antibody：Cardiac muscle troponin I, NT-proBNP, D bis- are used respectively
Aggressiveness sterling distinguishes immune mouse, and the monoclonal for preparing specific high-affinity using the method for preparing monoclonal antibody of standard resists
Body cell strain, the monoclonal antibody cell line to being obtained carries out pairing screening, according to pairing result and affinity data preferably go out for
The monoclonal antibody cell line of kit；
Step 2：The preparation of monoclonal antibody：Anti- cardiac troponin is prepared and purified using the ascites production technology of standard
Three kinds of mouse resource monoclonal antibodies such as I antibody, anti-NT-proBNP antibody and anti-D dimer antibody, be stored in after packing-
20 DEG C standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate of pH 9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：Rare-earth fluorescent microballoon mark Antibodies to cardiac troponin I, anti-NT-proBNP antibody and
Three kinds of preparations of coupled complex such as anti-DDi antibody：Three kinds of antibody-microspheres coupled complexes are individually coupled, operation
It is as follows：Choose from 2 anti-cardiac muscle troponin I monoclonal antibodies of the monoclonal cell cell line of different epitopes, press
According to mass ratio 1:1 by 2mg cardiac muscle troponin Is monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, Ran Houyu
The rare-earth fluorescent microballoon mixing of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM, 4 DEG C of reactions are added
4 hours；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C of closings are overnight；
Then use the buffer solution of 50mM Tris-HCL, pH7.4 to be washed 3 times using centrifugal process, be resuspended in the 50mM Tris-HCL of 100 μ l
In buffer solution (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation is prepared respectively
Anti- NT-proBNP antibody-microspheres coupled complex and anti-DDi antibody-microspheres coupled complex, 4 DEG C of lucifuges
Save backup.
The nitrocellulose filter for being coated with detection line and nature controlling line of the present invention is obtained by following steps：
Step 1：Using with anti-cardiac muscle troponin I used on pad, anti-NT-proBNP and anti-D dimerization
The different cell line of three kinds of cell strain of monoclonal antibody such as body, prepares and purifies anti-myocardium myo using the ascites production technology of standard
Three kinds of mouse resource monoclonal antibodies such as calcium protein I antibody, anti-NT-proBNP antibody and anti-D dimer antibody, obtain with
Labelled antibody pairing monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 2：Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted with coating dilution respectively
To 1-3mg/ml, film liquid amount is 1-3 μ l/cm to concentration, and they are sprayed on into nitric acid fibre as detection line is parallel with nature controlling line
It is coated with the plain film of dimension, detection line and nature controlling line are subsequently placed in baking oven at intervals of 3-7mm, 37 DEG C dry 2 hours.
Sample pad of the present invention is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%Triton X-
100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
A kind of realized present invention also offers kit as described above cardiac muscle troponin I, NT-proBNP,
The three-in-one preparation method of D dimer, it is characterised in that comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read, it is described glimmering
Light immunochromatographiassays assays instrument is a kind of Systems for optical inspection, and detection range is respectively：cTnI：0.20-40ng/mL、NT-
proBNP:100-20000pg/mL、D-Dimer：0.1-10ug/mL；
Step 3：Sample-adding：Take 100 μ L plasma samples or 150 μ L whole blood samples are added in a pipe buffer solution, fully mix, hang down
It is straight to be added dropwise at 100 μ L mixed liquors to test card sample-adding；It is careful not to suck bubble during sampling；
Step 4：Detection, can be detected, automatic test using automatic test or immediately test both of which：By test card
Insert on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically,
Immediately test：After test card room temperature places 10min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
The present invention provides myocardium myo prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Calcium protein I/NT-proBNP/D dimer is three-in-one to determine agent box, while being adapted to blood plasma and whole blood sample, and is adapted to
Clinically single part detection, relative to qualitative colloid gold reagent, cardiac muscle troponin I, N- akrencephalon in energy quantitative determination sample
The content of natriuretic peptide precursor and D dimer is quick, sensitive with easy to operate, reaction with more specific Clinical significance of MG
The advantages of spending height, high specificity, be adapted to Site Detection and be economical and practical.
Brief description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 3 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 4 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 5 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 6 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 7 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Specific embodiment：
The present invention is further illustrated with reference to the accompanying drawings and examples：
As shown in Figure 1, present invention firstly provides a kind of cardiac muscle troponin I/NT-proBNP/D dimerization
Body is three-in-one to determine agent box, and test card is provided with box, and the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, knot
Pad 3, nitrocellulose filter 4 and adsorptive pads 5 are closed, the cardiac muscle anti-respectively of rare-earth fluorescent microballoon mark is wherein adsorbed with pad 3
Three kinds of monoclonal antibodies such as Troponin I, anti-NT-proBNP and anti-D dimer, the rare-earth fluorescent microballoon it is straight
Footpath is 150nm, and rare-earth fluorescent microballoon includes rare earth lanthanide Eu3+, the stabilization under ground state, in the excitation source effect of 337nm
Under launch the fluorescence of wavelength 615nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, and pin is derived from respectively
The monoclonal antibody of the epitopes such as cardiac muscle troponin I, NT-proBNP, the D dimer different to 2-6 is thin
Born of the same parents' strain.
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 200nm；The rare-earth fluorescent microballoon preferably comprises dilute
Native lanthanide series europium (Eu)；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 different epitopes on pad
Monoclonal cell cell line.
Embodiment 1：
The three-in-one each composition for determining test card in agent box of cardiac muscle troponin I/NT-proBNP/D dimer
Part can be obtained by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris buffer solutions,
In the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, be subsequently placed in baking oven, 37 DEG C dry 2 hours.2nd, fluorescent microsphere is adsorbed
The preparation of the pad 3 of labelled antibody：
Glass fibre membrane is soaked in (X-100 containing 1.0%Triton, 2.5% in 150mM Tris-HCL treatment fluids
BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby.Glass fibre membrane is placed on Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare-earth fluorescent microballoon with Bio-Jet Quanti300 noncontacts
The cardiac muscle troponin I of mark, three kinds of coupled complexes of NT-proBNP and D dimer are sprayed onto glass fibre after mixing
On film, 37 DEG C drying 1 hour after be obtained.；
The aldehyde radical of rare-earth fluorescent Nano microsphere：5mg rare-earth fluorescent Nano microspheres are taken, with 20mM, the carbonate of pH9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Rare earth Eu3+Antibodies to cardiac troponin I, anti-NT-proBNP antibody and anti-D- that fluorescent microsphere is marked
Three kinds of preparations of coupled complex such as homodimeric antibody：Three kinds of antibody-microspheres coupled complexes are individually coupled, and operation is such as
Under：Choose from 2 anti-cardiac muscle troponin I monoclonal antibodies of the monoclonal cell cell line of different epitopes, according to
Mass ratio 1:1 by 2mg cardiac muscle troponin Is monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with it is upper
The rare-earth fluorescent microballoon mixing of aldehyde radical is stated, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM, 4 DEG C of reactions 4 are added
Hour；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C of closings are overnight；
Then use the buffer solution of 50mM Tris-HCL, pH7.4 to be washed 3 times using centrifugal process, be resuspended in the 50mM Tris-HCL of 100 μ l
In buffer solution (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation is prepared respectively
Anti- NT-proBNP antibody-microspheres coupled complex and anti-DDi antibody-microspheres coupled complex, 4 DEG C of lucifuges
Save backup.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using with three kinds of Dan Ke such as cardiac muscle troponin I used on pad, NT-proBNP and D dimer
The different cell line of grand antibody cell strain, prepared using the ascites production technology of standard and purify Antibodies to cardiac troponin I,
Three kinds of mouse resource monoclonal antibodies such as anti-NT-proBNP antibody and anti-D dimer antibody, obtain being matched with labelled antibody
Monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively to arrive
1.5mg/ml, film liquid amount is 1.5 μ l/cm, and they are sprayed on into nitrocellulose filter as detection line is parallel with nature controlling line
On be coated with, at intervals of 3mm between three detections line and with nature controlling line, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling
The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 that are coated with detection line and nature controlling line, obtain test paper big after assembling
Plate, it is wide to cut into 4mm as requested, loads in plastic clip and form test card test paper.
The equipment and raw material selected in above steps preferably following raw material：
The species specificity of cardiac muscle troponin I, NT-proBNP, D dimer etc. three matches somebody with somebody antagonist；Myocardium myo calcium egg
The three special quality control product such as white I, NT-proBNP, D dimer：Landau laboratory diagnosis Co., Ltd of Britain；Rare-earth fluorescent is micro-
Ball：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore Products；Bovine serum albumin(BSA)
(BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007), fluorescence immunity analyzer parameter
Setting：After test card technological parameter is set on fluorescence immunity analyzer, the above-mentioned test card for assembling is taken, used respectively
0.2nd, 1,5,10,20, the 30, cTnI of 40ng/mL, 100,1000,2000,5000,10000,15000, the NT- of 20000pg/mL
ProBNP, 0.1,0.2,0.5,1,2,5, the D-Dimer calibration objects of 10ug/mL, be measured with test card, obtain each calibration object
Fluorescence intensity level, result is input in the parameter of analyzer, complete analyzer parameter setting.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 200 parts of latex enhancing immune turbidimetry definite value samples
Sheet, wherein 100 parts of plasma sample, 100 parts of whole blood sample, cardiac muscle troponin I/NT-proBNP/D dimer content
Distributed area is respectively：cTnI：0.20-40ng/mL、NT-proBNP:100-20000pg/mL、D-Dimer：0.1-10ug/
mL。。
Preparation method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read；
Step 3：Sample-adding：Take 100 μ L plasma samples or 150 μ L whole blood samples are added in a pipe buffer solution, fully mix, hang down
It is straight to be added dropwise at 100 μ L mixed liquors to test card sample-adding, it is careful not to suck bubble during sampling；Step 4：Detection, can be using automatic
Test or immediately test both of which are detected, automatic to test：Test card is inserted the carrying of dry type fluorescence immunity analyzer
On device, by feeler switch, instrument will be scanned analysis detection to test card automatically, test immediately：Test card room temperature places 10min
Afterwards, on the carrier of insertion dry type fluorescence immunity analyzer, test immediately is clicked on.
Test result analysis：After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by preparation method,
And analyze testing result.
Result of the test：
As shown in accompanying drawing 2-7, the detected value with experimental system as Y-axis, as X-axis draw and dissipate by the test value with contradistinction system
Point diagram, and carry out correlation analysis.Clinical sample detection is less than to 200 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit for preparing
Can be good, it is suitable for clinical detection, meet the differentiation needs of the different detection occasions of different clients.
The present invention provides myocardium myo prepared by a kind of fluorescence immune chromatography technology by the use of rare earth element as mark substance
Calcium protein I/NT-proBNP/D dimer is three-in-one to determine agent box, is adapted to blood plasma and whole blood sample, and be adapted to clinic
Upper single part detection, relative to qualitative colloid gold reagent, cardiac muscle troponin I, N- akrencephalon in energy simultaneous quantitative detection sample
Natriuretic peptide precursor, three kinds of contents of material of D dimer, it is fast with easy to operate, reaction with more specific Clinical significance of MG
Speed, sensitivity high, high specificity, be adapted to Site Detection and it is economical and practical the advantages of.
Claims (7)
1. a kind of three-in-one measure kit of cardiac muscle troponin I/NT-proBNP/D dimer, is provided with test card,
It is characterized in that the test card is sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and water suction
Pad, before anti-cardiac muscle troponin I, anti-N- akrencephalon natriuretic peptide that rare-earth fluorescent microballoon is marked respectively are wherein adsorbed with pad
Body, three kinds of monoclonal antibodies of anti-D dimer-microballoon coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon,
Rare-earth fluorescent microballoon containing one or more in rare earth lanthanide, in the excitation source of 300-400nm make by the stabilization under ground state
Launch the fluorescence that wave-length coverage is 550-650nm under；The monoclonal antibody is the monoclonal antibody for mixing after purification, point
The list for epitopes such as 2-6 different cardiac muscle troponin I, NT-proBNP, D dimers is not derived from
Clonal antibody cell line.
2. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 1
Agent box, it is characterised in that the diameter of the rare-earth fluorescent microballoon of the pad is 120-200nm；The rare-earth fluorescent microballoon doping
There is rare earth lanthanide Eu3+。
3. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 2
Agent box, it is characterised in that rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+；The three of rare-earth fluorescent microballoon mark on pad
Antibody is planted to derive from for 2 monoclonal cell cell lines of different epitopes.
4. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 1
Agent box, it is characterised in that the pad is obtained using following steps：Glass fibre membrane is soaked at 150mM Tris-HCL
In reason liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4
Hour, it is standby, glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, it is non-with Bio-Jet Quanti300
Contact cardiac muscle troponin I, NT-proBNP and D dimer three that the quantitation nozzle that declines marks rare-earth fluorescent microballoon
Kind of coupled complex is sprayed onto on glass fibre membrane after mixing, and 37 DEG C of drying are obtained after 1 hour.
5. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 4
Agent box, it is characterised in that before the cardiac muscle troponin I/N- akrencephalon natriuretic peptides of the rare-earth fluorescent microballoon mark on pad
The three-in-one three kinds of monoclonal antibodies determined in agent box of body/D dimer are obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：Cardiac muscle troponin I, NT-proBNP, D dimer are used respectively
Sterling distinguishes immune mouse, and the monoclonal antibody for preparing specific high-affinity using the method for preparing monoclonal antibody of standard is thin
Born of the same parents' strain, the monoclonal antibody cell line to being obtained carries out pairing screening, is preferably gone out for reagent according to pairing result and affinity data
The monoclonal antibody cell line of box；
Step 2：The preparation of monoclonal antibody：Anti- cardiac muscle troponin I is prepared and purified using the ascites production technology of standard to resist
Three kinds of mouse resource monoclonal antibodies such as body, anti-NT-proBNP antibody and anti-D dimer antibody, are stored in -20 after packing
It is DEG C standby；
Step 3：Rare earth Eu3+The aldehyde radical of fluorescent microsphere：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate buffer of pH9.5
Liquid, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbonic acid of 100 μ l
In salt buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, dark reaction 4 hours, are washed using same centrifugal process at room temperature
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：Rare earth Eu3+The Antibodies to cardiac troponin I of fluorescent microsphere mark, anti-NT-proBNP antibody and anti-
Three kinds of preparations of coupled complex such as DDi antibody：Three kinds of antibody-microspheres coupled complexes are individually coupled, and operation is such as
Under：Choose from 2 anti-cardiac muscle troponin I monoclonal antibodies of the monoclonal cell cell line of different epitopes, according to
Mass ratio 1:1 by 2mg cardiac muscle troponin Is monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with it is upper
The rare-earth fluorescent microballoon mixing of aldehyde radical is stated, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM, 4 DEG C of reactions 4 are added
Hour；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C of closings are overnight；
Then use the buffer solution of 50mM Tris-HCL, pH7.4 to be washed 3 times using centrifugal process, be resuspended in the 50mM Tris-HCL of 100 μ l
In buffer solution (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation is prepared respectively
Anti- NT-proBNP antibody-microspheres coupled complex and anti-DDi antibody-microspheres coupled complex, 4 DEG C of lucifuges
Save backup.
6. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 1
Agent box, it is characterised in that the nitrocellulose filter for being coated with detection line and nature controlling line is obtained by following steps：
Step 1：Using with three kinds of lists such as cardiac muscle troponin I used on pad, NT-proBNP and D dimer
The different cell line of clonal antibody cell line, is prepared and purified using the ascites production technology of standard anti-cardiac muscle troponin I and resist
Three kinds of mouse resource monoclonal antibodies such as body, anti-NT-proBNP antibody and anti-D dimer antibody, obtain matching somebody with somebody with labelled antibody
To monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 2：Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively to arrive
1.5mg/ml, film liquid amount is 1.5 μ l/cm, and they are sprayed on into nitrocellulose filter as detection line is parallel with nature controlling line
On be coated with, detection line and nature controlling line are subsequently placed in baking oven at intervals of 3mm, 37 DEG C dry 2 hours.
7. a kind of cardiac muscle troponin I/three-in-one measure of NT-proBNP/D dimer according to claim 1
Agent box, it is characterised in that the sample pad is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%Triton
X-100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
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Application publication date: 20170613 |