CN108333357A - A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits - Google Patents
A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits Download PDFInfo
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- CN108333357A CN108333357A CN201810135422.8A CN201810135422A CN108333357A CN 108333357 A CN108333357 A CN 108333357A CN 201810135422 A CN201810135422 A CN 201810135422A CN 108333357 A CN108333357 A CN 108333357A
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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
The invention discloses a kind of magnetic bead time-resolved fluoroimmunoassay quantitatively to detect CK MB kits, includes CK MB monoclonal antibody solutions, cleaning solution and the enhancement solution of the immunomagnetic beads of coating CK MB monoclonal antibodies, CK MB calibration objects solution, europium label.The immunomagnetic beads of the described coating CK MB monoclonal antibodies be 1 ~ 3 μm of the diameter with modified with functional group super-paramagnetic bead respectively with CK MB monoclonal antibody covalent coupling objects.The sensitivity of the high sensitivity of the present invention, CK MB is 1ng/mL, and serum (slurry) sample need not dilute；Detection time is short, and 30min goes out report；Sample requirements are few, and a loading only needs 50 μ L；Mating full-automatic Timed-resolved fluoroimmunoassay instrument, easy to operate, no human error simultaneously saves manpower.The space of reagent strip is rationally utilized in this kit so that reagent strip structure is compacter, more easily transports, easy to use, and it is easy to operate, stability is good.
Description
Technical field
The present invention relates to external reagent detection technique fields, specifically, the present invention relates to a kind of magnetic bead time resolution is glimmering
Light immune quantitative detects CK-MB kits.
Background technology
Creatine kinase (Creatine Kinase, CK) is typically found in the thin of the tissues such as the heart, muscle and brain of animal
It is a significant energy tune for having direct relation with intracellular energy operating, contraction of muscle, ATP regeneration in endochylema and mitochondria
Enzyme is saved, the phosphoryl that turns that it is reversibly catalyzed between creatine and ATP reacts.CK is made of two subunits of M and B, and is divided into this
Three most basic isodynamic enzymes：Muscularity (CK-MM), brain type (CK-BB), hydridization type (CK-MB).
In human body cardiac muscular tissue contain a large amount of enzyme, including creatine kinase (CK), creatine kinase isozyme (CK-MB),
Lactic dehydrogenase (LDH) etc., under the influence of lesion, different myocardium enzymes will produce apparent abnormal change, and CK-MB is special
Property highest, diagnosis acute myocardial infarction on be a kind of effectively index.What if patient increased and declined with CK-MB activity
Sequentiality changes, and peak value is more than 2 times of the reference value upper limit, and when can be explained without other reasons, is considered as AMI.CK-MB diagnosis are saturating
The sensibility and specificity of wall-shaped myocardial infarction is high, and 2~4h starts to increase after the onset of AMI, arrives peaking for 24 hours, and 48
~72h restores normal, if not restoring, shows infarct sustainable development.
Currently, the method for clinical detection CK-MB mainly has：Immunodepression, enzyme-linked immunization, immunochromatographic method, chemistry
Luminescence method.Immunodepression is easy to be interfered by CM-BB, huge CK, limits the application of this method.Enzymoimmunoassay has
The reagent term of validity is long, specificity is good, result can be promoted with the features such as Instrument measuring, but since sensitivity is relatively low, institute
It the shortcomings of can quantitative determining narrow range and Instrument measuring narrow range with label enzyme-to-substrate, limits it and is quantitatively surveyed in skeptophylaxis
Application in fixed.Immunochromatographic method is although easy to operate, but sensitivity and accuracy are poor.Although chemiluminescence immunoassay sensitivity
Height, but detection time is long, and instrument and equipment is expensive, is not suitable for basic hospital use.
In order to solve the above technical problems, the present invention provides a kind of, the time resolved fluoro-immunoassay based on magnetic particle tries
Agent box and preparation method thereof and detection method can detect CK-MB contents in sample, and testing result is more accurate and reliable, has
Higher detection sensitivity and specificity, and reached preferable performance parameter.Diagnosis rate will be greatly improved, will be examined in AMI early stages
It plays an increasingly important role in disconnected, observation of curative effect and Index for diagnosis.
Invention content
It is a kind of of low cost, easy to operate, accurate, sensitive the purpose of the present invention is overcoming the deficiencies of the prior art and provide
Magnetic bead time-resolved fluoroimmunoassay quantitatively detect CK-MB kits.
To achieve the above object, present invention employs following technical solutions：
A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits, including coating CK-MB monoclonal antibodies
CK-MB monoclonal antibody solutions, cleaning solution and the enhancement solution of immunomagnetic beads, CK-MB calibration objects solution, europium label.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits, the coating CK-MB monoclonals
The immunomagnetic beads of antibody are that the super-paramagnetic bead of 1~3 μm of the diameter with modified with functional group is covalent with CK-MB monoclonal antibodies respectively
Conjugate, wherein used magnetic bead is carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic beads, chain
Enzyme avidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, one kind in hydrophobic magnetic bead or several
Kind.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits, the preparation of the immunomagnetic beads
Method is：It draws in carboxyl magnetic bead to centrifuge tube, centrifuge tube is placed in magnetic frame, magnetic bead and buffering are carried out using magnetic frame
The separation of liquid washs magnetic bead 3~5 times with 2- (N- morpholines) ethanesulfonic acid of 0.1M pH6.0；In the magnetic bead good to above-mentioned washing
2- (N- morpholines) ethanesulfonic acid of 0.1M pH6.0 is added, 1- (3- the dimethylamino-propyls) -3- ethyls carbon two of 20mg/mL is added
Imide hydrochloride solution and 20mg/ml n-hydroxysuccinimides, room temperature concussion reaction；Activated magnetic bead is placed in magnetic frame
On, supernatant is abandoned, activated magnetic bead is collected；Monoclonal antibody is added, room temperature persistently rotates incubation 3~5 hours, and the reaction time takes
Certainly in the dentate of magnetic bead and concentration；Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.It can also be added and contain
There are the Tris-HCl buffer solutions of 5%BSA 0.1M pH 8.0, magnetic bead is resuspended, closes the activated carboxyl of non-conjugated monoclonal antibodies
Site is reacted 30~60 minutes；Magnetic bead can also be placed on magnetic frame, abandon supernatant, immunomagnetic beads are added and preserve liquid；The guarantor
Liquid storage is containing 5% (w/v) BSA, 10% (w/v) trehalose, the Tris- of the 0.05M pH 8.0 of 0.1% (v/v) Tween-20
HCl buffer solutions.
The preparation method of the CK-MB monoclonal antibody solutions of the described europium label is：According to CK-MB antibody and Eu3+Matter
Amount is than being 2:1, using DTTA-Eu as marker, under the carbonate buffer solution of 0.05M pH8.0, DTTA-Eu and CK- is added
MB monoclonal antibodies are dissolved in the carbonate buffer solution of 0.05M pH8.0 after mixing, room temperature concussion is overnight；Use sephadex column
Peak pipe is collected after purification；By the above-mentioned europium labeling antibody dilution prepared, make final concentration of 0.005~0.010g/ of CK-MB antibody
L。
The formula of the cleaning solution is：0.8~1.5%Tween-20,0.02%Proclin300, pH 7.2~
7.50.05M Tris-Hcl buffer solutions.
The formula of the enhancement solution is：0.03% sodium acetate, 0.0002~0.0009% β-NTA, 0.0024%TOPO,
0.08% acetic acid, 0.1% absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.
The preparation method of the CK-MB calibration objects is：With containing 1.5%Tween-20,0.02%Proclin300, pH 7.5
0.02M PBS buffer solution is by CK-MB antigen diluents to 0,1,5,25,100,300ng/ml.
The excitation wavelength of the fluorescent material is 300~350nm, and launch wavelength is 500~650nm.
The testing principle that magnetic bead time-resolved fluoroimmunoassay of the present invention quantitatively detects CK-MB kits is sandwich
Method, is preinstalled with CK-MB immunomagnetic beads in the reacting hole of reagent strip, when test, first by sample to be tested (serum or blood plasma) or school
Quasi- product are added in the reacting hole of reagent, add europium mark CK-MB antibody, and oscillation incubation 20min forms immunomagnetic beads-antigen-europium
Labeling antibody compound.After washing, enhancement solution is added, under the action of exciting light, fluorescent material emits the light letter of certain wavelength
Number, identify that measured object is more in sample by Immunofluorescence test instrument, the fluorescence signal intensity of generation is stronger.By CK-MB calibration objects
Concentration and fluorescence signal value fitted dose-response curve, you can obtain CK-MB concentration in unknown sample by this measured value.
Compared with prior art, the present invention has the advantages that：
(1) magnetic separation technique is used, the immune complex of the formation Direct precipitation in externally-applied magnetic field, being not required to centrifugation can
Immune complex is detached with unbonded material.Due to magnetic bead has the bonded area of bigger and can disperse in the liquid phase fully instead
It answers, greatly improves detection range, shorten the reaction time, improve sensitivity.Magnetic bead is oriented by chemical group with antibody and is connected, greatly
The big precision for reducing pairing antibody dosage and improving detection.Since magnetic bead and antigen or antibody are covalent coupling, overcome
The unstability of physical absorption, therefore the immunomagnetic beads holding time is long and more stable.Technology automation easy to implement, overcomes
The board-like time resolution reagent of conventional microporous needs sample cumulative that could be detected to certain amount, realizes sample and detects immediately.
(2) Time-resolved fluoroimmunoassay is used, using lanthanide chelate europium, terbium, samarium, dysprosium as marker,
Using spectrally resolved, time resolution, dissociation-enhancing principle, there is wider excitation spectrum, relatively narrow emission spectrum, be conducive to drop
Low background improves sensitivity；Ultraviolet excitation has the sub- yield of higher amount, larger Stokes displacements, avoids excitation spectrum and glimmering
The advantages that optical emission spectroscopy and the spectrum of bio-matrix transmitting overlap, and fluorescence decay time is long, detects than conventional fluorescent substance
Range is wider, specific more preferable；In addition also there is marker to prepare, and easy, stability is good, "dead" pollution, detection repeatability
Well, the advantages that operating process is not short, very extensive by sample natural fluorescence interference and application range.
(3) mating full-automatic detecting instrument realizes automation mechanized operation, can detect CK-MB in one or more parts sample simultaneously
Content, sample dosage is few, and quickly (can go out result within 30 minutes) easy to operate provides important evidence for early diagnosis AMI, for disease
People has saved valuable time.
Description of the drawings
Fig. 1 is that magnetic bead time-resolved fluoroimmunoassay is quantitatively detected CK-MB kits and is detected using reagent strip, reagent strip
Structure schematic top plan view.
Fig. 2 is that magnetic bead time-resolved fluoroimmunoassay is quantitatively detected CK-MB kits and is detected using reagent strip, reagent strip
Structural schematic diagram.
1, instrument connection 1,3, fluorescent marker, 6, washing lotion, 7, washing lotion, 12, enhancement solution, 2,4,5,8,9,10,11,13 are
Preparation hole.
Specific implementation mode
CK-MB coatings magnetic bead of the present invention is the super-paramagnetic bead and albumen of 1~3 μm of the diameter with modified with functional group
Covalent coupling object, wherein used in magnetic bead have carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS
Magnetic bead, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic bead etc. are wherein
One or more.
Below in conjunction with the drawings and specific embodiments, the present invention will be described in detail.
Embodiment 1
In this embodiment, magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits by reagent strip and the schools CK-MB
Quasi- product composition.
Wherein, the shape of reagent strip is sector, is from left to right arranged in order 1~13 hole.1st, 2 holes are instrument connection, are used
In storage immunomagnetic beads, while it being also the reacting hole of reagent；3rd, 4 holes are fluorescent marker hole, for storing europium labelled antibody；The
6,7 holes are cleaning fluid apertures, for storing cleaning solution；8th, 9 be Sample Dilution fluid apertures, is used for stored samples dilution；12nd is
Enhance fluid apertures, for storing enhancement solution；5th, 10,11,13 be preparation hole.Magnet can be stored between 1st and 2 holes carries out magnetic point
From experiment, it is 300 μ l that the 1st and 2 holes, which can store liquid volume,；3rd, 4 holes can be dismantled from entire reagent strip as independent
Component, convenient for carrying out packing storage to fluorescent marker.3rd, 4,5 holes can store liquid volume be 400 μ l；6th, 7 holes
It is 3000 μ l that liquid volume, which can be stored,；It is 400 μ l that 8th~12 hole, which can store liquid volume,；13rd hole can store liquid
Volume is 600 μ l.
After reagent each component is respectively loaded in dedicated reagent strip hole, the opening of reagent strip will be sealed.Such as Fig. 1
Shown in Fig. 2.
The preparation process that the magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits is as follows, wherein
(1) preparation method of CK-MB immunomagnetic beads：
1. washing magnetic bead
It draws in 1mg 2 μm of carboxyl magnetic beads to 1.5ml centrifuge tubes of diameter, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carry out magnetic bead and buffer solution separation, with 2- (N- morpholines) ethanesulfonic acids (MES) of 1ml 0.1M pH6.0 wash magnetic bead 3~
5 times.
2. magnetic bead activates
The MES of 1ml 0.1M pH6.0 is added in the magnetic bead good to above-mentioned washing, the 1- (3- bis- of 20 μ L 20mg/mL are added
Methylaminopropyl) -3- ethyl carbodiimide hydrochlorides solution (EDC) and 20 μ L 20mg/ml n-hydroxysuccinimides (NHS),
Room temperature concussion reaction 0.5~1 hour.
3. the coupling of CK-MB antibody and magnetic bead
Above-mentioned activated magnetic bead is placed on magnetic frame, abandons supernatant, collects activated magnetic bead.0.05~1mgCK- is added
MB monoclonal antibodies, room temperature persistently rotate incubation 3~5 hours, and the reaction time depends on the dentate and concentration of magnetic bead.
4. closing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.Addition contains 5%BSA 0.1M pH8.0's
Tris-HCl buffer solutions, are resuspended magnetic bead, and closing is not coupled the activated carboxyl sites of CK-MB monoclonal antibodies, and reaction 30~
60min。
5. storing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, and 500 μ L immunomagnetic beads are added and preserve liquid.
The preservation liquid is containing 5% (w/v) BSA, 10% (w/v) trehalose, the 0.05M of 0.1% (v/v) Tween-20
The Tris-HCl buffer solutions of pH 8.0.
The immunomagnetic beads dilution prepared above-mentioned, make CK-MB monoclonal antibodies final concentration of 0.0025~
0.0100g/L, in packing to the 1st hole (instrument connection 1) of reagent strip.
(2) europium marks CK-MB preparation method for antibody：
1. the preparation of europium mark CK-MB monoclonal antibodies
(1) 1mg CK-MB monoclonal antibodies are taken out and are placed in 30KD ultra-filtration centrifuge tubes, 10000rpm centrifuges 10min, discards filter
Liquid.
(2) 200 μ L, 10000rpm centrifugation 10min of label buffer solution (0.05M pH9.0 carbonate buffer solutions) are added, abandon
Go filtrate.Repeat this operation 4~5 times.
(3) centrifuge tube filter membrane is inverted, 3000rpm centrifuges 6min, collects the antibody of concentration.200 μ L label bufferings are added
Liquid stands 3~5min, then centrifuge tube filter membrane is inverted, and 3000rpm centrifuges 6min, collects the CK-MB monoclonal antibodies of concentration.
(4) according to mass ratio CK-MB monoclonal antibodies：Europium chelate DTTA-Eu=2:1 ratio mixes well, and is put into
Rotary incubator reacts at room temperature 16~24 hours.
2. the purifying of europium mark CK-MB monoclonal antibodies
With SepHadex TM G-75 gel column purification europium mark CK-MB monoclonal antibodies, europium mark CK-MB monoclonal antibody preservative agents are then added
(15% (w/v) BSA+5% (w/v) Proclin300), makes BSA and Proclin300 final concentration of 0.3%, through 0.22 μm of filter membrane
Filtering, 4 DEG C store for future use.
By the above-mentioned europium mark CK-MB monoclonal antibodies dilution prepared, make final concentration of 0.005~0.010g/L of CK-MB monoclonal antibodies,
In packing to the 3rd hole of reagent strip.
The excitation wavelength of the fluorescent material europium is 340nm, launch wavelength 615nm.
(3) preparation method of cleaning solution：
It prepares and contains 0.8~1.5%Tween-20,7.2~7.5 0.05M Tris- of 0.02%Proclin300, pH
Hcl buffer solutions dispense prepared solution into washing lotion hole 6 and 7 with 2000 μ L of every hole.
(4) preparation method of enhancement solution：
Prepare containing 0.03% sodium acetate, 0.0002~0.0009% β-NTA, 0.0024%TOPO, 0.08% acetic acid,
0.1% absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.Prepared solution is dispensed with 400 μ L of every hole to examination
The 12nd hole of agent article (enhancing fluid apertures).
(5) calibration object preparation method：
The preparation method of the CK-MB calibration objects is：With containing 1.5%Tween-20,0.02%Proclin300, pH 7.5
0.02M PBS buffer solution is by CK-MB antigen diluents to 0,1,5,25,100,300ng/ml.
The making and detection of 2 reagent strip of embodiment
Reagent strip in the present embodiment, semi-finished product are assembled by following process：It is dispensed respectively the 1st, 3,6,7,12
50 μ LCK-MB immunomagnetic beads, 200 μ L europium mark CK-MB antibody, 2000 μ L cleaning solutions, 2000 μ L cleaning solutions, 400 μ L enhancement solutions, so
It is sealed, is coated on the sealing plate film for the product information mark of full-automatic fluorimetric analysis instrument scanning recognition with film sealing machine afterwards
Knowledge includes company standard curve, batch, date of manufacture, the term of validity.Finished product reagent strip, the CK-MB calibration objects dispensed and other
Assembling fittings are at kit.
Pattern detection：
1. being loaded：
Sample to be tested or calibration object are put into the Load System of full-automatic instrument, reagent strip is inserted into reagent clamp bar slot,
The product information of instrument automatic identification sealing plate film.50 μ L samples to be tested are added in reagent strip instrument connection 1,100 μ L are then added
Europium mark CK-MB antibody.
2. being incubated：
37 DEG C of concussions are incubated 20min after the completion of sample-adding.
3. washing：
After the completion of incubation, the automatic hole flushing of instrument 5 times, 200 holes μ L/ of each washing lotion.
4. enhancement solution is added：
After the completion of washing, 100 holes μ L/ of enhancement solution, 37 DEG C of incubation 3min are added.
5. detecting：
Reagent strip is pushed into darkroom, and instrument detects and goes out result automatically.
Full-automatic magnetic beads time-resolved fluorescence immunoassay instrument for test agent item includes sample loading system, bar code
Reading system, sample adding system, incubation system, fluorescent light source detecting system and automatic software analysis and Control system.
Magnetic bead time-resolved fluoroimmunoassay of the present invention quantitatively detects CK-MB kits, only needed when detecting sample by
Reagent strip is inserted into full-automatic instrument, and instrument is automatically performed sample-adding, incubation, Magneto separate, detection process, and whole process only needs 30min
Examining report can be read.
The comparison of embodiment 3 and commercial reagent box
Using commercially available creatine kinase assay kit (chemiluminescence particulate immunodetection) (the limited public affairs of Abbott Laboratories' trade
Take charge of) detect sample (CK-MB concentration ranges are 1~300ng/mL in the sample) in triplicate with the method in embodiment 1, it tests
The testing result correctness for demonstrate,proving the kit of the present invention, as a result see the table below 1.
1 pattern detection result of table
Compared with commercial reagent, this reagent deviation is respectively less than ± 5%, and the testing result of this reagent is accurate and reliable.
Using commercially available creatine kinase assay kit (chemiluminescence particulate immunodetection) (the limited public affairs of Abbott Laboratories' trade
Take charge of) 200 clinical samples are detected with the reagent in embodiment 1, testing result see the table below 2.
2 clinical sample testing result of table
This reagent is compared the testing results of 200 clinical serums with Abbott Laboratories reagent strips, positive coincidence rate 97.6%,
Total coincidence rate of negative match-rate 97.3%, the two is 97.5%.Illustrate magnetic bead time-resolved fluoroimmunoassay of the present invention
CK-MB reagent strips are quantitatively detected, there is following advantage：1) sensitivity of high sensitivity, CK-MB is 1ng/mL, serum (slurry) sample
Originally it need not dilute；2) detection time is short, and 30min goes out report；3) sample requirements are few, and a loading only needs 50 μ L；4) mating
Full-automatic Timed-resolved fluoroimmunoassay instrument, easy to operate, no human error simultaneously saves manpower.5) examination is rationally utilized in this kit
The space of agent item so that reagent strip structure is compacter, more easily transports, easy to use, and easy to operate, stability
It is good.
Claims (9)
1. a kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits, it is characterised in that including being coated with CK-MB Dan Ke
The immunomagnetic beads of grand antibody, CK-MB monoclonal antibody solutions, cleaning solution and the enhancement solution of CK-MB calibration objects solution, europium label.
2. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
State coating CK-MB monoclonal antibodies immunomagnetic beads be 1 ~ 3 μm of the diameter with modified with functional group super-paramagnetic bead respectively with
CK-MB monoclonal antibody covalent coupling objects, wherein used magnetic bead is carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, toluene sulphur
Acyl group magnetic bead, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, is dredged NHS magnetic beads
One or more of hydromagnetic pearl.
3. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
The preparation method for stating immunomagnetic beads is：It draws in carboxyl magnetic bead to centrifuge tube, centrifuge tube is placed in magnetic frame, magnetic force is utilized
Frame carries out the separation of magnetic bead and buffer solution, with the 2- of 0.1M pH6.0（N- morpholines）Ethanesulfonic acid washs magnetic bead 3 ~ 5 times；To above-mentioned
The 2- of 0.1M pH6.0 is added in the magnetic bead washed（N- morpholines）1- (the 3- dimethylaminos third of 20mg/mL are added in ethanesulfonic acid
Base) -3- ethyl carbodiimide hydrochlorides solution and 20mg/ml n-hydroxysuccinimides, room temperature concussion reaction；It will be activated
Magnetic bead is placed on magnetic frame, abandons supernatant, collects activated magnetic bead；Monoclonal antibody is added, it is small that room temperature persistently rotates incubation 3 ~ 5
When, the reaction time depends on the dentate and concentration of magnetic bead；Magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.
4. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 3 quantitatively detects CK-MB kits, which is characterized in that institute
It states immunomagnetic beads and is additionally added the Tris-HCl buffer solutions containing 5%BSA 0.1M pH 8.0, magnetic bead is resuspended, closing is not coupled Dan Ke
The activated carboxyl site of grand antibody is reacted 30 ~ 60 minutes.
5. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 4 quantitatively detects CK-MB kits, which is characterized in that institute
It states immunomagnetic beads to be placed on magnetic frame, abandons supernatant, immunomagnetic beads are added and preserve liquid；The preservation liquid is containing 5%（w/v）BSA,
10% （w/v）Trehalose, 0.1%（v/v）The Tris-HCl buffer solutions of the 0.05M pH 8.0 of Tween-20.
6. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
The preparation method of CK-MB monoclonal antibody solutions for stating europium label is：According to CK-MB antibody and Eu3+Mass ratio be 2:1, it will
DTTA-Eu is as marker, and under the carbonate buffer solution of 0.05M pH8.0, DTTA-Eu and CK-MB monoclonal antibodies is added,
The carbonate buffer solution of 0.05M pH8.0 is dissolved in after mixing, room temperature concussion is overnight；Peak is collected after purification with sephadex column
Pipe；By the above-mentioned europium labeling antibody dilution prepared, make final concentration of 0.005 ~ 0.010g/L of CK-MB antibody.
7. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
The formula for stating cleaning solution is：7.2 ~ 7.5 0.05M Tris- of the .5% of 0 .8 ~ 1 Tween-20,0.02%Proclin300, pH
Hcl buffer solutions.
8. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
The formula for stating enhancement solution is：0.03% sodium acetate, 0.0002 ~ 0.0009% β-NTA, 0.0024% TOPO, 0.08% acetic acid, 0.1%
Absolute ethyl alcohol, 0.05% Triton X-100 aqueous solution.
9. magnetic bead time-resolved fluoroimmunoassay as described in claim 1 quantitatively detects CK-MB kits, which is characterized in that institute
The preparation method for stating CK-MB calibration objects is：With containing 1.5% Tween-20,7.5 0.02M PBS of 0.02%Proclin300, pH
Buffer solution is by CK-MB antigen diluents to 0,1,5,25,100,300ng/ml.
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| CN109541224A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection syphilis helicoid antibody kit and preparation method thereof |
| CN109541226A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of pepsinogen Cgene/II bigeminy check reagent box and preparation method thereof |
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Application publication date: 20180727 |