CN102680697B - Detect kit and the preparation and application thereof of Troponin I - Google Patents
Detect kit and the preparation and application thereof of Troponin I Download PDFInfo
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- CN102680697B CN102680697B CN201110057358.4A CN201110057358A CN102680697B CN 102680697 B CN102680697 B CN 102680697B CN 201110057358 A CN201110057358 A CN 201110057358A CN 102680697 B CN102680697 B CN 102680697B
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Abstract
The present invention relates to the kit and preparation and application thereof that detect Troponin I, the kit wherein detecting Troponin I comprises base plate, one end described base plate to the other end is provided with the sample pad connected successively, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper, cross-linking agent pad bag is by the cross-linking agent of fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody, described nitrocellulose filter is provided with test strip and Quality Control band, described test strip is arranged at the side near bonding pad and contains troponin I resisting monoclonal antibody, described Quality Control band is arranged at the side away from bonding pad and contains sheep anti-mouse igg.It adopts portable fluorescence detector just can realize the high-sensitive detecting kit quantitatively detected, and it uses simply, detection speed is fast, cost is low, can be used for bedside diagnosis.
Description
Technical field
The present invention relates to a kind of method of protein detection and kit, be specifically related to kit detecting Troponin I and preparation method thereof, also relate to the method detecting Troponin I with this kit.
Background technology
Myocardial injury markers refers to the biochemical that can measure in blood circulation and can responsive, reflecting myocardium damage (exception) and the order of severity thereof specifically mark, and thus it may be used for the examination of myocardial damage (exception), diagnosis, evaluation prognosis and follow-up care effect.Under normal circumstances, myocardial injury markers is main or exist only in heart (cardiac muscle), is discharged in a large number under heart or Cardiovascular abnormality situation by heart.Cardiac marker mainly comprises myoglobins, troponin (cTnI and cTnT), isoenzymes of creatine kinase (CK-MB) etc.Wherein cTnI with cTnT by US and European heart disease association consistent be chosen as diagnosing acute miocardial infarction high specific and hypersensitivity make a definite diagnosis mark.Early stage at myocardial cell injury, be free on intracytoplasmic cTnI and cTnT and discharge fast, in serum/plasma, 3-6h raises, and 8-14h peaks, and 1-2 week is down to normally.Thus the detection for early stage cTnI/cTnT contributes to for the quality time is won in the treatment of patient, and also has very large meaning to more late medical patient after morbidity.In recent years, research shows, also has the remarkable rising of cTnT when Skeletal muscle injury and renal function failure, cTnI then can not, thus cTnI is more suitable for the diagnosis marker as miocardial infarction, and specificity is stronger.
The common method of current detection cTnI mainly contains enzyme linked immunosorbent assay (ELISA) (ELISA), chemoluminescence method, colloidal gold immunochromatographimethod (GICA) etc.The detection sensitivity of enzyme linked immunosorbent assay (ELISA) is higher, but operating process is relatively complicated, detects required time longer; Need special instrument and equipment as microplate reader come with the use of, operating personnel need through professional training; Needed for detection costly, the detection of single part can not be realized.Chemoluminescence method precision and sensitivity good, but need specialized equipment, and instrument and reagent price are all costly, are unwell to small hospital and medical center.Colloidal gold immunochromatographimethod is simple to operate, and detect required time short, do not need professional and technical personnel to operate, testing cost is low, but this method can only realize qualitative or half-quantitative detection, and detection sensitivity is lower, the Troponin I under super low concentration can not be detected.
Summary of the invention
For the limitation of prior art in promoting the use of, the invention provides a kind of portable fluorescence detector that adopts and just can realize the high-sensitive detecting kit quantitatively detected, it uses simply, detection speed is fast, cost is low, can be used for bedside diagnosis.
The kit of detection Troponin I of the present invention comprises base plate, one end described base plate to the other end is provided with the sample pad connected successively, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper, cross-linking agent pad bag is by the cross-linking agent of fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody, described nitrocellulose filter is provided with test strip and Quality Control band, described test strip is arranged at the side near bonding pad and contains troponin I resisting monoclonal antibody, described Quality Control band is arranged at the side away from bonding pad and contains sheep anti-mouse igg.
Preferably, described sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper are overlapped in respective junction respectively.
Preferably, described cross-linking agent pad covers on described sample pad and bonding pad.
Preferably, described sample pad and bonding pad are docking together, and described cross-linking agent pad covers on described sample pad and bonding pad.
The present invention also provides the preparation method of described kit, comprises the steps:
(1) nano SiO 2 particle is prepared: prepare nano SiO 2 particle by the water-in-oil microemulsion system containing cyclohexane, n-hexyl alcohol, TritonX-100;
(2) fluorescent silicon dioxide nano particle is prepared: by 3-aminopropyl trimethoxysilane by described silica nanoparticle surface amination, then pass through mode rare-earth fluorescent chelate on it is surface-crosslinked of covalent cross-linking;
(3) fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent is prepared: be oxidized by mark troponin I resisting monoclonal antibody with sodium metaperiodate, again the fluorescent silicon dioxide nano particle of described antibody and surface amination is mixed, covalent reaction occurs and forms fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent;
(4) cross-linking agent pad is prepared: be coated on glass fibre by described fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent and make cross-linking agent pad;
(5) nitrocellulose filter is prepared: troponin I resisting monoclonal antibody and sheep anti-mouse igg arranged respectively with spray film instrument and be coated in the test strip of nitrocellulose filter and control on band, fully dry at 37 DEG C.
(6) successively sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter and thieving paper are affixed on Polyvinylchloride base plate with being interconnected.
Preferably, in described step (1), be the cyclohexane of 3: 1: 1, TritonX-100, n-hexyl alcohol mixing by ratio, under agitation add ultrapure water, ammoniacal liquor and tetraethyl orthosilicate successively, stir 24h, close with acetone, adopt absolute ethyl alcohol and milli-Q water, be i.e. obtained nano SiO 2 particle;
In described step (2), 3-aminopropyl trimethoxysilane is adopted to carry out amination the nano SiO 2 particle of step (1) gained, 2h is reacted with adding BHHCT after absolute ethanol washing again, with being suspended in 50mmol/L after absolute ethanol washing, pH value is the Tris of 7.8, adds EuCl
3make its chelating on the BHHCT of particle surface, more namely obtain fluorescent silicon dioxide nano particle through absolute ethanol washing;
In described step (3); sodium metaperiodate is adopted to be aldehyde radical by the hydroxyl oxidize of mark troponin I resisting monoclonal antibody sugar chain molecule; again the described fluorescent silicon dioxide nano particle of this antibody and surface amination is mixed, covalent reaction occurs and forms fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent.
Preferably, in described step (4) and (5), described fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent is sprayed on after on glass fibre, with after freeze dryer freeze drying as cross-linking agent pad; With Biodot point film instrument, bag is sprayed onto on the test strip of nitrocellulose filter and control band with troponin I resisting monoclonal antibody and sheep anti-mouse igg, fully dry.
Preferably, further described Polyvinylchloride base plate is cut into single part test strips, single part test strips is loaded in supporting kit bar card, kit bar card and drying agent are put into aluminium foil bag, sealing, the final fluorescent silicon dioxide nano particle kit obtaining detection Troponin I.
The present invention also provides a kind of method adopting described kit measurement Troponin I, comprises the following steps:
(1) sample drop to be added in described sample pad and to drip dilution;
(2) Troponin I in described sample forms compound with fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody specific binding in the process of described cross-linking agent pad;
(3) described compound combines with the troponin I resisting monoclonal antibody in shortage relative to described compound and the sheep anti-mouse igg opposite sex successively in the process through described nitrocellulose filter;
(4) fluorescent value is measured with fluorescence detector;
(5) if the fluorescent value of Quality Control band is greater than preset value, then show that testing result is effective; If the fluorescent value of Quality Control band is less than preset value, then show sample fails or experimental implementation wrong, testing result is invalid;
(6) if the fluorescent value of test strip and Quality Control band is all greater than described preset value, showing to there is Troponin I in sample, is the positive; If the fluorescent value of test strip is less than described preset value, the fluorescent value of Quality Control band is greater than preset value, shows to there is not Troponin I in sample, is feminine gender.
The present invention also provides the method for the kit measurement Troponin I described in another kind of employing, comprises the following steps:
(1) sample drop to be added in described sample pad and to drip dilution;
(2) Troponin I in described sample forms compound with fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody specific binding in the process of described cross-linking agent pad;
(3) described compound combines with the troponin I resisting monoclonal antibody in shortage relative to described compound and the sheep anti-mouse igg opposite sex successively in the process through described nitrocellulose filter;
(4) fluoroscopic image is gathered with uviol lamp;
(5) if fluorescence appears in Quality Control band, then show that testing result is effective; If there is not fluorescence in Quality Control band, then show sample fails or experimental implementation wrong, testing result is invalid;
(6) if test strip and the equal Quality Control band of Quality Control band, showing to there is Troponin I in sample, is the positive; If fluorescence does not appear in test strip and fluorescence appears in Quality Control band, showing to there is not Troponin I in sample, is feminine gender.
Beneficial effect of the present invention is as follows.
This kit is cheap, and preparation process is simple, and detection speed is fast, within 15 minutes, gets final product judged result.Adopt portable fluorescence detector, the accurate quantification of thing can be analyzed by realize target.It is easy to operate, can carry out Site Detection, and operating personnel do not need through training; Highly sensitive, specificity is good, and result is accurate, conveniently promotes the use of.
Bonding pad in this kit plays buffer action.Make to wrap and fully dissolved by (especially freeze-drying) nanoparticle label thing on cross-linking agent pad, discharge, otherwise it is residual and cause detection signal and weaken to have part.
Accompanying drawing explanation
Fig. 1 is the structural representation of kit of the present invention.Wherein, 1, base plate; 2, sample pad; 3, cross-linking agent pad; 4, bonding pad; 5, nitrocellulose filter; 6, thieving paper; 7, test strip; 8, Quality Control band.
Fig. 2 is the result schematic diagram detecting Troponin I with kit of the present invention.Wherein Fig. 2 A is fluorescence shooting results under ultraviolet light, and Fig. 2 B is rear by fluorescence detector detects is made quantitative test curve map; Wherein the photoluminescence line of top is test strip, and the photoluminescence line of below is Quality Control band.
Embodiment
Below in conjunction with accompanying drawing, specific embodiment of the invention scheme is described.
In one embodiment of the invention, kit comprises base plate, and the one end described base plate to the other end is provided with the sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, the thieving paper that connect successively.Cross-linking agent pad bag is by the cross-linking agent of fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody, described nitrocellulose filter is provided with test strip and Quality Control band, described test strip is arranged at the side near bonding pad and containing troponin I resisting monoclonal antibody, described Quality Control band is arranged at the side away from bonding pad and contains sheep anti-mouse igg.
In a preferred embodiment in accordance with this invention, with reference in accompanying drawing 1, described sample pad and bonding pad are docking together, and described cross-linking agent pad covers on described sample pad and bonding pad.
In a preferred embodiment in accordance with this invention, kit is prepared according to the following steps.(1) silicon dioxide is prepared: by 30mL cyclohexane, 10mLTritonX-100 (Triton X-100), the mixing of 10mL n-hexyl alcohol; add appropriate ultrapure water, ammoniacal liquor and tetraethyl orthosilicate (TEOS) under magnetic stirring successively; after stirring 24h; acetone is closed; adopt absolute ethyl alcohol and milli-Q water several times, nano SiO 2 particle suspension can be obtained.(2) fluorescent silicon dioxide nano particle is prepared: get above-mentioned 1mL nano SiO 2 particle suspension and adopt 3-aminopropyl trimethoxysilane to carry out amination; 2h is reacted with adding appropriate BHHCT (chlorosulfonyl-adjacent diphenyl benzene) after absolute ethanol washing again; be suspended in 50mmol/LTris7.8 with after absolute ethanol washing, add EuCl
3(europium chloride) makes its chelating on the BHHCT of particle surface, more namely obtains fluorescent silicon dioxide nano particle through absolute ethanol washing.(3) preparation of fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent: adopt 0.5mol/L sodium metaperiodate to be aldehyde radical by the hydroxyl oxidize of 0.4mg mark troponin I resisting monoclonal antibody sugar chain molecule; again the fluorescent silicon dioxide nano particle of this antibody and 2mg surface amination is mixed, covalent reaction occurs and forms fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent.(that is, the Fc of antibody holds oxidized generation aldehyde radical, can be cross-linked, thus be directed and be fixed to particle surface with the amino of fluorescent silicon dioxide nano grain surface.) appropriate fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent is sprayed on glass fibre by (4), as cross-linking agent pad after freeze dryer freeze drying, with Biodot point film instrument, bag is sprayed onto on the test strip of nitrocellulose filter and control band with 2mg/mL troponin I resisting monoclonal antibody and 2mg/mL sheep anti-mouse igg, fully dry.(5) cross-linking agent pad, nitrocellulose filter and undressed sample pad, bonding pad, thieving paper are affixed on PVC base plate.(6) the above-mentioned PVC base plate pasted is placed on cutting cutter, cuts into single part test strips.(7) single part test strips is loaded in supporting kit bar card.(8) kit bar card and drying agent are put into aluminium foil bag, sealing, normal temperature is preserved.(9) test test strips to be checked, investigate its sensitivity, specificity and stability.The final fluorescent silicon dioxide nano particle test strips finished product obtaining detection Troponin I.
Of the present invention another preferred embodiment in, after described fluorescent silicon dioxide nano particle adopts water-in-oil microemulsion system to synthesize blank nano SiO 2 particle, then modify Rare Earth Chelate in its surface (as BHHCT-Eu by the mode of covalent cross-linking
3+), under uviol lamp, can observe the rare-earth fluorescent nano particle of intense fluorescence.
Described mark anti-troponin I antibody is purchased from the monoclonal antibody (lot number M8010509) of Fitzgerald company of the U.S., describedly catch use (anti-troponin I antibody is fixed at the detection line place of test strips, for catching the troponin molecule in sample during chromatography) anti-troponin I antibody refers to monoclonal antibody (the lot number M8010521 being purchased from Fitzgerald company of the U.S., M0110609, M0110510), three is sprayed on nitrocellulose filter as test strip by after 2: 2: 1 mixings.In sensitivity experiment, cTnI is purchased from the standard antigen (lot number A01101802) of Fitzgerald company of the U.S..
The method adopting kit of the present invention to carry out detecting and result judge as follows, see accompanying drawing 2.From aluminium foil bag, take out Test paper card, lie against on operator's console, 70 microlitre measuring samples are added in well, then add 50 microliter of buffer liquid, insert fluorescence detector after 15min and measure fluorescent value.If there is the Troponin I that will detect to exist in sample, then the fluorescent value at test strip place can be greater than 10, and the fluorescent value simultaneously controlling band also can be greater than 10, and now result is positive; If the Troponin I that will not detect in sample, then the fluorescent value at test strip place is less than 10, but the fluorescent value controlling band place is greater than 10, and now result is negative; If control band place fluorescent value to be less than 10, then product is invalid.
In another embodiment of detection method of the present invention, the mode judged result of fluorescence imaging under employing ultraviolet light.Under test strips can being placed uviol lamp, the digital camera with optical filter is adopted to gather fluoroscopic image.On fluoroscopic image, if test strip all has fluorescent bands with control band, then result is positive; If only fluorescent bands appears in control cincture, then result is negative; If test strip does not all occur fluorescent bands with control band, then product is invalid.
Under described ultraviolet light, fluorescence imaging is under centre wavelength is the uviol lamp of 312nm and 360nm, adopts the fluorescence shooting that camera lens carries out with the digital camera of optical filter (filter range 584-664nm).
The excitation wavelength of described fluorescence detector is 365nm, and emission wavelength is the quantitative instrument that can carry out fluorescent scanning to kit bar card of 625nm.
Prepare in the embodiment of kit at of the present invention another; under magnetic agitation, 30mL cyclohexane, 10mLTritonX-100,10mL n-hexyl alcohol is added to the triangular flask with magnetite; 500 μ L ultrapure waters, 60 μ L ammoniacal liquor and 100 μ LTEOS are added successively again under stirring; equal-volume acetone capping is added after stirring 24h; use absolute ethyl alcohol and milli-Q water several times again; finally be suspended in 1mL absolute ethyl alcohol, obtain the nano SiO 2 particle suspension that particle diameter that milky turbidity liquid is synthesis is about 80nm.Again this lmL nano SiO 2 particle suspension is transferred in the round bottom bottle with magnetite; under magnetic agitation, add 3-aminopropyl trimethoxysilane silica nanoparticle surface is modified upper amido functional group; reaction reacts 2h with adding 120 μ LBHHCT (4mg/mL) after absolute ethanol washing again after spending the night; after absolute ethanol washing, settling flux is in 50mmol/LTris7.8, adds 30 μ LEuCl
3(0.04mol/L) make its chelating on the BHHCT of particle surface, more namely obtain fluorescent silicon dioxide nano particle with ethanol washing.After this again this fluorescent silicon dioxide nano particle microballoon is carried out amination thus further with antibody linked, makes it have biologically active.Being aldehyde radical with the hydroxyl oxidize first adopting 0.5mol/L sodium metaperiodate by 0.4mg troponin I resisting monoclonal antibody sugar chain molecule time antibody linked; again the fluorescent silicon dioxide nano particle of this antibody and the above-mentioned surface amination of 2mg is mixed; reaction is spent the night, fluorescent silicon dioxide nano particle and antibody generation covalent reaction and form fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent.Must spray even for this cross-linking agent on the glass fibers, every milliliter is sprayed 16 square centimeters, encloses aluminium foil bag, save backup under room temperature after freeze drying.
Nitrocellulose filter draws two parallel bands with machine, two band interval 8mm, the band wherein near glass fibre one end is sprayed troponin I resisting monoclonal antibody (being diluted to 2mg/mL with 10mmol/LpH7.4PBS) and is formed detection zone; Another band sprays the anti-formation control band of sheep anti mouse two, dries 6h at 37 DEG C, and inclosure aluminium foil bag room temperature preservation under dry environment is for subsequent use.
On one side plastic plate, nitrocellulose filter (the long 30cm of parallel coated antibody is posted prior to central authorities, wide 2.5cm), stick application of sample pad glass fibre (long 30cm more successively, wide 1.4cm), be sprayed with cross-linking agent pad glass fibre membrane (the long 30cm of the antibody linked thing of fluorescent silicon dioxide nano particle, wide 0.8cm), bonding pad glass fibre (long 30cm, wide 0.8cm), finally stick thieving paper (long 30cm, wide 3.4cm), equal overlapping 0.2cm between film with film, to make it closely be connected each other, makes immunity-chromatography test cardboard.With cutting cutter, the plastic plate pasted longitudinally is cut into the wide immuno-chromatographic test paper strip of 4mm again, loads in paper slip draw-in groove, then be encapsulated into aluminium foil bag, save backup under room temperature.
In the embodiment that another uses kit to detect of the present invention, take out the paper slip card of packed encapsulation, add 70 μ L serum samples in well, then add 50 μ L damping fluid (10mmol/LTris-HCl9.1,0.5%BSA, 0.4%NaCl, 0.25%, 0.6%Triton-X100), only need chromatography 15min, then test card is inserted fluorescence paper slip detector and carry out fluorescence signal acquisition, realize quantitative test.If the fluorescence at detection zone and control cincture place is read value and is all greater than 10 on film, be then illustrated as positive findings, the content that the fluorescence of detection zone reads to be worth higher then Applications of Cardiac Markers cTnI is higher; If only have the fluorescence at control cincture place to read value be greater than 10, then illustrating in sample to be tested do not have cTnI, is negative findings.If the fluorescence signal at detection zone and control cincture place reads value be all less than 10, illustrate that detection is invalid.The sensitivity of this detection method is 2ng/mL, in batch and batch between repeatability better.
Claims (7)
1. one kind is detected the kit of Troponin I, described kit comprises base plate, one end described base plate to the other end is provided with the sample pad connected successively, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper, it is characterized in that, cross-linking agent pad bag is by the cross-linking agent of fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody, described nitrocellulose filter is provided with test strip and Quality Control band, described test strip is arranged at the side near bonding pad and contains troponin I resisting monoclonal antibody, described Quality Control band is arranged at the side away from bonding pad and contains sheep anti-mouse igg,
Described sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper are overlapped in respective junction respectively;
Described cross-linking agent pad covers on described sample pad and bonding pad;
Described sample pad and bonding pad are docking together, and described cross-linking agent pad covers on described sample pad and bonding pad; Described link paves buffer action, makes the nanoparticle label thing be coated on cross-linking agent pad fully dissolve, discharge, and the material of described bonding pad is glass fibre.
2. prepare a method for kit described in claim 1, it is characterized in that, comprise the steps:
(1) nano SiO 2 particle is prepared: prepare nano SiO 2 particle by the water-in-oil microemulsion system containing cyclohexane, n-hexyl alcohol, TritonX-100;
(2) fluorescent silicon dioxide nano particle is prepared: by 3-aminopropyl trimethoxysilane by described silica nanoparticle surface amination, then pass through mode rare-earth fluorescent chelate on it is surface-crosslinked of covalent cross-linking;
(3) fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent is prepared: be oxidized by mark troponin I resisting monoclonal antibody with sodium metaperiodate, again the fluorescent silicon dioxide nano particle of described antibody and surface amination is mixed, covalent reaction occurs and forms fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent;
(4) cross-linking agent pad is prepared: be coated on glass fibre by described fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent and make cross-linking agent pad;
(5) nitrocellulose filter is prepared: troponin I resisting monoclonal antibody and sheep anti-mouse igg arranged respectively with spray film instrument and be coated in the test strip of nitrocellulose filter and control on band, fully dry at 37 DEG C;
(6) successively sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter and thieving paper are affixed on Polyvinylchloride base plate with being interconnected.
3. method according to claim 2, it is characterized in that, in described step (1), be the cyclohexane of 3: 1: 1, TritonX-100, n-hexyl alcohol mixing by ratio, under agitation add ultrapure water, ammoniacal liquor and tetraethyl orthosilicate successively, stir 24h, close with acetone, adopt absolute ethyl alcohol and milli-Q water, i.e. obtained nano SiO 2 particle; In described step (2), 3-aminopropyl trimethoxysilane is adopted to carry out amination the nano SiO 2 particle of step (1) gained, 2h is reacted with adding BHHCT after absolute ethanol washing again, with being suspended in 50mmol/L after absolute ethanol washing, pH value is the Tris of 7.8, adding EuCl3 makes its chelating on the BHHCT of particle surface, more namely obtains fluorescent silicon dioxide nano particle through absolute ethanol washing; In described step (3); sodium metaperiodate is adopted to be aldehyde radical by the hydroxyl oxidize of mark troponin I resisting monoclonal antibody sugar chain molecule; again the described fluorescent silicon dioxide nano particle of this antibody and surface amination is mixed, covalent reaction occurs and forms fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent.
4. method according to claim 3, it is characterized in that, in described step (4) and (5), described fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody cross-linking agent is sprayed on after on glass fibre, with after freeze dryer freeze drying as cross-linking agent pad; With Biodot point film instrument, bag is sprayed onto on the test strip of nitrocellulose filter and control band with troponin I resisting monoclonal antibody and sheep anti-mouse igg, fully dry.
5. method according to claim 2, it is characterized in that, further described Polyvinylchloride base plate is cut into single part test strips, single part test strips is loaded in supporting kit bar card, kit bar card and drying agent are put into aluminium foil bag, sealing, the final fluorescent silicon dioxide nano particle kit obtaining detection Troponin I.
6. adopt a method for kit measurement Troponin I according to claim 1, it is characterized in that, comprise the following steps:
(1) sample drop to be added in described sample pad and to drip dilution;
(2) Troponin I in described sample forms compound with fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody specific binding in the process of described cross-linking agent pad;
(3) described compound combines with the troponin I resisting monoclonal antibody in shortage relative to described compound and the sheep anti-mouse igg opposite sex successively in the process through described nitrocellulose filter;
(4) fluorescent value is measured with fluorescence detector;
(5) if the fluorescent value of Quality Control band is greater than preset value, then show that testing result is effective; If the fluorescent value of Quality Control band is less than preset value, then show sample fails or experimental implementation wrong, testing result is invalid;
(6) if the fluorescent value of test strip and Quality Control band is all greater than described preset value, showing to there is Troponin I in sample, is the positive; If the fluorescent value of test strip is less than described preset value, the fluorescent value of Quality Control band is greater than preset value, shows to there is not Troponin I in sample, is feminine gender.
7. adopt a method for kit measurement Troponin I according to claim 1, it is characterized in that, comprise the following steps:
(1) sample drop to be added in described sample pad and to drip dilution;
(2) Troponin I in described sample forms compound with fluorescent silicon dioxide nano particle-troponin I resisting monoclonal antibody specific binding in the process of described cross-linking agent pad;
(3) described compound combines with the troponin I resisting monoclonal antibody in shortage relative to described compound and the sheep anti-mouse igg opposite sex successively in the process through described nitrocellulose filter;
(4) fluoroscopic image is gathered with uviol lamp;
(5) if fluorescence appears in Quality Control band, then show that testing result is effective; If there is not fluorescence in Quality Control band, then show sample fails or experimental implementation wrong, testing result is invalid;
(6) if test strip and Quality Control band all occur fluorescence, showing to there is Troponin I in sample, is the positive; If fluorescence does not appear in test strip and fluorescence appears in Quality Control band, showing to there is not Troponin I in sample, is feminine gender.
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| CN103217535A (en) * | 2013-03-18 | 2013-07-24 | 杭州德安奇生物工程有限公司 | Immunochromatography detection card for detecting troponin I |
| CN104730251B (en) * | 2014-11-28 | 2016-11-23 | 威海纽普生物技术有限公司 | Troponin I detection kit and detection method |
| CN104730231B (en) * | 2015-03-26 | 2016-06-08 | 北京乐普医疗科技有限责任公司 | A kind of sample buffer for fluorescence immunoassay detection by quantitative and application thereof |
| CN105486859B (en) * | 2015-11-20 | 2019-03-15 | 润和生物医药科技(汕头)有限公司 | A kind of novel improved immuno-chromatographic test paper strip and its preparation and application |
| CN106198954A (en) * | 2016-07-21 | 2016-12-07 | 王雅 | A kind of sample buffer for fluorescence immunoassay detection by quantitative |
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| CN201464478U (en) * | 2009-03-05 | 2010-05-12 | 艾博生物医药(杭州)有限公司 | Improved detection device |
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