CN109239031A - Settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit - Google Patents
Settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit Download PDFInfo
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- CN109239031A CN109239031A CN201811048833.XA CN201811048833A CN109239031A CN 109239031 A CN109239031 A CN 109239031A CN 201811048833 A CN201811048833 A CN 201811048833A CN 109239031 A CN109239031 A CN 109239031A
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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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
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
A kind of settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit, belongs to time resolution Immunofluorescence test technical field.The purpose of the present invention is use lateral immunochromatographic method (Lateral flow immunoassay, LFIA MYBPC3 concentration in blood) is measured, result can be gone out in 15 minutes, simple device is only needed, the settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit that quick diagnosis AMI is required is able to satisfy.The present invention includes test card, it is that treated sample pad, the bonding pad for being adsorbed with MYBPC3 antibody-fluorescent microballoon, the nitrocellulose filter for being coated with detection line and nature controlling line are sequentially mutually overlapped on bottom plate, and water absorption pad, test paper plate is formed after assembling, the test strips of 3-4mm wide are then cut into, test strips are packed into plastic shell and form test card.The present invention is simple, and quickly, MYBPC3 concentration in quantitative detection blood is clinically widely used for early diagnosing AMI, will there is very big market demand.
Description
Technical field
The invention belongs to time resolution Immunofluorescence test technical fields.
Background technique
Acute myocardial infarction (Acute myocardial infarction, AMI) has become the main original of human death
Cause, due to the feature that AMI morbidity is anxious, the death rate is high, the early diagnosis of AMI and in time treatment are the key that reduce the AMI death rate
Place.Currently, diagnosis AMI is mainly according to typical clinical manifestation, the change of electrocardiogram and lab index detection.About 25%
Patient AMI fall ill early clinic symptom it is unobvious, about 50% patient's AMI electrocardiogram is without typical change, in this case, inspection
Thought-read injury of muscle Specific marker AMI early stage is made a definite diagnosis just play the role of it is key, need to develop a kind of high sensitivity,
Specificity is good, clinical diagnosis coincidence rate is high, fast and easily detects myocardial damage specific index kit.
Clinically often detect Creatine kinase MB isozyme (Creatine kinase-MB, CK-MB) in blood, troponin
T(Cardiac troponin T, cTnT), Troponin I (Cardiac troponin I, cTnI) cardiac injury marker,
Determine AMI.
CK-MB is primarily present in cardiac muscle cell, is one of common myocardial injury markers, CK-MB is after the onset of AMI
Increase in 4 8h, for 24 hours reach to peak value, couple of days restores normal.CK-MB although sensitivity with higher in serum, but cardiac muscle with
CK-MM has a cross reaction in skeletal muscle, and specificity is not high, " window phase " it is short (start that the raised time is later and the duration compared with
It is short), CK-MB is not to determine myocardial tissue damage specific index.
Summary of the invention
The purpose of the present invention is measured using lateral immunochromatographic method (Lateral flow immunoassay, LFIA)
MYBPC3 concentration in blood can go out as a result, only need simple device in 15 minutes, be able to satisfy what quick diagnosis AMI was required
Settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit.
The present invention includes test card, is sequentially mutually to overlap treated sample pad on bottom plate, be adsorbed with MYBPC3
The bonding pad of antibody-fluorescent microballoon, the nitrocellulose filter and water absorption pad for being coated with detection line and nature controlling line, form after assembling
Test paper plate is then cut into the test strips of 3-4mm wide, and test strips are packed into plastic shell and form test card.
It is coated with MYBPC3 antibody on nitrocellulose filter of the present invention and forms detection line and anti-igg antibody formation nature controlling line.
Fluorescent microsphere surface modification of the present invention functional group is one kind of carboxyl, hydroxyl or epoxy group, diameter 100-
500nm。
Fluorescent microsphere of the present invention marks lanthanide chelate, including europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) element
One of chelate;The present invention preferentially selects Europium chelate (Europium Chelate), and fluorescence exciting wavelength is
420nm, launch wavelength are 615nm.
When the present invention is detected with time-resolved fluorescence (TRFIA) analyzer, acted at exciting light (wavelength 350-430nm)
Afterwards, be delayed 100-400uS, then measures test card fluorescence (wavelength 600-650nm) intensity.
MYBPC3 antibody-fluorescent microballoon of the present invention selects Europium chelate fluorescent marker carboxyl modified microballoon, sub- with carbon two
After amine (EDC) and N- hydroxy thiosuccinimide (Sulfo-NHS) processing carboxyl modified fluorescent microsphere, MYBPC3 antibody coupling
Onto above-mentioned fluorescent microsphere, MYBPC3 antibody and fluorescent microsphere weight ratio are 1:50-1:5, with spray film instrument that MYBPC3 antibody-is glimmering
Light microballoon is sprayed on bonding pad, after dry, is sealed up for safekeeping spare；MYBPC3 antibody is selected to identify myocardium type MYBPC3 epitope,
Without combining skeletal muscle type MYBPC3 antigen.
It is calibration object that the present invention, which purifies MYBPC3, with TRFIA analysis-e/or determining various concentration MYBPC3 calibration object in test paper
Fluorescence intensity is presented in card detection line, prepares MYBPC3 concentration standard curve, establishes MYBPC3 concentration standard card, be input to
TRFIA analyzer establishes TRFIA analyzer and shows MYBPC3 concentration detection system in sample automatically, for detecting serum, blood
MYBPC3 concentration in slurry and whole blood.
The present invention is simple, quickly, MYBPC3 concentration in quantitative detection blood, and for early diagnosing AMI, clinically
It is widely used, will there is very big market demand.For judging acute myocardial infarction.
Detailed description of the invention
Fig. 1 is lateral immunochromatographic method test strips side view；
Fig. 2 is on rear side of Package casing to immunochromatographic method test card schematic diagram；1 is bottom plate in Fig. 1 and Fig. 2, and 2 be sample pad, and 3 are
Bonding pad, 4 be nitrocellulose filter, and 5 be detection line, and 6 be nature controlling line, and 7 be water absorption pad, and 8 be sample window, and 9 be detection window,
10 be plastic shell；
Fig. 3 is MYBPC3 concentration standard curve；
Fig. 4 is that MYBPC3 concentration sensitive is write music line；
Fig. 5 is MYBPC3 protein structure schematic diagram.
Specific embodiment
CTnT and cTnI (cTn) are existed only in cardiac muscle cell, can be used determination of immunological methods, be examined myocardial damage
Disconnected tool is highly sensitive and is specifically used for clinical diagnosis AMI.It is released into blood within cTnT 3-6 hours after myocardial damage, reaches within 12 hours
Peak increases multiple up to 30 200 times, can maintain in blood 14 days or so, so its maximum efficient diagnosis window is wide to 2
- 14 days hours.After myocardial damage, cTnT is released into blood compared with cTnl earlier, is easier to be detected, and blood level is higher, to inspection
The value of thought-read injury of muscle also may be higher.
AMI ideal marker is to be released quickly against in blood after myocardial infarction from cardiac muscle cell, and cardiac myosin
Binding protein C (Myosin-Binding Protein C, Cardiac-Type, MYBPC3 or cMyBP-C) more meets this
Standard.
People's MYBPC3 gene is located on No. 11 chromosomes (11p11.2), contains 35 exons, encodes 1273 amino acid
Residue forms MYBPC3 albumen, belongs to III type fibronectin superfamily and immunoglobulin superfamily, connects egg containing 3 III type fibres
White structural domain (Fibronectintype3, FN3) and 8 immunoglobulin like domain (Immunoglobulin, Ig), Pro-
Ala structural domain (PAdomain), the combined area (Mdomain) Myosin (S2).FN3 is the layer that the antiparallel β chain of two panels is formed
Shape structure, what it is containing FN3 is mostly receptor protein；Ig is made of about 70-110 amino acid, is two layers of antiparallel β-pleated sheet chain
At layer structure, see MYBPC3 protein structure schematic diagram.
MYBPC3 participate in sarcomeric protein structure and function, the C10 structural domain of MYBPC3 in conjunction with myosin fiber, and
C8~C10 structural domain combination titin (Titin) has stable sarcomere structure function.MYBPC3 C1 and C2 structural domain it
Between have a specific region, M domain is in conjunction with myosin (Myosin) neck and MYBPC3 phosphorylation position
(Phosphorylation sites), the work as the protein kinase that the cAMP protein kinase relied on and calmodulin rely on
With site, the specific block of phosphorylation cardiac muscle adjusts myocardial contractive power, and MYBPC3 is not only involved in the maintenance of myocardial structural, also participates in
Intracellular messengers transmitting, influences the easypro contracting campaign of myofilament.
There are 3 kinds of different MyBP-C isomers in body, encoded by different genes, but 3 kinds of MyBP-C isomers ammonia
Base acid sequence has very high homology compared with skeletal muscle type MyBP-C, and the end N- of heart-type MyBP-C has unique domain
(C0) and there is specific antigen epitope, heart-type MyBP-C can be gone out with immunological method specific assay.
MYBPC3 is one of distinctive thick filament structural proteins of cardiac muscle cell, and when AMI occurs, serum MYBPC3 concentration rises fastly
Height, mainly due to MYBPC3 have height solubility and to proteolytic enzyme it is very sensitive, in myocardial ischemia, proteolysis
Enzyme is activated and hydrolyzes thick filament albumen, and MYBPC3 dephosphorylation is simultaneously released into blood, causes in serum MYBPC3 concentration in short-term
It is interior sharply to increase.
MYBPC3 is released into blood for 2 hours after AMI generation, reaches peak within 4-6 hours, at 12 hours, MYBPC3 restored to base
Line is horizontal.
The study found that can measure cTNT/I level when needing 3-9mg myocardial necrosis and increase；And 0.07mg myocardial necrosis
When, MYBPC3 level can be measured and increased.Since MYBPC3 molecular weight is big, in AMI extreme early, complete MYBPC3 and its hydrolysis
Product continues largely to be released into blood, can be higher than cTNT/I several times or even decades of times, be easy to detect, and it is dense to measure MYBPC3 in blood
Degree can become extreme early diagnosis AMI sensitivity and specific index.
AMI is acute disease, needs quick diagnosis and treatment.The present invention uses lateral immunochromatographic method (Lateral flow
Immunoassay, LFIA) MYBPC3 concentration in measurement blood, it can go out in 15 minutes as a result, only need simple device, be able to satisfy
Quick diagnosis AMI is required.
Kit of the present invention is using the long feature of Europium chelate (Europium Chelate) fluorescence lifetime, and delay measures,
That is time-resolved fluorescence method (Time-resolved fluoroimmunoassay, TRFIA), in conjunction with lateral immunochromatography technique
(Lateral flow immunoassay, LFIA) realizes quantitative detection MYBPC3.Kit of the present invention mainly has detection
MYBPC3 test card, by sequentially mutually overlap joint sample pad, bonding pad, nitrocellulose filter and water absorption pad composition try on bottom plate
Paper slip, dress become test card in a plastic housing.Preparation method includes: MYBPC3 antibody coupling Europium chelate fluorescent microsphere;
MYBPC3 antibody-fluorescent microballoon sprays on bonding pad, and MYBPC3 antibody sprays on nitrocellulose filter；MYBPC3 is added to calibrate
After product to MYBPC3 test card, is detected with time-resolved fluorescence (TRFIA) analyzer, establish TRFIA analyzer and show automatically
Show MYBPC3 concentration detection system.
The present invention selects Europium chelate fluorescence, and fluorescence lifetime is long, is measured using delay, and background signal is discharged；Increase detection
Time improves detection sensitivity.
The present invention includes test card, is sequentially mutually to overlap treated sample pad on bottom plate, be adsorbed with MYBPC3
The bonding pad of antibody-fluorescent microballoon, the nitrocellulose filter and water absorption pad for being coated with detection line and nature controlling line, form after assembling
Test paper plate is then cut into the test strips of 3-4mm wide, and test strips are packed into plastic shell and form test card.
It is coated with MYBPC3 antibody on nitrocellulose filter of the present invention and forms detection line and anti-igg antibody formation nature controlling line.
Fluorescent microsphere surface modification of the present invention functional group is one kind of carboxyl, hydroxyl or epoxy group, diameter 100-
500nm。
Fluorescent microsphere of the present invention marks lanthanide chelate, including europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) element
One of chelate;The present invention preferentially selects Europium chelate (Europium Chelate), and fluorescence exciting wavelength is
420nm, launch wavelength are 615nm.
When the present invention is detected with time-resolved fluorescence (TRFIA) analyzer, acted at exciting light (wavelength 350-430nm)
Afterwards, be delayed 100-400uS, then measures test card fluorescence (wavelength 600-650nm) intensity.
MYBPC3 antibody-fluorescent microballoon of the present invention selects Europium chelate fluorescent marker carboxyl modified microballoon, sub- with carbon two
After amine (EDC) and N- hydroxy thiosuccinimide (Sulfo-NHS) processing carboxyl modified fluorescent microsphere, MYBPC3 antibody coupling
Onto above-mentioned fluorescent microsphere, MYBPC3 antibody and fluorescent microsphere weight ratio are 1:50-1:5, with spray film instrument that MYBPC3 antibody-is glimmering
Light microballoon is sprayed on bonding pad, after dry, is sealed up for safekeeping spare；MYBPC3 antibody is selected to identify myocardium type MYBPC3 epitope,
Without combining skeletal muscle type MYBPC3 antigen.
It is calibration object that the present invention, which purifies MYBPC3, with TRFIA analysis-e/or determining various concentration MYBPC3 calibration object in test paper
Fluorescence intensity is presented in card detection line, prepares MYBPC3 concentration standard curve, establishes MYBPC3 concentration standard card, be input to
TRFIA analyzer establishes TRFIA analyzer and shows MYBPC3 concentration detection system in sample automatically, for detecting serum, blood
MYBPC3 concentration in slurry and whole blood.
One, establishes TRFIA immunochromatography detection MYBPC3 test card
TRFIA immunochromatographyassay assay MYBPC3 kit of the present invention is sequentially mutual on bottom plate containing test card
Treated sample pad is overlapped, MYBPC3 antibody-fluorescent microballoon bonding pad is adsorbed with, is coated with MYBPC3 antibody (detection line)
With anti-igg antibody (nature controlling line) nitrocellulose filter and water absorption pad, test paper plate is formed after assembling, is then cut into 3-5mm wide examination
Test strips are fitted into plastic shell and form test card by paper slip.
The Europium chelate fluorescent microsphere adsorbed on the bonding pad, diameter range 100-500nm are further preferably, selected
It is 300nm with fluorescent microsphere diameter.
The fluorescent microsphere marks lanthanide series, including any one in europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd)
Or it is several.Further preferably, selected lanthanide series is Europium chelate (Europium Chelate), is stablized under ground state,
Under the effect of 420nm excitation light source, wavelength 615nm fluorescence can be launched.
It is described to be mixed with fluorescent microsphere coupled antibody for 3 MYBPC3 monoclonal antibodies, identify myocardium type MYBPC3 antigen
Epitope does not combine skeletal muscle type MYBPC3 antigen；The antibody of the detection line is anti-myocardium type MYBPC3 polyclonal antibody.
Prepare MYBPC3 monoclonal antibody-fluorescent microsphere
(1) MYBPC3 monoclonal antibody is added in antibody purification and concentration centrifugal column (Innova Biosciences), normally
Bright book step, purification and concentration MYBPC3 antibody adjust MYBPC3 MAb concentration to 1.0mg/ with phosphate buffer
ml；
(2) Europium chelate fluorescent marker carboxyl modified microballoon is washed with 50mmol MES buffer (pH6.0), 10mmol carbon is added
Diimine (EDC) and 20mmol N- hydroxy thiosuccinimide (Sulfo-NHS) react 20 minutes at room temperature.
(3) microballoon is washed with above-mentioned MES buffer, after redissolving microballoon with 100mmol phosphate buffer (pH7.5), added
Enter MYBPC3 antibody after purifying, make the mass ratio 1:40 of MYBPC3 antibody and microballoon, at room temperature, reacts 2 hours.
(4) reaction is terminated with 200mM Tris (pH7.5).
(5) with 0.2% BSA, 0.01% Tween-20,50mmol phosphate buffer (pH7.5) washing microballoon, settling flux
MYBPC3 antibody-fluorescent microballoon.
Preparation absorption MYBPC3 monoclonal antibody-fluorescent microsphere bonding pad
Contain 0.1%BSA, 10% Sucrose, 0.2% PEG, 0.2%PVP, 0.02% with 10mM Tris buffer (pH8.0)
Tween20,0.01% NaN3 redissolves MYBPC3 antibody-fluorescent microballoon to needing concentration.The present invention selects glass fibre to combine
Pad, soaking exist, 10mmolTris buffer (pH8.0), contain 0.2% PVP, 0.2% PEG, 10% Sucrose, 0.02%
Tween-20,0.01% NaN3), at room temperature, 1 hour, then 37 °C, dry 5 hours.Above-mentioned glass fibre membrane is placed on Bio-
On DotXYZ3060 three-dimensional specking platform, with the non-contact quantitation nozzle that declines, 5ul-15ul/cm speed, by MYBPC3 monoclonal
Antibody-fluorescent microballoon is sprayed on glass fibre membrane, 37 °C, is dried 4 hours, and addition desiccant is sealed up for safekeeping spare.
It is prepared with the nitrocellulose filter of detection line and nature controlling line
Contain 0.1%BSA, 5% Sucrose, 0.01% Tween20,0.01% NaN3 with 10mM Tris buffer (pH8.0)
MYBPC3 polyclonal antibody and anti-mouse IgG antibody are dissolved to concentration is needed, and nitrocellulose filter is placed on Bio-DotXYZ3060
On three-dimensional specking platform, with the non-contact quantitation nozzle that declines, 0.8-1.5ul/cm speed, by MYBPC3 antibody and anti-mouse IgG
Antibody is sprayed on nitrocellulose filter, is respectively formed detection line and nature controlling line, and line-to-line is divided into 4-6mm, and 37 °C, drying 2 is small
When, addition desiccant is sealed up for safekeeping spare.
Assemble TRFIA immunochromatographyassay assay MYBPC3 test card
Pre- reason sample pad is successively pasted on bottom plate plate, is adsorbed with MYBPC3 antibody-fluorescent microballoon bonding pad, has detection line and matter
Line nitrocellulose filter and water absorption pad are controlled, test paper plate is obtained, is cut into 4mm wide test strips as requested, test strips are packed into and are moulded
Expect that shell forms test card.
Two, establish TRFIA analyzer detection MYBPC3 concentration systems
TRFIA analyzer is a kind of Systems for optical inspection, can be after exciting light (350-430nm) effect, and be delayed 100-400uS,
Fluorescence (600-650nm) intensity is measured again.It is 0.05-200ng/mL to MYBPC3 detection range.
Calibration object is put into equilibrium at room temperature, MYBPC3 test card is taken out, lays flat;20ul calibration object is taken, is added in sample well,
Reaction 15-20 minutes is measured detection line and nature controlling line fluorescence intensity in test card, is prepared calibration object concentration with TRFIA analyzer
With fluorescence intensity relation curve, after further TRFIA analyzer concentration and fluorescence intensity relevant parameter are set, TRFIA points are obtained
Analyzer shows MYBPC3 concentration detection system automatically.
Increase serum (20ul), blood plasma (20ul) or whole blood (40 μ 1) react 15-20 into MYBPC3 test card sample well
Minute, with TRFIA analyzer, MYBPC3 concentration automatic display system is selected, detects serum, MYBPC3 in blood plasma or whole blood.
In conjunction with specific experiment, the principle of the invention and result are described further, are listed below multistep experimentation of the present invention
Process.
One, TRFIA immunochromatographyassay assay MYBPC3 kit is prepared
(1) time-resolved fluoroimmunoassay chromatography measurement MYBPC3 test card composition
Test card operation is assembled in humidity less than 30%, stablizes 30 °C of room and carries out.The side MYBPC3 is measured to immune chromatography test paper
Item (Fig. 1) includes bottom plate and sequentially covers along the floor length direction on bottom plate and sample pad (22mm), bonding pad
(10mm), nitrocellulose filter (25mm), blotting paper (30mm);Wherein, nitrocellulose filter covers the position among bottom plate,
On have the detection line formed by anti-MYBPC3 antibody coating and the nature controlling line formed by sheep anti-mouse igg antibody coating separately,
Detection line is located at bonding pad side, and nature controlling line is close to blotting paper side;Bonding pad is coated with MYBPC3 antibody-Europium chelate fluorescence
Microballoon coating；Between blotting paper, nitrocellulose filter, bonding pad and sample pad, is successively contacted with adjacent regions and part is heavy
It is folded, form test paper plate.
In the embodiment of the present invention, detection line is arranged in parallel with nature controlling line, and the distance between detection line and nature controlling line are 5mm.
Blotting paper is located at nitrocellulose filter close to nature controlling line one end, and blotting paper one end and nitrocellulose filter have part
Overlapping, length of overlapped part 2mm；Blotting paper overlaps above nitrocellulose filter.
Bonding pad is located at nitrocellulose filter close to detection line one end；And bonding pad one end and nitrocellulose membrane part weight
It is folded, length of overlapped part 2mm；Bonding pad overlaps above nitrocellulose filter.
Sample pad is located at the outside of bonding pad, and partly overlaps with bonding pad, length of overlapped part 5mm;Sample pad
It overlaps above bonding pad.
The above-mentioned test paper plate that is assembled into is cut into width as 4mm test strips.
Above structure test strips are fitted into composition test card (Fig. 2) in plastic shell, shell includes pedestal and Ka Gai, Ka Gai
On be provided with sample-adding window (8) detection window (9), expose test strips regional area；Sample-adding window is opened on sample pad (2)
Portion, exposed portion sample pad area；Watch window is opened on nitrocellulose filter (4) top, with expose whole detection lines (5) and
Nature controlling line (6).
The present invention selects glass fibre Fusion5 sample pad (GE Healthcare), and soaking is in 20mmolTris buffer
(pH7.5) contain 0.02% Tween-20,0.01% NaN3, at room temperature, 1 hour, then 37 °C, dry 5 hours, dry closing is standby
With.The present invention selects glass fibre GFDX bonding pad (EMD Millipore), and soaking exists, 10mmolTris buffer
(pH8.0), contain 0.2% PVP, 0.2% PEG, 10% Sucrose, 0.02% Tween-20,0.01% NaN3), at room temperature, 1
Hour, then 37 °C, it dries 5 hours, dry closing is spare.
(2) in MYBPC3 monoclonal antibody chemical coupling to Europium chelate fluorescent microsphere
In the embodiment of the present invention, Europium chelate (Europium Chelate) fluorescent marker carboxyl modified particle is selected
(Carboxylated Fluorescent Microspheres), is purchased from Ocean NanoTech, and it is glimmering that Europium chelate can be selected
Signal carboxyl modified particle diameter is 100-500nm.
It in the embodiment of the present invention, selects MYBPC3 monoclonal antibody (Invitrogen), with InnovaBiosciences public affairs
It takes charge of Antibody Concentration and purification centrifugal column (AbSelect Antibody Concentration and Clean-Up Kit) is pre-
MYBPC3 antibody is handled, steps are as follows:
1. add 500ulMYBPC3 antibody into Antibody Concentration and purification centrifugal column,
2. centrifugation 15000g 1-4 minutes, reduces MYBPC3 antibody to 100ul
3. removing liquid in collecting pipe, add 400ul20mmol phosphate buffer (pH7.5) to Antibody Concentration and purification centrifugal column
In
4. 15000g 1-4 minutes, it is long-pending to 100ul to reduce liquid in centrifugal column for centrifugation
5. 3-4 step is repeated, 6 times or more
6. about 100ulMYBPC3 antibody in Antibody Concentration and purification centrifugal column is sucked out
7. measuring MYBPC3 antibody concentration, 50mmol phosphate buffer (pH7.5) dilutes MYBPC3 antibody to needing concentration
In the embodiment of the present invention, with EDC and Sulfo-NHS MYBPC3 antibody coupling to Europium chelate fluorescent marker carboxyl modified
On microballoon (diameter 300nm), steps are as follows:
1. taking 0.4mlCarboxylated Fluorescent Microspheres (10mg/ml), diameter 300nmm, it is added
In 1.5ml centrifuge tube, centrifugation, 10000rpm 5 minutes, removes supernatant
2. adding 1.0ml 50mmolMES buffer (pH6.0) into centrifuge tube, suspended microspheres
3. centrifugation, 10000rpm 5 minutes, remove supernatant
4. repeating 2-3 step 2 time
5. adding 0.5ml50mmol MES buffer (pH6.0) carbodiimide containing 10mmol (EDC) and 20mmol N- hydroxyl sulphur again
It for succinimide (Sulfo-NHS) into centrifuge tube, is mixed, 25 °C, reacts 20 minutes
6. centrifugation, 10000rpm 5 minutes, remove reaction solution
7. 1.0ml 50mmol phosphate buffer (pH7.5) is added into centrifuge tube
8. centrifugation, 10000rpm 5 minutes, remove supernatant, repeat 8-9 step 1 time
9. the pretreated MYBPC3 antibody (0.3mg/ml) of 0.4ml is added to be added in above-mentioned centrifuge tube, 25 °C, react 2 hours
10. 50ul of 0.2M Tris (pH7.5) is added into centrifuge tube, 25 °C, react 30 minutes
11. centrifugation, 10000rpm 5 minutes, remove supernatant,
12. 0.2% BSA of 1.0ml is added, 0.01% Tween-20 50mmol phosphate buffer (pH7.5) is into centrifuge tube
13. centrifugation, 10000rpm 5 minutes, remove supernatant, repeat 13-14 step 2 time
14. containing 0.1%BSA, 10% Sucrose, 0.2% PEG, 0.2%PVP, 0.02% with 10mM Tris buffer (pH8.0)
Tween20, for 0.01% NaN3 suspension MYBPC3 antibody-fluorescent microballoon to concentration is needed, 4 °C of storages are spare.
(3) is sprayed on MYBPC3 antibody to bonding pad and nitrocellulose filter
1. by above-mentioned preparation MYBPC3 antibody-Europium chelate fluorescent microsphere liquid (0.2mg/ml), with Bio-DotXYZ3060 instrument,
With the non-contact quantitation nozzle mode that declines, MYBPC3 monoclonal antibody-fluorescent microsphere is sprayed onto glass fibre by 10ul/cm speed
It on film, 37 °C, dries 2 hours, addition desiccant is sealed up for safekeeping spare.
2. containing 0.2%BSA, 5% Sucrose, 0.01% Tween20,0.01% with 10mM Tris buffer (pH8.0)
NaN3 dissolves MYBPC3 polyclonal antibody (2.2mg/ml) and anti-mouse IgG antibody (0.5mg/ml), and nitrocellulose filter is placed on
Bio-DotXYZ3060 instrument, with the non-contact quantitation nozzle mode that declines, 1.0ul/cm speed, by MYBPC3 antibody and anti-mouse IgG
Antibody is sprayed on nitrocellulose filter, and line-to-line 37 °C, is dried 2 hours every 5mm, and addition desiccant is sealed up for safekeeping spare.
Two .TRFIA analyzers detect MYBPC3 concentration process
(1) MYBPC3 test card operating process
When carrying out quantitative detection MYBPC3 using MYBPC3 test card, calibration solution and sample liquid are added on sample pad window
(20ul), whole blood 40ul, under capillary phenomenon effect, sample liquid is to the swimming of water absorption pad direction, when containing in sample liquid
When MYBPC3 is moved to bonding pad, it is glimmering to form MYBPC3-MYBPC3 antibody-in conjunction with MYBPC3 antibody-fluorescent microballoon by MYBPC3
Light particles compound, as chromatography acts on, compound is moved forward, and is reached at nitrocellulose filter detection line, with coating
MYBPC3 antibody is further combined with formation MYBPC3 antibody-MYBPC3-MYBPC3 antibody-fluorescent microsphere compound is gathered in inspection
On survey line；And unbonded MYBPC3 antibody-fluorescent microballoon continues to move forward, when reaching nature controlling line, be coated with herein dynamics with
MYBPC3 antibody-fluorescent microballoon combines, and it is poly- to occur dynamics-MYBPC3 antibody-fluorescent microsphere compound at nature controlling line
Collection, entire reaction are completed in 10-20 minutes, and detection line and nature controlling line should all generate corresponding fluorescence signal, use TRFIA points
Analyzer is detected.
(2) establishes TRFIA analyzer and shows detection MYBPC3 concentration schedule automatically
1. establishing MYBPC3 concentration standard curve
Purifying MYBPC3 is diluted with PBS, various concentration MYBPC3 calibration object (0,5,10,20,50,100,200ng/ml) is made,
Add 20ulMYBPC3 calibration object to MYBPC3 immune chromatography test card sample window position, then plus 150ul experiment liquid (PBS,
PH7.4,0.02% Tween20) sample window position is arrived, film layer analysis reaction is carried out, after 15 minutes, with TRFIA analyzer, choosing
Determine excitation wavelength 420nm, Detection wavelength 615nm, postpone minute 200us, measures test strips card detection line and nature controlling line is glimmering
Luminous intensity.Using MYBPC3 calibration object concentration as ordinate, calibration object fluorescence intensity is abscissa, prepares MYBPC3 calibration object concentration
Standard curve obtains equation, y=2.8922x -8.0125, R2=0.9929, it sees Fig. 3, MYBPC3 is obtained by this standard curve
Concentration standard card, as the basis for carrying out quantitative analysis to MYBPC3 concentration contained in sample.
。
Input above-mentioned standard snaps into TRFIA analyzer, establishes automatic running system, TRFIA analyzer by dividing accordingly
Analysis software calculates MYBPC3 concentration in sample to be tested automatically.
0-5ng/ml MYBPC3 calibration object fluorescence intensity is measured, 1-5ng/mlMYBPC3 calibration object fluorescence intensity is linear
Relationship, TRFIA analysis-e/or determining 1ng/ml MYBPC3 calibration object fluorescence intensity (0.97) and 0mg/ml MYBPC3 calibration object are glimmering
Luminous intensity (0.43) differs about 2 times, determines that this kit TRFIA analysis-e/or determining MYBPC3 susceptibility is 1ng/ml.
。
With MYBPC3 concentration in MYBPC3 immune chromatography test card detection serum
Add 20ul blood serum sample to measurement MYBPC3TRFIA chromatographic test paper card sample-adding window position, carries out film layer analysis and react, 15 points
Zhong Hou measures MYBPC3 concentration in blood serum sample with TRFIA analyzer automatic checkout system, as a result as follows:
。
Claims (7)
1. a kind of settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit, it is characterised in that: including test card, be
Treated sample pad is sequentially mutually overlapped on bottom plate, the bonding pad for being adsorbed with MYBPC3 antibody-fluorescent microballoon, is coated with
The nitrocellulose filter and water absorption pad of detection line and nature controlling line, form test paper plate after assembling, are then cut into the examination of 3-4mm wide
Test strips are packed into plastic shell and form test card by paper slip.
2. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, feature exist
In: it is coated with MYBPC3 antibody on nitrocellulose filter and forms detection line and anti-igg antibody formation nature controlling line.
3. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, feature exist
In: fluorescent microsphere surface modification functional group is one kind of carboxyl, hydroxyl or epoxy group, diameter 100-500nm.
4. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, feature exist
In: fluorescent microsphere marks lanthanide chelate, including one in europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) element chelate
Kind;The present invention preferentially selects Europium chelate (Europium Chelate), and fluorescence exciting wavelength is 420nm, and launch wavelength is
615nm。
5. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, feature exist
In: when being detected with time-resolved fluorescence (TRFIA) analyzer, after exciting light (wavelength 350-430nm) effect, be delayed 100-
400uS, then measure test card fluorescence (wavelength 600-650nm) intensity.
6. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, it is characterised in that:
MYBPC3 antibody-fluorescent microballoon selects Europium chelate fluorescent marker carboxyl modified microballoon, with carbodiimide (EDC) and N- hydroxyl
After thiosuccimide (Sulfo-NHS) handles carboxyl modified fluorescent microsphere, MYBPC3 antibody coupling to above-mentioned fluorescent microsphere
On, MYBPC3 antibody and fluorescent microsphere weight ratio are 1:50-1:5, and MYBPC3 antibody-fluorescent microballoon is sprayed at knot with spray film instrument
It closes on pad, after dry, seals up for safekeeping spare；MYBPC3 antibody is selected to identify myocardium type MYBPC3 epitope, without combining skeletal muscle
Type MYBPC3 antigen.
7. settling time resolved fluorometric immunochromatographyassay assay MYBPC3 kit according to claim 1, it is characterised in that:
Purifying MYBPC3 is calibration object, is presented in test card detection line with TRFIA analysis-e/or determining various concentration MYBPC3 calibration object
Fluorescence intensity prepares MYBPC3 concentration standard curve, establishes MYBPC3 concentration standard card, be input to TRFIA analyzer, establishes
TRFIA analyzer shows MYBPC3 concentration detection system in sample automatically, for detecting serum, MYBPC3 in blood plasma and whole blood
Concentration.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004323A (en) * | 2019-12-03 | 2020-04-14 | 长春恒晓生物科技有限责任公司 | Preparation of cardiac myosin binding protein C antibody and detection method |
| CN111024956A (en) * | 2019-12-31 | 2020-04-17 | 江苏美克医学技术有限公司 | Time-resolved fluorescence immunochromatography kit for detecting PTX3 |
| CN111487230A (en) * | 2020-04-20 | 2020-08-04 | 中国农业科学院烟草研究所 | Ralstonia solanacearum detection device |
| CN111505267A (en) * | 2020-04-28 | 2020-08-07 | 上海泰辉生物科技有限公司 | Time-resolved immunoassay detection system and detection method |
| CN113514634A (en) * | 2020-04-09 | 2021-10-19 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting Adipo/SHBG |
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| CN113533714A (en) * | 2020-04-21 | 2021-10-22 | 武汉市朗典精医生物科技有限公司 | Preparation of MCM5/NMP22 double-index immunochromatography kit |
| CN113533713A (en) * | 2020-04-21 | 2021-10-22 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting THBS2/CA19-9 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997026534A1 (en) * | 1996-01-15 | 1997-07-24 | Finnish-Russian Joint Biotechnology Laboratory | Detection of cardiac muscle necrosis by immunoassay and appropriate antibodies therefor |
| CN106248927A (en) * | 2016-07-21 | 2016-12-21 | 上海奥普生物医药有限公司 | The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method |
| CN107121548A (en) * | 2016-02-25 | 2017-09-01 | 深圳市迈科龙生物技术有限公司 | Quantitatively detect test paper, preparation method and the detection method of tumor markers |
| CN107505459A (en) * | 2017-07-03 | 2017-12-22 | 广州瑞博奥生物科技有限公司 | Quantitatively detect people H FABP time-resolved fluoroimmunoassay chromatograph test strip, kit and preparation method thereof |
| CN108195815A (en) * | 2018-01-31 | 2018-06-22 | 李翀 | Time-resolved fluoroimmunoassay chromatography detects test strips, kit and the method for AMH |
| CN108254563A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof |
| CN108254562A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of MYO and preparation method thereof |
-
2018
- 2018-09-10 CN CN201811048833.XA patent/CN109239031A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997026534A1 (en) * | 1996-01-15 | 1997-07-24 | Finnish-Russian Joint Biotechnology Laboratory | Detection of cardiac muscle necrosis by immunoassay and appropriate antibodies therefor |
| CN107121548A (en) * | 2016-02-25 | 2017-09-01 | 深圳市迈科龙生物技术有限公司 | Quantitatively detect test paper, preparation method and the detection method of tumor markers |
| CN106248927A (en) * | 2016-07-21 | 2016-12-21 | 上海奥普生物医药有限公司 | The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method |
| CN108254563A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof |
| CN108254562A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of MYO and preparation method thereof |
| CN107505459A (en) * | 2017-07-03 | 2017-12-22 | 广州瑞博奥生物科技有限公司 | Quantitatively detect people H FABP time-resolved fluoroimmunoassay chromatograph test strip, kit and preparation method thereof |
| CN108195815A (en) * | 2018-01-31 | 2018-06-22 | 李翀 | Time-resolved fluoroimmunoassay chromatography detects test strips, kit and the method for AMH |
Non-Patent Citations (2)
| Title |
|---|
| KYOUNGHWAN LEE等: "Orientation of Myosin Binding Protein C in the Cardiac Muscle Sarcomere Determined by Domain-Specific Immuno-EM", 《JOURNAL OF MOLECULAR BIOLOGY》 * |
| THOMAS E. KAIER: "Direct Comparison of Cardiac Myosin-Binding Protein C With Cardiac Troponins for the Early Diagnosis of Acute Myocardial Infarction", 《CIRCULATION》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004323A (en) * | 2019-12-03 | 2020-04-14 | 长春恒晓生物科技有限责任公司 | Preparation of cardiac myosin binding protein C antibody and detection method |
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| CN113514642A (en) * | 2020-04-09 | 2021-10-19 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting IGFBP4/SHBG |
| CN111487230A (en) * | 2020-04-20 | 2020-08-04 | 中国农业科学院烟草研究所 | Ralstonia solanacearum detection device |
| CN113533714A (en) * | 2020-04-21 | 2021-10-22 | 武汉市朗典精医生物科技有限公司 | Preparation of MCM5/NMP22 double-index immunochromatography kit |
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| CN113533716A (en) * | 2020-04-22 | 2021-10-22 | 武汉市朗典精医生物科技有限公司 | Preparation of double-index immunochromatography kit for detecting sST2/Gal3 |
| CN111505267A (en) * | 2020-04-28 | 2020-08-07 | 上海泰辉生物科技有限公司 | Time-resolved immunoassay detection system and detection method |
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| CN114441766A (en) * | 2021-12-27 | 2022-05-06 | 迪亚莱博（张家港）生物科技有限公司 | Fluorescent immunochromatographic test strip for quantitatively detecting anti-PLA 2R antibody and preparation method thereof |
| CN114441766B (en) * | 2021-12-27 | 2023-12-29 | 迪亚莱博（张家港）生物科技有限公司 | Fluorescent immunochromatography test strip for quantitatively detecting anti-PLA 2R antibody and preparation method thereof |
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