CN107389941A - A kind of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box and its application method - Google Patents
A kind of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box and its application method Download PDFInfo
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- 102000004420 Creatine Kinase Human genes 0.000 title claims abstract description 38
- 108010042126 Creatine kinase Proteins 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 108010044467 Isoenzymes Proteins 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 34
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- 238000010521 absorption reaction Methods 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
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- 238000003556 assay Methods 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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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
- G01N33/53—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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
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- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box and its application method; the kit has some hole positions being arranged side by side; the reagent that the part hole position includes has CK MB magnetic particles reagent, CK MB labeling reactions, Sample dilution, calibration object, magnetic bead cleaning fluid and preexciting liquid, and each reagent distinguishes sealed storage in corresponding hole site using heat-sealing membrane technology.Kit of the present invention is detected using chemiluminescence instrument, the POCTization of the cost degradation of chemiluminescence instrument, simplification and reagent, the quantitative detection for causing creatine kinase isozyme is become into easy.
Description
Technical field
The present invention relates to a kind of chemical luminescent analysis reagent kid, more particularly to a kind of creatine kinase isozyme magnetic particle
Chemical luminescent analysis reagent kid and its application method.
Background technology
CK-MB is that (another two is CK-BB to a kind of one of three isomers of creatine kinase (CK, apoenzyme of muscle metabolism)
And CK-MM).CK-MB molecular weight is 84000, and it is made up of Liang Ge subunits：M subunits represent muscle, and B subunits represent big
Brain.When acute myocardial infarction AMI (AMI) occurs, CK-MB comes across in blood circulation and illustrates that myocardium receives damage.CK-
Quickly rise reaches peak (in 12 hours) to MB, can drop to normal level (36-72 hours) afterwards.The elevated width of CK-MB
Degree, the amplitude declined can reveal that time of myocardial damage, help to carry out Noninvasive Reperfu- sion and myocardial infarction area
Assess.
The product is based on magnetic microparticle chemiluminescence technology, using the bar magnet extractive technique of patent, coordinates new ripple STBS complete certainly
Dynamic chemical illumination immunity analysis instrument uses, it is contemplated that for quantitatively detecting the content of the creatine kinase isozyme in human serum, blood plasma.
The method of conventional detection creatine kinase isozyme clinical at present has colloidal gold method, micro-fluidic and chemoluminescence method etc..
Wherein, colloidal gold method operating method flexible and convenient is quick, but sensitivity is relatively low, and specificity is poor；Other microflow control technique due to
Runner control is complicated, the high expensive singly tested, the more difficult popularization under current state.Chemoluminescence method high sensitivity, set
Paramagnetic particles reach the purpose of fast reaction.But general chemical light irradiating machine is due to having the fluid path purging system of complexity, machine
It is general huger, even if making to diminish by optimization design, but cause the regular maintenance of machine to become difficult simultaneously.
The content of the invention
The present invention proposes a kind of creatine kinase isozyme magnetic microparticle chemiluminescence to solve above mentioned problem of the prior art
Immue quantitative detection reagent box and its application method.
Quantitative detection of the invention for existing creatine kinase isozyme needs high sensitivity, and (lowest detection is not higher than
0.3ng/ml), specificity is high, is loaded onto the problem of Turnaround Time is short, there is provided a kind of creatine kinase isozyme magnetic particle chemistry
Luminous immue quantitative detection reagent box and its detection method, can be detected using chemiluminescence instrument, chemiluminescence instrument it is low into
The POCTization of this change, simplification and reagent, becomes easy by the quantitative detection for causing creatine kinase isozyme.
To achieve the above object, the present invention uses following technical scheme：
The first aspect of the invention is to provide a kind of creatine kinase isozyme magnetic microparticle chemiluminescence quantitative detecting reagent
Box, including the reagent strip being made up of some hole positions being arranged side by side, the reagent that the reagent strip includes have the examination of CK-MB magnetic particles
Agent, CK-MB labeling reactions.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the CK-MB
Magnetic particle reagent is containing the magnetic microsphere for being marked with anti-CK-MB monoclonal antibodies.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the CK-MB
Labeling reaction is the anti-CK-MB monoclonal antibodies containing acridinium ester label.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the reagent
Bar magnet set is provided with a hole position on bar.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the reagent
Box also includes calibration object, magnetic bead cleaning fluid and preexciting liquid.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the magnetic bead
Cleaning fluid is the Tris-HCl buffer solutions containing tween, preservative.
Further, it is described pre- sharp on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box
Lotion is containing hydrogen peroxide, salpeter solution.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the calibration
Product are the BSA protein solutions containing a certain amount of CK-MB antigens.
Further, on the creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, the reagent
No. 0 position on bar is provided with bar magnet set；No. 3 positions are provided with CK-MB magnetic particle reagents；5th, 7,8, No. 9 positions are provided with magnetic bead cleaning fluid；No. 6
Position is provided with CK-MB labeling reactions；No. 10 positions are provided with preexciting liquid.
The second aspect of the invention is to provide a kind of kit and carries out creatine kinase isozyme magnetic particle chemistry hair
The use method that light quantitatively detects, during detection, reagent strip is placed in and is incubated in storehouse, the incubation storehouse is arranged on conveyer belt, institute
Conveying band connection horizontal displacement motor is stated, the end of the conveyer belt is provided with assay readings module；And use can be with respect to bar magnet set
The updraft type magnetic bead transfer rifle moved up and down, which is realized, takes bar magnet set, absorption magnetic bead, magnetic bead elution, magnetic bead to mix on the reagent strip
Even, bar magnet set plays de- action；
The updraft type magnetic bead shifts rifle, including extraction rifle pipette tips that interference be connected can be covered with the bar magnet, positioned at described
It is used for the bar magnet for adsorbing magnetic bead on extraction rifle pipette tips axial line, the bar magnet is driven by extraction motor and relatively described extraction rifle rifle
Head moves up and down, and the extraction motor is installed on the transfer rifle top, and the extraction motor passes through motor shaft and the magnetic
Rod connects；
Step 1, project is selected：According to samples selection CK-MB projects, in reagent strip inserting instrument reaction channel, click on
Start；
Step 2, barcode scanning：The Quick Response Code on instrument scanning reagent strip sealing aluminum membrane surface, confirms the information such as lot number, calibration curve
Whether match with the principal curve in software；
Step 3, puncture：Reagent strip runs to puncture position, and puncture module carries out puncture.
Step 4, bar magnet set is taken：Reagent strip runs to bar magnet set position, and instrument takes bar magnet set.
Step 5, it is loaded：Sample loading gun gets tip heads, draws 50uL samples, is added in No. 4 holes of reagent strip；
Step 6, magnetic bead extracts：Bar magnet set plus magnetic, extract the magnetic bead in No. 3 holes；
Step 7, the first step is incubated：Magnetic bead is extracted to No. 4 holes, carries out first step reaction；
Step 8, magnetic bead extracts：After the completion of the first step is incubated, bar magnet set plus magnetic, the magnetic bead in No. 4 holes is extracted；
Step 9, magnetic bead cleans：Magnetic bead is extracted to No. 5 holes and carries out first time cleaning；
Step 10, second step is incubated：Magnetic bead is extracted to 6 labelled notations work fluid apertures, carries out second step reaction；
Step 11, magnetic bead cleans：Magnetic bead is extracted to 7,8, No. 9 holes and cleaned three times；
Step 12, detect：Magnetic bead is extracted to No. 10 preexciting fluid apertures, reagent strip runs to reading position, reading module and hidden
Light shield is fallen, and adds exciting liquid into No. 10 holes, while photomultiplier collects luminous signal；
Step 13, result of calculation：Relative luminous intensity, is substituted into curve by software calibration curve according to corresponding to every batch of reagent
In, calculate concentration corresponding to sample.
Further, in the application method of the kit, the extraction rifle pipette tips can be with the inwall of bar magnet set
Or its outer wall interference connection.
Further, in the application method of the kit, the extraction rifle is by the length travel that is arranged on substrate
Motor driving moves up and down.
It is further preferred that in the application method of the kit, the output shaft of the length travel motor is provided with
Screw rod, the screw rod are provided with nut, and the nut is fixedly connected with the extraction rifle.
The present invention uses above-mentioned technical proposal, compared with prior art, has the following technical effect that：
1) using updraft type magnetic bead transfer rifle transfer magnetic bead technology, bar magnet is deep into liquid internal rapid extraction magnetic bead；
Using disposable bar magnet set, prevent magnetic bead from polluting bar magnet, while cause magnetic bead elution to become easy；
2) using comprising the total overall reaction liquid in addition to exciting liquid, the complicated liquid-way system of instrument is avoided so that instrument is small
Typeization becomes prone to safeguard simultaneously, while has saved cost；
3) transient chemical luminescence technology is used, more than common phot-luminescence, ensure that the high sensitivity of test, is saved
Time；The detection mode of wall scroll list test, realizes project POCTization；
4) to be swift in response using 0.5-1nm magnetic bead, machine is to report result from sample, and total time was at 15 minutes
It is interior；
5) each reagent is solved using heat-sealing membrane technology difference sealed storage in corresponding hole site, effectively prevent reagent string hole
Determine transportation problem；
6) range of linearity：Kit detection range of the present invention is 0.3-300ng/ml；Linearly：Line taking range limit is (about
Sample 300ng/ml) is diluted, and it is R=0.992 to calculate linear regression coefficient correlation；
7) HOOK effects：Concentration≤10000ng/ml sample is without hook effect；
8) methodology contrasts：Totally 183 clinical samples, with this kit and kit is listed while has been tested, two
The result of person enters Correlation series statistics, coefficient R=0.99.
Brief description of the drawings
Fig. 1 is the structural representation of reagent strip of the present invention；
Fig. 2 is the structural representation that updraft type magnetic bead of the present invention shifts rifle；
Fig. 3 is the cross section structure schematic diagram that updraft type magnetic bead of the present invention shifts rifle；
Fig. 4 is that updraft type magnetic bead of the present invention shifts rifle location state diagram of bar magnet and bar magnet set when drawing magnetic bead；
Fig. 5 is that updraft type magnetic bead of the present invention shifts rifle location state diagram of bar magnet and bar magnet set when eluting magnetic bead；
Fig. 6 is that the present invention uses theoretical concentration and the straight linear regression figure for determining concentration；
Fig. 7 is to use kit of the present invention with dependency graph when being detected with reference reagent to sample simultaneously；
Wherein, each reference is：
1- horizontal displacement motors, 2- conveyer belts, 3- are incubated storehouse, 4- reagent strips, 5- bar magnet sets, 6- length travel motors, 7-
Substrate, 8- screw rods, 9- nuts, 10- extraction motors, 11- transfer rifles, 12- extraction rifle pipette tips, 13- assay readings modules, 14- electricity
Arbor, 15- bar magnets, 16- magnetic beads.
Embodiment
The present invention is described in more detail below by specific embodiment, for a better understanding of the present invention,
But following embodiments are not intended to limit the scope of the invention.
Embodiment 1
As shown in figure 1, present embodiments provide a kind of creatine kinase isozyme magnetic microparticle chemiluminescence quantitative detecting reagent
Box, has some hole positions being arranged side by side, and the reagent that part hole position includes has CK-MB magnetic particles reagent, CK-MB mark reactions
Thing, the kit also include calibration object, magnetic bead cleaning fluid and preexciting liquid, and each reagent seals storage respectively using heat-sealing membrane technology
It is stored in corresponding hole site, effectively prevent reagent string hole, solve transportation problem.
It is provided with a hole position on reagent strip and is used to shift the matching used bar magnet set of rifle with special updraft type magnetic bead, is
When quantitatively being detected using kit progress creatine kinase isozyme magnetic microparticle chemiluminescence, shifted with updraft type magnetic bead transfer rifle
Magnetic bead technology, bar magnet is deep into liquid internal rapid extraction magnetic bead；Bar magnet set on the kit uses disposable bar magnet set,
Prevent magnetic bead from polluting bar magnet, while cause magnetic bead elution to become easy.
In addition, in the present embodiment creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, CK-MB magnetic is micro-
Grain reagent is containing the magnetic microsphere for being marked with anti-CK-MB monoclonal antibodies；CK-MB labeling reactions are containing acridinium ester label
Anti- CK-MB monoclonal antibodies；Magnetic bead cleaning fluid is the Tris-HCl buffer solutions containing tween, preservative；Preexciting liquid is to contain
There are hydrogen peroxide, salpeter solution；Calibration object is the BSA protein solutions containing a certain amount of CK-MB antigens.
As the optimal technical scheme of the present embodiment, examination is quantitatively detected in the creatine kinase isozyme magnetic microparticle chemiluminescence
In agent box, No. 0 position on the reagent strip is provided with bar magnet set；No. 3 positions are provided with CK-MB magnetic particle reagents；5th, 7,8, No. 9 positions are provided with
Magnetic bead cleaning fluid；No. 6 positions are provided with CK-MB labeling reactions；No. 10 positions are provided with preexciting liquid.
Embodiment 2
Present embodiments provide a kind of preparation side of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box
Method, specifically comprise the following steps：
Step 1, the preparation of creatine kinase isozyme calibration product：
Creatine kinase isozyme antigen is configured to calibration product liquid with 2%BSA and freezed, is assigned with company standard product
Value, concentration is respectively 10,60ng/mL；
Step 2, the preparation of the creatine kinase isozyme antibody of biotin labeling
1mg creatine kinase isozyme antibody is taken, 16-24h is no less than in 4 DEG C of dialysis with PB buffer solutions；Will be anti-after dialysis
Body adds 0.05mg biotins, adds PB buffer solutions, mixes after 25 ± 1 DEG C of constant incubator, stand reaction 120 ±
10min；Using PB buffer solutions in 4 DEG C of dialysis biotinylated antibody 16-24h；
Step 3, CK-MB magnetic beads/acridine value mark preserves liquid and prepared
Under room temperature condition, Tris, sodium chloride and HCl are added into the purified water of certain volume, is added after being completely dissolved
Tween-20 and %NaN3, BSA is eventually adding to being completely dissolved, after all the components are completely dissolved, purified water is settled to 1L；
Step 4, magnetic bead cleaning fluid is prepared
Under room temperature condition, into the purified water of certain volume add 1.212g Tris, 9.0g sodium chloride, 0.4mL HCl,
0.2mL Tween-20,5.0mL 20%NaN3 etc., purified water is settled to 1L；
Step 5, prepared by biotinylated antibody coating magnetic bead
20mg Streptavidin MagneSpheres are drawn, are cleaned twice with the magnetic bead cleaning fluid not less than 2 times of volumes of magnetic bead, Ran Houyong
CK-MB magnetic beads preserve liquid and magnetic bead are redissolved to not less than 5mg/mL.Magnetic bead and above-mentioned biotinylated antibody are mixed, magnetic bead is placed in
37 ± 2 DEG C of constant temperature oscillation case, 100rmp/min shake up reaction 16-24h.Reacted magnetic bead is removed, with not less than 2 times reactions
The magnetic bead cleaning fluid of volume cleans 4 times, and magnetic bead liquid is preserved into liquid with CK-MB magnetic beads/acridine value mark is diluted to aimed concn, 2-
8 DEG C of preservations are stand-by；
Step 6, the preparation of creatine kinase isozyme antibody-acridinium ester conjugate
1mg CK-MB labelled antibodies are taken, with PB buffer solutions in 4 DEG C of dialysis 16-24h.The antibody dialysed is buffered with PB
Antibody is diluted to 1.5mg/ml by liquid, takes the acridinium ester of target usage amount, is placed in after antibody is mixed with acridinium ester incubated
25 ± 2 DEG C of case, stand 120 ± 10min of reaction；Purified using G-50 chromatographic columns, purified acridinium ester label antibody is used
CK-MB acridines value mark preserves liquid and is diluted to aimed concn, and 2-8 DEG C of preservation is stand-by；
Step 7, preexciting liquid is prepared
Under room temperature condition, 44mlH2O2 and 0.55mL concentrated nitric acids are added into the purified water of certain volume, after being well mixed
It is 2 ± 0.1 to detect pH value, and purified water is settled to 1L；
Step 8, assemble：Using heat-sealing membrane technology, mentioned reagent is assembled into box, lucifuge is stored in 2~8 DEG C.
Kit made from the present embodiment is checked, it is main to include following aspect：
(1) PE：Reagent strip, which visually observes, to be cleaned, good without obvious impurity, sealer in each hole of reagent strip；
(2) accuracy：Inner quality control product L, M, H are determined, measured value should be in allowed band；
(3) it is linear：Dose-response curve correlation coefficient r absolute value in 0.3-300ng/mL concentration ranges is not less than
0.99；
(4) sensitivity for analysis：Kit assay sensitivity is not higher than 0.3ng/mL；
(5) precision：Criticize internal difference：Same batch test-strips parallel determination accuracy reference material CV (n=10), it is desirable to accurate
Spend (CV%) and be not higher than 8%；Difference between batch：Each parallel determination accuracy reference material CV (n=3*10) of three different batches, it is desirable to essence
Density (CV%) is not higher than 10%；
(6) stability：37 DEG C are placed 7 days, and measured value should meet above-mentioned requirements.
Embodiment 3
Present embodiments provide a kind of user of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box
Method, storehouse 3 is incubated as shown in Fig. 2 creatine kinase isozyme magnetic microparticle chemiluminescence quantitative detecting reagent bar 4 is placed in during detection
It is interior, it is incubated storehouse 3 and is arranged on conveyer belt 2, conveyer belt 2 connects horizontal displacement motor 1, and the end of conveyer belt 2 is provided with assay readings
Module 13；And the updraft type magnetic beads transfer rifles that can be moved up and down with respect to bar magnet set 5 are used to realize taking bar magnet set, drawing for kit
Magnetic bead, magnetic bead elution, magnetic bead mixes, bar magnet set plays de- action；Specifically comprise the following steps：
Step 1, project is selected：According to samples selection CK-MB projects, in reagent strip inserting instrument reaction channel, click on
Start；
Step 2, barcode scanning：The Quick Response Code on instrument scanning reagent strip sealing aluminum membrane surface, confirms the information such as lot number, calibration curve
Whether match with the principal curve in software；
Step 3, puncture：Reagent strip runs to puncture position, and puncture module carries out puncture；
Step 4, bar magnet set is taken：Reagent strip runs to No. 0 bar magnet set position, and bar magnet set is taken by shifting rifle；
Step 5, it is loaded：Sample loading gun gets tip heads, draws 50uL samples, is added in No. 4 holes of reagent strip；
Step 6, magnetic bead extracts：Bar magnet set plus magnetic, extract the magnetic bead in No. 3 holes；
Step 7, the first step is incubated：Magnetic bead is extracted to No. 4 holes, carries out first step reaction；
Step 8, magnetic bead extracts：After the completion of the first step is incubated, bar magnet set plus magnetic, the magnetic bead in No. 4 holes is extracted；
Step 9, magnetic bead cleans：Magnetic bead is extracted to No. 5 holes and carries out first time cleaning；
Step 10, second step is incubated：Magnetic bead is extracted to 6 labelled notations work fluid apertures, carries out second step reaction；
Step 11, magnetic bead cleans：Magnetic bead is extracted to 7,8, No. 9 holes and cleaned three times；
Step 12, detect：Magnetic bead is extracted to No. 10 preexciting fluid apertures, reagent strip runs to reading position, reading module and hidden
Light shield is fallen, and adds exciting liquid into No. 10 holes, while photomultiplier collects luminous signal；
Step 13, result of calculation：Relative luminous intensity, is substituted into curve by software calibration curve according to corresponding to every batch of reagent
In, calculate concentration corresponding to sample.
Embodiment 4
As shown in Fig. 2 the present embodiment provides one kind carries out creatine kinase isozyme magnetic microparticle chemiluminescence using kit
Quantitatively detect updraft type magnetic bead transfer rifle, including can with bar magnet cover 5 interference be connected extraction rifle pipette tips 12, positioned at extract rifle rifle
It is used for the bar magnet 15 for adsorbing magnetic bead 16 on first 12 axial line, magnetic bead transfer is carried out using the updraft type magnetic bead transfer rifle of the present embodiment
When, bar magnet set 5 is extracted by extracting rifle pipette tips 12,5 carrying magnetics or mistake are then covered by the relative displacement bar magnet of bar magnet 15
Magnetic is gone, is then shifted by the absorption magnetic bead 16 of bar magnet set 5.
As shown in figure 3, extraction motor 10 is installed on transfer rifle 11 top, and extracts motor 10 and pass through motor shaft 14 and bar magnet
15 connections, bar magnet 15 is installed in the lower end of motor shaft 14, by extracting moving up and down for the controlled motor axle 14 of motor 10, then band
The bar magnet 15 for moving its lower end moves up and down, the bottom away from or close to bar magnet set 5 so that the bottom carrying magnetic of bar magnet set 5 or mistake
Magnetic is gone, the transfer for magnetic bead 16.
On the updraft type magnetic bead transfer rifle of the present embodiment, extraction rifle pipette tips 12 can cover 5 inwall or its outer wall with bar magnet
Interference connects.Specifically, extract rifle pipette tips 12 to move down with transfer rifle 11 is overall, extraction rifle pipette tips 12 are cylindrical in shape, and can be designed
The internal diameter of extraction rifle pipette tips 12 is slightly less than the external diameter of bar magnet set 5, the interference of extraction rifle pipette tips 12 is packed tightly the outer wall in bar magnet set 5
On, during packing tightly, due to being acted on by the downward power of extraction rifle pipette tips 12, certain deformation occurs for bar magnet set 5；Or design carries
The external diameter of pipette tips of taking arms 12 covers 5 internal diameter slightly larger than bar magnet, the interference of extraction rifle pipette tips 12 is close on the inwall of bar magnet set 5,
During fitting, due to being acted on by the downward power of extraction rifle pipette tips 12, certain shape occurs in extrusion process for bar magnet set 5
Become.
It is arranged at as shown in Fig. 2 being incubated storehouse 3 on conveyer belt 2, conveyer belt 2 connects horizontal displacement motor 1, by horizontal displacement
Motor 1 drives conveyer belt 2 to run, and then drives incubation storehouse 3 to be thereon moved to relevant position, the end of conveyer belt 2 is additionally provided with
The assay readings module 13 tested and analyzed for the reagent after being mixed to magnetic bead.
Rifle is shifted please continue to refer to updraft type magnetic bead as shown in Figure 2, and transfer rifle 11 is by longitudinal position for being arranged on substrate 7
Move the driving of motor 6 to move up and down, substrate 7 is fixed on the top position of conveyer belt 2, and length travel motor 6 is fixedly mounted on base
Plate 7, and be connected with transfer rifle 11, for driving transfer rifle 11 is overall to move up and down.Preferably, length travel motor 6
Output shaft is provided with screw rod 8, and screw rod 8 is provided with nut 9, and nut 9 is fixedly connected with transfer rifle 11, i.e., length travel motor 6 is logical
Crossing screw rod 8 drives the transfer rifle 11 on screw rod 8 to move up and down.
Embodiment 5
A kind of method shifted using updraft type magnetic bead transfer rifle to the magnetic bead in kit is present embodiments provided,
Specifically comprise the following steps：
Step 1：Take bar magnet set
Bar magnet set 5 is moved to the underface of transfer rifle 11, moves downward transfer rifle 11,12 extraction rifle pipette tips probe into 5
In bar magnet set；Under the extruding of transfer rifle 11, bar magnet set 5 links together with the extraction interference of rifle pipette tips 12；Make 11 extraction rifles again
Move up, while extract rifle pipette tips 12 and moved up with bar magnet set 5, take out bar magnet set 5；
Step 2：Draw magnetic bead
After having taken bar magnet set 5, the aperture that will draw magnetic bead on reagent strip 4 is moved to the underface of transfer rifle 11,
Drive bar magnet 15 to be moved down into the bottom of bar magnet set 5 by extraction motor 10, the bottom belt of bar magnet set 5 is magnetic；Then make
Transfer rifle 11 moves down, and the bottom of bar magnet set 5 is insinuated into the liquid containing magnetic bead 16, then make transfer rifle slow about 11
Mobile, magnetic bead can be drawn onto the bottom of bar magnet set 5 by bar magnet 15；Finally, move up transfer rifle 11, while extract rifle pipette tips 12
Moved up with bar magnet set 5, magnetic bead 16 is also moved to outside liquid with bar magnet set 5；When drawing magnetic bead, bar magnet 15 and bar magnet set 5
Location status it is as shown in Figure 4：
Step 4：Elute magnetic bead
After drawing magnetic bead 16, the aperture that will receive magnetic bead 16 on reagent strip 4 is moved to the underface of transfer rifle 11,
Transfer rifle 11 is moved down, the bar magnet for being adsorbed with magnetic bead 16 set 5 is insinuated into the liquid of magnetic bead 16 to be received, by extraction electricity
Machine 10 drives bar magnet 15 to move up, and the bottom of bar magnet set 5 is lost magnetism；Then make transfer rifle about 11 by a small margin faster
Motion, magnetic bead 16 is eluted in liquid；When eluting magnetic bead, the location status of bar magnet 15 and bar magnet set 5 is as shown in Figure 5：
Step 4：Magnetic bead mixes
After eluting magnetic bead 16, the aperture that will mix magnetic bead 16 on reagent strip 4 is run to the underface of transfer rifle 11,
The aperture that magnetic bead 16 will be mixed refers to the aperture during being incubated, and in the state of nonmagnetic in 5 bottoms of bar magnet set, makes transfer
Rifle 11 moves downward, by bar magnet set 5 bottom be insinuated into the liquid that will mix magnetic bead 16, make transfer rifle about 11 by a small margin
Faster move, the magnetic bead 16 in liquid is mixed into holding suspended state；
Step 5：Bar magnet set is beaten de-
When no longer needing bar magnet to cover 5, the bar magnet set putting hole on reagent strip 4 is moved to the underface of transfer rifle 11,
Bar magnet 15 is driven to move downward by extraction motor 10, until bar magnet set 5 is de- from top in extraction rifle pipette tips 12 so that bar magnet set 5
Drop in the bar magnet set putting hole on reagent strip 4.
In the magnetic bead transfer method of the present embodiment kit, when step 4 magnetic bead mixes, the position of bar magnet 15 and bar magnet set 5
Configuration state is bottom of the lower end of bar magnet 15 away from bar magnet set 5, bar magnet is covered 5 bottoms and loses magnetism, i.e., elutes magnetic bead with step 3
When bar magnet 15 and bar magnet set 5 location status it is identical.
When quantitatively being detected using kit of the present invention progress creatine kinase isozyme magnetic microparticle chemiluminescence, using updraft type
Magnetic bead transfer rifle transfer magnetic bead technology, liquid internal rapid extraction magnetic bead is deep into by bar magnet；Using disposable bar magnet set, prevent
Magnetic bead pollutes bar magnet, while causes magnetic bead elution to become easy；Using the reagent for including the total overall reaction liquid in addition to exciting liquid
Box, avoid the complicated liquid-way system of instrument so that instrument miniaturization becomes prone to safeguard simultaneously, while has saved cost；Detection
Using transient chemical luminescence technology, more than common phot-luminescence, the high sensitivity of test is ensure that, has saved the time；Wall scroll
The detection mode singly tested, realize project POCTization；Kit to be swift in response using 0.5-1nm magnetic bead, from sample
Upper machine is to report result, and total time is in 15 minutes；Each reagent using heat-sealing membrane technology difference sealed storage in corresponding hole site,
Reagent string hole is effectively prevent, solves transportation problem；
The performance test of embodiment 6：
1st, detection sensitivity
1.1st, blank test limit
Method：Blank test limit, i.e. LOB, refer to the highest result that may detect that in dummy.Dummy is connected
Continuous detection 3 days, detects 20, totally 60 dummy data daily, and employment source calibration product do calibration curve and calculate concentration, according to
Arrange from small to large, it is exactly blank test limit to select the 95th percentile.Detect shown in the following table 1-2 of data：
The dummy of table 1 detects (RLU)
The dummy of table 2 detects-calculated concentration and sorts from small to large
Interpretation of result：The 95th percentile=60 × 95%=57 is selected, the result of the 57th digit is 0.03ng/ml, then
LOB is not higher than 0.08ng/ml.
1.2 sensitivity for analysis
Method：Sensitivity for analysis refers to the detectable minimum measured concentration of detection method, also referred to as minimum detection limit or
Minimum concentrations.60 dummy data of detection more than choosing, calculate the average value of luminous valueWith standard deviation S D.
CalculateShow that its corresponding concentration value is minimum detection limit from calibration object curve.
Interpretation of result：CalculateConcentration 0.045ng/ml is calculated, then LOD is not higher than
0.1ng/ml。
1.3 Functional Sensitivity
Method：Less than the sample of reference range (0.3ng/ml), concentration value is about 0.4ng/ml, uses Sample Dilution for selection
Liquid diluted concentration gradient, the accuracy of various concentrations gradient is detected respectively, it is considered that CV is just above 10% concentration value
Functional Sensitivity.
Testing result：
The confirmation of the Functional Sensitivity of table 3
Interpretation of result：As can be seen that when diluted concentration is 0.1ng/ml, CV is still less than 10%, it is believed that work(
Energy sensitivity is not higher than 0.2ng/ml.
2nd, the range of linearity is verified
Method：Use the high concentration sample of negative human serum gradient dilution definite value, diluted concentration covering 0.3-300ng/
Ml, upper limit lower limit each more than 20%, diluted sample is detected with CK-MB reagents；Straight line line is made using theoretical concentration and measure concentration
Property return, linearly dependent coefficient r >=0.975, straight line regression slope is in the range of 1 ± 0.05.
Detect data：
The range of linearity of table 4
The straight linear tropic relation of theoretical concentration and measure concentration, as shown in Figure 6.
Interpretation of result：In the range of 0.14-368ng/ml, slope disclosure satisfy that requirement with r values, therefore the range of linearity meets
0.3-300ng/ml。
3rd, methodology compares
Method：183 parts of heparin lithium plasma samples are collected, it is same with this reagent and reference reagent Beckman Dxi800CK-MB
When it is detected, observation and reference reagent segmentation coincidence rate and correlation.
Testing result：
Coincidence rate wherein less than 6.3ng/ml is 96.7%, and the coincidence rate more than 6.3ng/ml is 95.7%, is always met
Rate is 96.2%.
As shown in fig. 7, do correlation fitting with Beckman Dxi800 CK-MB testing results, coefficient correlation 0.99,
More than 0.95.
Kit detection range of the present invention is 0.3-300ng/ml；Linearly：Line taking range limit (about 300ng/ml)
Sample is diluted, and it is R=0.99 to calculate linear regression coefficient correlation；Methodology contrasts：Totally 183 clinical samples, are tried with this
Agent box and kit is listed while has been tested, both results enter Correlation series statistics, coefficient R=0.99.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (13)
1. a kind of creatine kinase isozyme magnetic microparticle chemiluminescence immue quantitative detection reagent box, it is characterised in that if including by dry doubling
The reagent strip for the hole position composition that row is set, the reagent that the reagent strip includes have CK-MB magnetic particles reagent, CK-MB mark reactions
Thing.
2. kit according to claim 1, it is characterised in that the CK-MB magnetic particles reagent is anti-containing being marked with
The magnetic microsphere of CK-MB monoclonal antibodies.
3. kit according to claim 1, it is characterised in that the CK-MB labeling reactions are containing acridinium ester mark
The anti-CK-MB monoclonal antibodies of note.
4. kit according to claim 1, it is characterised in that be provided with bar magnet set in the hole position on the reagent strip.
5. kit according to claim 1, it is characterised in that the kit is also comprising calibration object, magnetic bead cleaning fluid
And preexciting liquid.
6. kit according to claim 5, it is characterised in that the magnetic bead cleaning fluid is containing tween, preservative
Tris-HCl buffer solutions.
7. kit according to claim 5, it is characterised in that the preexciting liquid is containing hydrogen peroxide, salpeter solution.
8. kit according to claim 5, it is characterised in that the calibration object is containing a certain amount of CK-MB antigens
BSA protein solutions.
9. kit according to claim 5, it is characterised in that No. 0 position on the reagent strip is provided with bar magnet set；No. 3
Position is provided with CK-MB magnetic particle reagents；5th, 7,8, No. 9 positions are provided with magnetic bead cleaning fluid；No. 6 positions are provided with CK-MB labeling reactions；No. 10
Position is provided with preexciting liquid.
10. one kind kit as described in claim any one of 1-9 carries out creatine kinase isozyme magnetic microparticle chemiluminescence and quantified
The application method of detection, it is characterised in that during detection, reagent strip (4) is placed in and is incubated in storehouse (3), be incubated storehouse (3) are set
It is placed on conveyer belt (2), conveyer belt (2) the connection horizontal displacement motor (1), the end of the conveyer belt (2) is provided with analysis
Reading module (13)；And realized using rifle can be shifted with respect to the updraft type magnetic bead that bar magnet set (5) move up and down on the reagent strip
Bar magnet set, absorption magnetic bead, magnetic bead elution, magnetic bead mixing, bar magnet set is taken to beat de- act；
The updraft type magnetic bead shifts rifle, including can be connected with bar magnet set (5) interference extraction rifle pipette tips (12), positioned at institute
The bar magnet (15) for being used to adsorb magnetic bead (16) on extraction rifle pipette tips (12) axial line is stated, the bar magnet (15) is by extraction motor (10)
Drive and relatively described extraction rifle pipette tips (12) move up and down, the extraction motor (10) is installed on transfer rifle (11) top
End, and the extraction motor (10) is connected by motor shaft (14) with the bar magnet (15)；
Specific detection method comprises the following steps：
Step 1, project is selected：According to samples selection CK-MB projects, in reagent strip inserting instrument reaction channel, click starts；
Step 2, barcode scanning：Whether the Quick Response Code on instrument scanning reagent strip sealing aluminum membrane surface, confirm the information such as lot number, calibration curve
Match with the principal curve in software；
Step 3, puncture：Reagent strip runs to puncture position, and puncture module carries out puncture；
Step 4, bar magnet set is taken：Reagent strip runs to No. 0 bar magnet set position, takes bar magnet to cover 5 by shifting rifle；
Step 5, it is loaded：Sample loading gun gets tip heads, draws 50uL samples, is added in No. 4 holes of reagent strip；
Step 6, magnetic bead extracts：Bar magnet set plus magnetic, extract the magnetic bead in No. 3 holes；
Step 7, the first step is incubated：Magnetic bead is extracted to No. 4 holes, carries out first step reaction；
Step 8, magnetic bead extracts：After the completion of the first step is incubated, bar magnet set plus magnetic, the magnetic bead in No. 4 holes is extracted；
Step 9, magnetic bead cleans：Magnetic bead is extracted to No. 5 holes and carries out first time cleaning；
Step 10, second step is incubated：Magnetic bead is extracted to 6 labelled notations work fluid apertures, carries out second step reaction；
Step 11, magnetic bead cleans：Magnetic bead is extracted to 7,8, No. 9 holes and cleaned three times；
Step 12, detect：Magnetic bead is extracted to No. 10 preexciting fluid apertures, reagent strip is run to reading position, reading module light shield
Fall, add exciting liquid into No. 10 holes, while photomultiplier collects luminous signal；
Step 13, result of calculation：Software calibration curve according to corresponding to every batch of reagent, relative luminous intensity is substituted into curve,
Calculate concentration corresponding to sample.
11. the application method of kit according to claim 10, it is characterised in that the extraction rifle pipette tips (12) can be with institute
State inwall or its outer wall interference connection of bar magnet set (5).
12. the application method of kit according to claim 10, it is characterised in that the transfer rifle (11) is by being arranged at base
Length travel motor (6) driving on plate (7) moves up and down.
13. the application method of kit according to claim 12, it is characterised in that the length travel motor (6) it is defeated
Shaft is provided with screw rod (8), and the screw rod (8) is provided with nut (9), the nut (9) and the fixed company of the transfer rifle (11)
Connect.
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| CN108333357A (en) * | 2018-02-09 | 2018-07-27 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits |
| CN109490549A (en) * | 2018-10-19 | 2019-03-19 | 迪瑞医疗科技股份有限公司 | The chemical luminescent analysis reagent kid and preparation method thereof of creatine kinase isozyme in a kind of detection serum/plasma |
| CN110736740A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | homogeneous phase chemiluminescence POCT detection method and device using same |
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| CN109490549A (en) * | 2018-10-19 | 2019-03-19 | 迪瑞医疗科技股份有限公司 | The chemical luminescent analysis reagent kid and preparation method thereof of creatine kinase isozyme in a kind of detection serum/plasma |
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