CN104360073A - Troponin diagnosis test paper strip for coupled immunomagnetic beads - Google Patents
Troponin diagnosis test paper strip for coupled immunomagnetic beads Download PDFInfo
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- CN104360073A CN104360073A CN201410597902.8A CN201410597902A CN104360073A CN 104360073 A CN104360073 A CN 104360073A CN 201410597902 A CN201410597902 A CN 201410597902A CN 104360073 A CN104360073 A CN 104360073A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention relates to a troponin diagnosis test paper strip for coupled immunomagnetic beads. The structure of the troponin diagnosis test paper strip is that an NC (nitrocellulose) film is arranged between a combined pad and an absorption pad and the NC film is an area in which a blood sample to be detected is dropped. The troponin diagnosis test paper strip is characterized in that the immunomagnetic beads are accommodated in the NC film; the immunomagnetic beads are magnetic beads with carboxyl on the surfaces, and are antibody-coupled immunomagnetic beads formed after coupling with a monoclonal antibody in an activated buffer solution. In an optimal scheme, the using amount of the antibody-coupled immunomagnetic beads and the blood sample to be detected are as follows: 4-5 microliters of 16A11 antibody-coupled immunomagnetic beads, 4-5 microliters of 810 antibody-coupled immunomagnetic beads, 2.5-3.5 microliters of blood sample to be detected, and 142-152 microliters of mixed buffer solution. The troponin diagnosis test paper strip for coupled immunomagnetic beads disclosed by the invention overcomes the defects in the prior art, so that troponin can be simply and quickly detected, the detection result is more accurate, the application field is wider, and the utilization rate for antibody raw materials is higher.
Description
Technical field
The present invention relates to a kind of medical science testing tool, be specifically related to a kind of troponin diagnosis test paper of coupling immunomagnetic beads.
Background technology
Troponin is a kind of protein molecule regulating myocardial contraction, is made up of TnC (TnC), TnT (TnT) and Troponin I (TnI) three subunits.Along with the development of cardiac biomarkers application, troponin has become the first-selected mark of diagnosing myocardial infarction (MI).The development of the clinical fast inspection (POCT) of this mark, can allow patient obtain treatment suitable in time.For the TnT test card of Roche, the critical value of cTnT is 0.1ng/ml, and the sensing range of test card is 0.1-2ng/ml, and detection time is 8-12 minute.
The immunological method of cTnT and cTnI is diagnosed mainly to include colloidal gold immunity chromatography and fluorescence method for POCT at present.These two kinds of methods can both adopt diagnosis test paper.
Colloidal gold diagnosis test strips prepared by colloidal gold immunity chromatography, its testing result is by being observed visually colour developing phenomenon.But the concentration of troponin cannot change accurate quantitative analysis by color.In addition, the connection of gold nano and antibody adopts passive adsorption method usually, although this reaction is simple and quick, the Fab of uncontrollable antibody holds the directivity at nano grain surface, and reduce antigen capture efficiency, the utilization factor of antagonist raw material also has a certain impact.
Fluorescence method and colloidal gold immunity chromatography similar, for gold nano grain, when fluorescent microsphere flows on diagnosis test paper, due to Microsphere Size comparatively large (200nm and more than), easily produce reunion; In flow process, microballoon-antibody connector there occurs the deposition of part when not arriving T line, cause stronger non-specific adsorption; When detecting, because fluorescence detector can only collect the light intensity signal on test strips surface, cannot reflect completely the situation after blood sample all flows through T line, making testing result not accurate.
Although semiconductor-quantum-point good light stability, emission band are narrower, quantum yield is higher, luminescence is adjustable, its potential bio-toxicity and interval photism (optical flare) limit the application in field of biological detection.
The crystal height crystallization degree of rare earth up-conversion luminescent material on the luminous power impact of material significantly.But improve operation and cost that high crystallization degree can increase synthetic material greatly.Supporting fluorescence detector requires very high to the wavelength coverage of exciting light, and the emissive power of conventional exciting light is on the low side, the manufacturing cost that result in a whole set of testing product comprising luminescent material and detecting device is higher, limits large-scale production and the popularization and application of such material.
Summary of the invention
The object of this invention is to provide a kind of improvement, the troponin diagnosis test paper of coupling immunomagnetic beads.This new diagnosis test paper overcomes the deficiencies in the prior art, can detect troponin quickly and easily, and testing result is more accurate, and application is wider, and the utilization factor of antagonist raw material is higher.
The technical scheme completing foregoing invention task is, a kind of troponin diagnosis test paper of coupling immunomagnetic beads, the structure of this diagnosis test paper is, NC film (nitrocellulose membrane) is provided with between pad and absorption pad, this NC film is the region instilling blood sample to be detected, it is characterized in that, in described NC film, containing immunomagnetic beads; This immunomagnetic beads refers to, iron nano-particle that diameter is about 200nm, that wrapped up by polystyrene, and there is carboxyl on its surface, and in activation buffer with monoclonal antibody coupling after the coupling that the forms immunomagnetic beads of antibody.
The coupling immunomagnetic beads of antibody, can referred to as " magnetic reactance ", and wherein, described coupled antibody can be 16A11 and/or 810.According to the difference of coupled antibody, can be called: 16A11 magnetic reactance or 810 magnetic reactances.
The described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are:
16A11 magnetic reactance 4-5 μ l, 810 magnetic reactance 4-5 μ l, blood sample 2.5-3.5 μ l, cocktail buffer 142-152 μ l to be measured.The concentration of immunomagnetic beads is 4mg/ml, and wherein the concentration of antibody on magnetic bead is not less than 10 μ g/mg, and cocktail buffer concentration is not higher than 0.1M.
Wherein, cocktail buffer is the NC film instilling this diagnosis test paper before detection together with blood sample to be measured.Unlike the prior art: magnetic reactance both can be fixed in the NC film of this diagnosis test paper; Also by after the outer mixed reaction of magnetic reactance and blood sample, damping fluid, can be directly loaded onto on NC film.
The application recommends following optimal proportion:
16A11 magnetic reactance 4.6 μ l, 810 magnetic reactance 4.6 μ l, blood sample 3 μ l, cocktail buffer 138 μ l to be measured.
The composition of above-mentioned cocktail buffer is: 2%BSA+1% tween+2.5% sucrose+0.3% PVP-K30, is dissolved in 10mM pH7.4 phosphate buffer.
In described test strips, NC choice of membrane pore size scope is: millipore N135, Sartorius N95.Sartorius N95 for magnetic reactance flowing and do not reunite and have good effect.
In described test strips, adsorptive pads thickness range of choice: GE Whatman CF6, prompt peaceful H1.When adopting H1, the detection signal difference of antigen under 0 ~ 1ng/ml concentration is obvious, and suggestion adopts.
The application recommends: described test strips, and its absorption pad, connection gap between pad and NC film control to be positioned at distance pad 42.3mm place at 2mm, T line position.
The preparation method of the troponin diagnosis test paper of above-mentioned coupling immunomagnetic beads, step is as follows:
Step (1)-step be (7) test strips make:
(1). test strips is 32 DEG C of dried overnight;
(2). taken out from refrigerator by NC film, clip suitable length puts equilibrium at room temperature at least 2h;
(3). join the antibody of desired concn, the 19C7 of 2mg/ml; Method: 19C7 antibody buffer dilutes;
(4). NC film is attached on base plate;
(5). leave standstill after first cleaning a circulation with rare NaOH before line, then clean a circulation with rare HCl, after leaving standstill, clean a circulation with pure water; Pen machine configures: 1.5 μ l/cm, with pencil by good for the T wire tag pulled;
(6). 32 ~ 37 DEG C of dried overnight;
(7). dry end, paste sample pad, adsorptive pads, AR film, slitting mounted box is for subsequent use subsequently;
Step (8)-step (9). be magnetic bead method for coating:
(8). activation:
Using 0.01M 2-(N-morpholino) ethanesulfonic acid buffer (MES buffer) as activation buffer, get the magnetic bead (solids content 10% that there is carboxyl on 10 μ l surfaces, Millipore company provides) add in centrifuge tube, MSE buffer washs, after being separated with magnetic separator, abandon supernatant; After washing 3 times, 242.5 μ l MES buffer are added in centrifuge tube, add freshly prepared 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide solution (EDC solution) 2.5 μ l and 0.25g/ml N-hydroxy-succinamide solution (NHS solution) 5 μ l again, vibration mixing, the carboxyl of room temperature backspin Transactivation magnetic bead surfaces, after reaction terminates, wash the activator removing reaction with MSE buffer, then wash magnetic bead with MSE buffer;
(9). coupling:
Using 0.02M BST buffer as the damping fluid of coupling process; Magnetic bead is washed with the BST buffer of 250 μ l; After having washed, add appropriate monoclonal antibody 16A11/810, then add BST buffer, keep liquor capacity in centrifuge tube to be 250 μ l; Vibration mixing, the carboxyl that magnetic bead surfaces is activated and the amino of antibody at room temperature revolving reaction; After coupling completes, magnetic separator separated and collected supernatant, is ready for use on detection coupling efficiency;
Step (10)-step is (11) for enclosure method:
(10). supernatant adds BST damping fluid and the glycine solution of 242.5 μ l, closed rotary after drawing in centrifuge tube;
(11). after magnetic separator divides and abandons supernatant, add the 1%(W/V of 250 μ l) BSA confining liquid (dilution of BST damping fluid), do not have the activated group of complete reaction to close to immunomagnetic beads surface;
(12). preserve:
BST buffer washs the magnetic bead closed, and is finally preserved by immunomagnetic beads and is resuspended in the immune microsphere conserving liquid of 250 μ l.
The troponin diagnosis test paper using method of coupling immunomagnetic beads of the present invention is as follows:
16A11 magnetic reactance 4.6 μ l, 810 magnetic reactance 4.6 μ l, blood sample 3 μ l to be measured, add 149.5 μ l cocktail buffer (2%BSA+1% tween+2.5% sucrose+0.3% PVP-K30, be dissolved in 10mM pH7.4 phosphate buffer) in, hybrid reaction adds the point sample mouth of test strips after 3 minutes.
Sample introduction 128 μ l, after 15 minutes, with Magnasense magnetic detector reading.
Illustrate:
1) cocktail buffer: 2%BSA (w/v)+1% Tween-20 (w/v)+2.5% sucrose (w/v)+0.3% PVP-K30 (w/v), is dissolved in 10mM pH7.4 phosphate buffer;
Wherein, BSA scope is 0.3 ~ 3%(w/v); Tween-20 scope is 0 ~ 2%(w/v); Sucrose scope 0.5 ~ 3% (w/v); PVP-K30 scope is 0. 1 ~ 1% (w/v);
2) detection time was unstable change before 15 minutes, and the numerical value detected when 15 minutes ~ 30 minutes is more stable.
More optimize and more particularly,
Step (1) described in test strips, (with reference to Fig. 1), its absorption pad, connection gap between pad and NC film control to be positioned at distance pad 42.3mm place at 2mm, T line position.
Step (2) described in " NC film is taken out from refrigerator ", be generally the refrigerator of 4 DEG C.
Step (3) described in 19C7 antibody buffer, it consists of: 10mM PBS ph7.4, containing 1% sucrose, the methyl alcohol of 3%.
Step (5) described in NaOH cleaning, be clean standing 10min after a circulation with the NaOH of 0.1M; Clean a circulation with 0.1M HCl again, after leaving standstill 10min, clean a circulation with pure water.
Step (5) described in line, during line, relative humid control is about 60%.
Step (5) described in leave standstill, recommend leave standstill 10min.
Step (5) described in clean with rare NaOH, be with the NaOH of 0.1M clean one circulation after leave standstill 10min;
Described cleans with rare HCl, is to clean a circulation with 0.1M HCl, cleans a circulation after leaving standstill 10min with pure water.
Step (6) described in 32 ~ 37 DEG C of dried overnight, be good with 32 DEG C.
Step (7) described in slitting mounted box for subsequent use, relative humidity controls about 35%.
Wherein,
1. NC choice of membrane pore size scope is: millipore N135, Sartorius N95.Sartorius N95 for magnetic reactance flowing and do not reunite and have good effect.
2. adsorptive pads thickness range of choice: GE Whatman CF6, prompt peaceful H1.When adopting H1, the detection signal difference of antigen under 0 ~ 1ng/ml concentration is obvious, and suggestion adopts.
Step (8) described in vibration mixing, be with vortex mixer vibration mixing.
Step (8) described in the carboxyl of room temperature backspin Transactivation magnetic bead surfaces, be generally 3-30 minute.
Wherein:
1) pH value of MES buffer is 5.0 ~ 6.4, the best results of wash-out magnetic bead when pH5.0.
2) during soak time 5 minutes, activation effect is best, and the connection amount of antibody had both met the needs of detection signal strength, saves antibody consumption again.
3) mol ratio of EDC and NHS is the concentration range of 1:2, EDC is 2 μm of ol ~ 2mmol in the concentration range of 1 μm of ol ~ 10mmol, NHS, and time wherein with EDC 2 μm of ol, NHS 4 μm of ol, activation effect is best.With reference to Fig. 2.
Step (9) described in vibration mixing, be with vortex mixer vibration mixing.
Wherein:
1) BST pH of cushioning fluid is greater than 4, and when selecting pH9, the coupling of antibody is best.
2) amount ranges of monoclonal antibody is 2 ~ 40 μ g, wherein during 10 μ g, can reach de-luxe compartment by efficiency and sensitive signal, economize in raw materials again.
3) the coupling revolving reaction time under room temperature should be greater than 1 hour, is best with 3 hours.With reference to Fig. 3.
The enclosure method that step is (11) described: be at room temperature revolving reaction 30 minutes.
Wherein:
1) according to the calculating of magnetic bead specific surface area, glycocoll consumption should be no more than total system.When volume is 7.5 μ l, can play the effect of adequate closure, consumption is most economical simultaneously.The concentration of glycocoll should be not less than 10mM, can the little site of adequate closure antibody when reaching 25mM, and magnetic reactance can be kept again not reunite.
2) the closed rotary glycocoll time is in 0.5 ~ 3 hour, and it is minimum that reaction in 0.5 hour closes impact to BSA afterwards.
3) concentration of BSA is 0.1 ~ 3%(w/v), 1% time, room temperature rotates and all can reach closed object in more than 15 minutes, and wherein room temperature 30 minutes is roughly the same with 2 ~ 10 degree of refrigeration effect of a day, is optimized parameter.With reference to Fig. 4.
Step (12) described BST buffer wash the magnetic bead closed, recommendation operation 4 times.
The composition of the immune microsphere conserving liquid that step is (12) described is: add in BST damping fluid in 0.1%BSA and 0.02% Sodium azide.
The troponin diagnosis test paper of coupling immunomagnetic beads of the present invention, overcomes the deficiencies in the prior art, can detect troponin quickly and easily, and testing result is more accurate, and application is wider, and the utilization factor of antagonist raw material is higher.
Magnetic signal detects more responsive, quantitatively can detect the sample of extremely low concentration.Using CRP: (the domestic magnetic detector that do not use is as the product of clinical fast inspection at present, and the product for analogy is the result that colloidal gold immunity chromatography and fluorescence method instrument detect).
Accompanying drawing explanation
Fig. 1 is the troponin diagnosis test paper structural representation of coupling immunomagnetic beads;
Fig. 2 affects histogram to antigen detection signal the magnetic bead surfaces activated carboxylic time;
Fig. 3 affects histogram on antigen detection signal after different quality antibody and magnetic bead coupling;
Fig. 4 is the dispersed effect of optimization figure of magnetic reactance.
Embodiment
Embodiment 1, a kind of troponin diagnosis test paper of coupling immunomagnetic beads, the structure of this diagnosis test paper is, is provided with NC film between pad and absorption pad, and this NC film is the region instilling blood sample to be detected, in described NC film, containing immunomagnetic beads; This immunomagnetic beads refers to: iron nano-particle that diameter is about 200nm, that wrapped up by polystyrene, and there is carboxyl on its surface, and in activation buffer with monoclonal antibody coupling after the coupling that the forms immunomagnetic beads of antibody.Wherein, described coupled antibody can be 16A11 and/or 810.According to the difference of coupled antibody, can be called: 16A11 magnetic reactance or 810 magnetic reactances.The described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4-5 μ l, 810 magnetic reactance 4-5 μ l, blood sample 2.5-3.5 μ l, cocktail buffer 138-152 μ l to be measured.Wherein, cocktail buffer is the NC film instilling this diagnosis test paper before detection together with blood sample to be measured.Unlike the prior art: magnetic reactance both can be fixed in the NC film of this diagnosis test paper; Also by after the outer mixed reaction of magnetic reactance and blood sample, damping fluid, can be directly loaded onto on NC film.
The preparation side of the troponin diagnosis test paper of above-mentioned coupling immunomagnetic beads:
Step (1)-step be (7) test strips make:
(1) .(is with reference to Fig. 1), absorption pad, connection gap between pad and NC film control to be positioned at distance pad 42.3mm place at 2mm, T line position; Test strips is 32 DEG C of dried overnight;
(2). taken out from 4 DEG C of refrigerators by NC film, clip suitable length puts equilibrium at room temperature at least 2h;
(3). join the antibody of desired concn, the 19C7 of 2mg/ml; Method: 19C7 antibody buffer(10mM PBS ph7.4, containing 1% sucrose, the methyl alcohol of 3%) dilution;
(4). NC film is attached on base plate;
(5). leave standstill 10min after first cleaning a circulation with 0.1M NaOH before line, then clean a circulation with 0.1M HCl, after leaving standstill 10min, clean a circulation with pure water; Pen machine configures: 1.5 μ l/cm, and with pencil by good for the T wire tag pulled, during line, relative humid control is about 60%;
(6). 32 ~ 37 DEG C of dried overnight; Be good with 32 DEG C.
(7). dry end, paste sample pad, adsorptive pads, AR film, slitting mounted box is for subsequent use subsequently; Relative humidity controls about 35%;
Step (8)-step (9). be magnetic bead method for coating: for 250 μ l systems
(8). activation:
Using 0.01M MES buffer as activation buffer, getting 10 μ l surfaces has the magnetic bead of carboxyl (solids content 10%, Millipore company provides) to add in 1.5ml centrifuge tube, and MSE buffer washs 3 times, after being separated, abandons supernatant upper clear supernate with magnetic separator; After washing 3 times, 242.5 μ l MES buffer are added in centrifuge tube, add freshly prepared EDC solution 2.5 μ l and 0.25g/ml NHS solution 5 μ l again, with vortex mixer vibration mixing, the carboxyl 3-30 minute of room temperature backspin Transactivation magnetic bead surfaces, after reaction terminates, wash the activator removing reaction with MSE buffer, then wash magnetic bead with MSE buffer; 2 times.With reference to Fig. 2.
(9). coupling:
Using 0.02M BST buffer as the damping fluid of coupling process; Wash magnetic bead 2 times with the BST buffer of 250 μ l, process is the same.After having washed, add appropriate monoclonal antibody 16A11/810, then add BST buffer, keep liquor capacity in centrifuge tube to be 250 μ l; Vortex mixer vibration mixing, the carboxyl that magnetic bead surfaces is activated and the amino of antibody at room temperature revolving reaction; After coupling completes, magnetic separator separated and collected supernatant upper clear supernate, is ready for use on detection coupling efficiency; With reference to Fig. 3.
Step (10)-step is (11) for enclosure method:
(10). supernatant upper clear supernate adds BST damping fluid and the glycine solution of 242.5 μ l, closed rotary after drawing in centrifuge tube;
(11). after magnetic separator divides and abandons supernatant upper clear supernate, add the 1%(W/V of 250 μ l) BSA confining liquid (dilution of BST damping fluid), do not have the activated group of complete reaction to close to immunomagnetic beads surface, revolving reaction 30 minutes under room temperature.With reference to Fig. 4.
(12). preserve: BST buffer washs the magnetic bead closed 4 times, finally immunomagnetic beads preservation is resuspended in the immune microsphere conserving liquid of 250 μ l.
Embodiment 2, substantially the same manner as Example 1, but the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4 μ l, 810 magnetic reactance 4 μ l, blood sample 2.5 μ l, cocktail buffer 142 μ l to be measured.
Embodiment 3, substantially the same manner as Example 1, but the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 5 μ l, 810 magnetic reactance 5 μ l, blood sample 3.5 μ l, cocktail buffer 152 μ l to be measured.
Embodiment 4, substantially the same manner as Example 1, but the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4 μ l, 810 magnetic reactance 5 μ l, blood sample 3.5 μ l, cocktail buffer 142 μ l to be measured.
Embodiment 5, substantially the same manner as Example 1, but the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 5 μ l, 810 magnetic reactance 4 μ l, blood sample 2.5 μ l, cocktail buffer 152 μ l to be measured.
Embodiment 6, substantially the same manner as Example 1, but the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4.6 μ l, 810 magnetic reactance 4.6 μ l, blood sample 3 μ l, cocktail buffer 147 μ l to be measured.
The composition of above-mentioned cocktail buffer is: 2%BSA+1% tween+2.5% sucrose+0.3% PVP-K30, is dissolved in 10mM pH7.4 phosphate buffer.
Embodiment 7 is substantially the same manner as Example 1, but the antibody of described coupling coupled by the immunomagnetic beads of antibody replaces with c reactive protein antibody (anti-CRP), for atherosclerotic detection.
Embodiment 8, substantially the same manner as Example 1, but the antibody of described coupling coupled by the immunomagnetic beads of antibody replaces with Procalcitonin antibody (anti-PCT), for distinguishing the detection of bacteriological infection and virus infections.
Embodiment 9, substantially the same manner as Example 1, but the antibody of described coupling coupled by the immunomagnetic beads of antibody replaces with LDL receptor antibody (anti-LDL), for the detection of coronary heart disease.
Claims (10)
1. a troponin diagnosis test paper for coupling immunomagnetic beads, the structure of this diagnosis test paper is, is provided with NC film between pad and absorption pad, this NC film is the region instilling blood sample to be detected, it is characterized in that, in described NC film, containing immunomagnetic beads; This immunomagnetic beads refers to, the iron nano-particle wrapped up by polystyrene, and there is carboxyl on its surface, and in activation buffer with monoclonal antibody coupling after the coupling that the forms immunomagnetic beads of antibody.
2. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 1, it is characterized in that, the composition of described cocktail buffer is: 2%BSA+1% tween+2.5% sucrose+0.3% PVP-K30, is dissolved in 10mM pH7.4 phosphate buffer.
3. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 1, is characterized in that, described coupled antibody, is 16A11 and/or 810; The described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4-5 μ l, and/or 810 magnetic reactance 4-5 μ l, blood sample 2.5-3.5 μ l, cocktail buffer 142-152 μ l to be measured.
4. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 3, it is characterized in that, the described coupling immunomagnetic beads of antibody and the ratio of blood sample to be detected are: 16A11 magnetic reactance 4.6 μ l, and/or 810 magnetic reactance 4.6 μ l, blood sample 3 μ l, cocktail buffer 138 μ l to be measured.
5. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 1, is characterized in that, the diameter that described immunomagnetic beads is is 200nm; The concentration of described immunomagnetic beads is 4mg/ml, and wherein the concentration of antibody on magnetic bead is not less than 10 μ g/mg, and cocktail buffer concentration is not higher than 0.1M.
6. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 1, is characterized in that, in described diagnosis test paper, NC choice of membrane pore size scope is: millipore N135, Sartorius N95; Described adsorptive pads thickness range of choice: GE Whatman CF6, or prompt peaceful H1.
7. according to the troponin diagnosis test paper of the coupling immunomagnetic beads one of claim 1-6 Suo Shu, it is characterized in that, described test strips, its absorption pad, connection gap between pad and NC film control to be positioned at distance pad 42.3mm place at 2mm, T line position.
8. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 7, is characterized in that, the troponin diagnosis test paper of described coupling immunomagnetic beads, refers to and prepare diagnosis test paper in accordance with the following methods:
Step (1)-step be (7) test strips make:
(1). test strips is 32 DEG C of dried overnight;
(2). taken out from refrigerator by NC film, clip suitable length puts equilibrium at room temperature at least 2h;
(3). join the antibody of desired concn, the 19C7 of 2mg/ml; Method: 19C7 antibody buffer dilutes;
(4). NC film is attached on base plate;
(5). leave standstill after first cleaning a circulation with rare NaOH before line, then clean a circulation with rare HCl, after leaving standstill, clean a circulation with pure water; Pen machine configures: 1.5 μ l/cm, with pencil by good for the T wire tag pulled;
(6). 32 ~ 37 DEG C of dried overnight;
(7). dry end, paste sample pad, adsorptive pads, AR film, slitting mounted box is for subsequent use subsequently;
Step (8)-step (9). be magnetic bead method for coating:
(8). activation:
Using 0.01M 2-(N-morpholino) ethanesulfonic acid buffer as activation buffer, getting 10 μ l surfaces has the magnetic bead of carboxyl (solids content 10%, Millipore company provides) to add in centrifuge tube, and MSE buffer washs, after being separated with magnetic separator, abandon supernatant; After washing 3 times, 242.5 μ l MES buffer are added in centrifuge tube, add freshly prepared 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide solution 2.5 μ l and 0.25g/ml N-hydroxy-succinamide solution (NHS solution) 5 μ l again, vibration mixing, the carboxyl of room temperature backspin Transactivation magnetic bead surfaces, after reaction terminates, wash the activator removing reaction with MSE buffer, then wash magnetic bead with MSE buffer;
(9). coupling:
Using 0.02M BST buffer as the damping fluid of coupling process; Magnetic bead is washed with the BST buffer of 250 μ l; After having washed, add appropriate monoclonal antibody 16A11/810, then add BST buffer, keep liquor capacity in centrifuge tube to be 250 μ l; Vibration mixing, the carboxyl that magnetic bead surfaces is activated and the amino of antibody at room temperature revolving reaction; After coupling completes, magnetic separator separated and collected supernatant, is ready for use on detection coupling efficiency;
Step (10)-step is (11) for enclosure method:
(10). supernatant adds BST damping fluid and the glycine solution of 242.5 μ l, closed rotary after drawing in centrifuge tube;
(11). after magnetic separator divides and abandons supernatant, add the 1%(W/V of 250 μ l) BSA confining liquid (dilution of BST damping fluid), do not have the activated group of complete reaction to close to immunomagnetic beads surface;
(12). preserve:
BST buffer washs the magnetic bead closed, and is finally preserved by immunomagnetic beads and is resuspended in the immune microsphere conserving liquid of 250 μ l.
9. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 8, is characterized in that,
Step (1) described in test strips, its absorption pad, connection gap between pad and NC film control to be positioned at distance pad 42.3mm place at 2mm, T line position;
Step (2) described in " NC film is taken out from refrigerator ", be the refrigerator of 4 DEG C;
Step (5) described in line, during line, relative humid control is 60%; Step (5) described in leave standstill, be standing 10min;
Step (6) described in dried overnight temperature be 32 DEG C;
Step (8) described in vibration mixing, be with vortex mixer vibration mixing; Step (9) described in vibration mixing, be with vortex mixer vibration mixing;
The (12) described BST buffer of step washs the magnetic bead closed, and is operation 4 times.
10. the troponin diagnosis test paper of coupling immunomagnetic beads according to claim 8 or claim 9, is characterized in that, step (1) described in test strips in, NC choice of membrane pore size scope is: millipore N135, Sartorius N95; Described adsorptive pads thickness range of choice: GE Whatman CF6 or prompt peaceful H1;
Step (8) described in wash-out, the pH value of MES buffer is 5.0 ~ 6.4; Described soak time is 5 minutes;
The mol ratio of described EDC and NHS is the concentration range of 1:2, EDC is 2 μm of ol ~ 2mmol in the concentration range of 1 μm of ol ~ 10mmol, NHS;
Step (9) described in BST pH of cushioning fluid when being 9; The amount ranges of monoclonal antibody is 10 μ g; The coupling revolving reaction time under room temperature should be 3 hours;
The enclosure method that step is (11) described, when glycocoll volume is 7.5 μ l; The concentration of glycocoll is 25mM; The closed rotary glycocoll time is 0.5 hour; The concentration of BSA is 0.1 ~ 3%(w/v); Room temperature rotational time is 30 minutes.
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