CN102680689A - Kit for detecting creatine kinase isoenzyme and preparation and use methods thereof - Google Patents
Kit for detecting creatine kinase isoenzyme and preparation and use methods thereof Download PDFInfo
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- CN102680689A CN102680689A CN2011100573495A CN201110057349A CN102680689A CN 102680689 A CN102680689 A CN 102680689A CN 2011100573495 A CN2011100573495 A CN 2011100573495A CN 201110057349 A CN201110057349 A CN 201110057349A CN 102680689 A CN102680689 A CN 102680689A
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Abstract
The invention relates to a kit for detecting creatine kinase isoenzymes and preparation and use methods thereof. The kit for detecting creatine kinase isoenzymes comprises a substrate, as well as a sample pad, a conjugate pad, a coupling pad, a nitrocellulose membrane and water absorbent paper, which are arranged from one end to the other end on the substrate, wherein the conjugate pad is coated with a conjugate of fluorescence silicon dioxide nanoparticle-anti-creatine kinase isoenzyme MB monoclonal antibody, the nitrocellulose membrane is provided with a test line and a quality control line, the test line is arranged on the side close to the coupling pad and contains an anti-creatine kinase isoenzyme MB monoclonal antibody, and the quality control line is arranged on the side away from the coupling pad and contains goat anti-mouse IgG (Immunoglobulin G). The kit is low in price and has simple preparation process, and works with a high detection speed to provide the detection result within 15 minutes. The portable fluorescence detection device is adopted to achieve the purpose of accurate quantification of a target analyte. The kit is convenient to operate, can be applied to on-site detection, and is suitable for operators who do not need training. The kit has the advantages of high sensitivity, good specificity and accurate result, and is convenient for generalization and application.
Description
Technical field
The present invention relates to a kind of method of protein detection and kit, be specifically related to kit that detects isoenzymes of creatine kinase and preparation method thereof, also relate to the method that detects isoenzymes of creatine kinase with this kit.
Background technology
The myocardial damage mark be meant the biochemical that in blood circulation, can measure and can be responsive, the mark of reflecting myocardium damage (unusually) specifically and the order of severity thereof, thereby it can be used for examination, diagnosis, evaluation prognosis and the follow-up care effect of myocardial damage (unusually).Under normal circumstances, the myocardial damage mark is main or exist only in heart (cardiac muscle), under heart or Cardiovascular abnormality situation, is discharged in a large number by heart.Cardiac marker mainly comprises myoglobins, troponin (cTn I and cTn T), isoenzymes of creatine kinase (CK-MB) etc.Wherein CK-MB is to the diagnostic sensitivity height, particularly detection of dynamic of myocardial necrosis, and nineteen ninety-five CK-MB is considered to the diagnosis index of acute myocardial infarction AMI.This mark is after suffering from myocardial infarction, and 2-4 hour just begins to raise, and peaks in 24-36 hour, recovers normal in 3-4 days, and the degree of rising is directly proportional with the area of infarct.But increasing of CK-MB also arranged during Skeletal muscle injury, thereby its specificity is also relatively poor.The dynamic monitoring of CK-MB is commonly used to monitoring and has or not infraction again.And the detection of this mark can combine the index as diagnosing myocardial infarction with the detection of other myocardium marks.
The common method of current detection isoenzymes of creatine kinase 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, and it is longer to detect required time; Need special instrument and equipment such as ELIASA to be used, operating personnel need pass through professional training; It is higher to detect required expense, can not realize that single part is detected.Chemoluminescence method precision and sensitivity are good, but need specialized equipment, and instrument and reagent price are all comparatively expensive, are unwell to small hospital and medical center.Colloidal gold immunochromatographimethod is simple to operate, and it is short to detect required time, does not need the professional and technical personnel to operate, and testing cost is low, but this method can only realize qualitative or half-quantitative detection, and detection sensitivity is lower, can not detect the isoenzymes of creatine kinase under the super low concentration.
Summary of the invention
To the limitation of prior art in promoting the use of, the present invention provides a kind of high-sensitive detecting kit that adopts portable fluorescence detector just can realize detection by quantitative, and it uses simply, detection speed is fast, cost is low, can be used for the bedside diagnosis.
The kit of detection isoenzymes of creatine kinase provided by the invention comprises base plate; Be provided with sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, the thieving paper that connects successively from an end to the other end on the said base plate; The cross-linking agent pad has encapsulated the cross-linking agent of fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody; Said nitrocellulose filter is provided with test strip and Quality Control band; Said test strip is arranged near a side of bonding pad and contains anti-isoenzymes of creatine kinase MM monoclonal antibody, and said Quality Control band is arranged at away from a side of bonding pad and contains sheep anti-mouse igg.
Preferably, said sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper are respectively in that the junction is overlapped separately.
Preferably, said cross-linking agent pad covers on said sample pad and the bonding pad.
Preferably, said sample pad and bonding pad are docking together, and said cross-linking agent pad covers on said sample pad and the bonding pad.
The present invention also provides the preparation method of said kit, comprises the steps:
(1) preparation nano SiO 2 particle: the water-in-oil microemulsion system with containing cyclohexane, n-hexyl alcohol, Triton X-100 prepares nano SiO 2 particle;
(2) preparation fluorescent silicon dioxide nano particle: with said nano SiO 2 particle surface amination, pass through mode rare-earth fluorescent chelate on it is surface-crosslinked of covalent cross-linking again with the 3-aminopropyl trimethoxysilane;
(3) preparation fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent: will resist the oxidation of isoenzymes of creatine kinase MB monoclonal antibody with sodium metaperiodate; Again with the fluorescent silicon dioxide nano particle mixing of said antibody and surface amination, covalent reaction takes place and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent;
(4) preparation cross-linking agent pad: said fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent is encapsulated and on spun glass, processes the cross-linking agent pad;
(5) preparation nitrocellulose filter: will resist isoenzymes of creatine kinase MM monoclonal antibody and sheep anti-mouse igg setting respectively and encapsulate on the test strip of nitrocellulose filter and control band with spray film appearance, 37 ℃ of abundant dryings down.
(6) successively sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter and thieving paper are affixed on the PVC base plate with interconnecting.
Preferably; In said step (1), be 3: 1: 1 cyclohexane, Triton X-100, n-hexyl alcohol mixing with ratio, under agitation add ultrapure water, ammoniacal liquor and tetraethyl orthosilicate successively; Stir 24h; With the acetone sealing, adopt the washing of absolute ethyl alcohol and ultrapure water, promptly make nano SiO 2 particle;
In said step (2); Adopt the 3-aminopropyl trimethoxysilane to carry out amination the nano SiO 2 particle of step (1) gained; With adding BHHCT reaction 2h behind the absolute ethanol washing again, using and being suspended in 50mmol/L, pH behind the absolute ethanol washing is 7.8 Tris, adds EuCl
3Make its chelating on the BHHCT of particle surface, promptly obtain the fluorescent silicon dioxide nano particle through absolute ethanol washing again;
In said step (3); Adopting sodium metaperiodate will resist the hydroxyl oxidize of isoenzymes of creatine kinase MB monoclonal antibody sugar chain molecule is aldehyde radical; Again with the said fluorescent silicon dioxide nano particle mixing of this antibody and surface amination, covalent reaction takes place and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent.
Preferably, in said step (4) and (5), after said fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent is sprayed on the spun glass, with after the freeze dryer freeze drying as the cross-linking agent pad; To resist isoenzymes of creatine kinase MM monoclonal antibody and sheep anti-mouse igg to be sprayed onto on the test strip and control band of nitrocellulose filter with Biodot point film appearance, fully dry.
Preferably; Further said PVC base plate is cut into single part of test strips; In packing single part test strips into kit bar card supporting; Kit bar card and drying agent are put into aluminium foil bag, seal, the final fluorescent silicon dioxide nano particle kit that obtains to detect isoenzymes of creatine kinase.
The present invention also provides a kind of method that adopts described kit measurement isoenzymes of creatine kinase, may further comprise the steps:
(1) adds to sample drop on the said sample pad and drip dilution;
(2) isoenzymes of creatine kinase in the said sample is through combining the formation compound in the process of said cross-linking agent pad with fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody specificity;
(3) said compound combines with the anti-isoenzymes of creatine kinase MM monoclonal antibody and the sheep anti-mouse igg opposite sex in shortage with respect to said compound in the process process of said nitrocellulose filter successively;
(4) measure fluorescent value with fluorescence detector;
(5) if the fluorescent value of Quality Control band, shows then that testing result is effective greater than preset value; If the fluorescent value of Quality Control band, shows then that sample fails or experimental implementation are wrong less than preset value, testing result is invalid;
(6) if the fluorescent value of test strip and Quality Control band all greater than said preset value, show to have isoenzymes of creatine kinase in the sample, positive; If the fluorescent value of test strip is less than said preset value, the fluorescent value of Quality Control band shows not have isoenzymes of creatine kinase in the sample greater than preset value, and is negative.
The present invention also provides the another kind of method that adopts described kit measurement isoenzymes of creatine kinase, may further comprise the steps:
(1) adds to sample drop on the said sample pad and drip dilution;
(2) isoenzymes of creatine kinase in the said sample is through combining the formation compound in the process of said cross-linking agent pad with fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody specificity;
(3) said compound combines with the anti-isoenzymes of creatine kinase MM monoclonal antibody and the sheep anti-mouse igg opposite sex in shortage with respect to said compound in the process process of said nitrocellulose filter successively;
(4) gather fluoroscopic image with uviol lamp;
(5), show that then testing result is effective if fluorescence appears in the Quality Control band; If fluorescence does not appear in the Quality Control band, show that then sample fails or experimental implementation are wrong, testing result is invalid;
(6) if the equal Quality Control band of test strip and Quality Control band show to have isoenzymes of creatine kinase in the sample, positive; Fluorescence appears in the Quality Control band if fluorescence does not appear in test strip, shows not have isoenzymes of creatine kinase in the sample, and is negative.
Beneficial effect of the present invention is following.
This kit is cheap, and the preparation process is simple, and detection speed is fast, gets final product judged result in 15 minutes.Adopt portable fluorescence detector, can realize the accurate quantification of target analytes.It is easy to operate, can carry out the scene and detect, and operating personnel need not pass through training; Highly sensitive, specificity is good, and the result is accurate, conveniently promotes the use of.
Bonding pad in this kit plays buffer action.Make to encapsulate (especially freeze-drying) nanoparticle label thing on the cross-linking agent pad and fully dissolve, discharge, otherwise it is residual and cause detection signal and weaken to have part.
Description of drawings
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 for detecting the synoptic diagram as a result of isoenzymes of creatine kinase with kit of the present invention.Wherein scheming A is fluorescence shooting results under the ultraviolet light, and figure B detects the back by fluorescence detector and made the quantitative test curve map; Wherein the photoluminescence line of top is a test strip, and the photoluminescence line of below is the Quality Control band.
Embodiment
Below in conjunction with accompanying drawing specific embodiments of the present invention is described.
In one embodiment of the invention, kit comprises base plate, is provided with sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, the thieving paper that connects successively from an end to the other end on the said base plate.The cross-linking agent pad has encapsulated the cross-linking agent of fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody; Said nitrocellulose filter is provided with test strip and Quality Control band; Said test strip is arranged near a side of bonding pad and contains anti-isoenzymes of creatine kinase MM monoclonal antibody, and said Quality Control band is arranged at away from a side of bonding pad and contains sheep anti-mouse igg.
In an embodiment preferred of the present invention, in accompanying drawing 1, said sample pad and bonding pad are docking together, and said cross-linking agent pad covers on said sample pad and the bonding pad.
In an embodiment preferred of the present invention, prepare kit according to the following steps.(1) preparation silicon dioxide: with 30mL cyclohexane, 10mL Triton X-100 (Triton X-100), 10mL n-hexyl alcohol mixing; Under magnetic agitation, add an amount of ultrapure water, ammoniacal liquor and tetraethyl orthosilicate (TEOS) successively; After stirring 24h; Absolute ethyl alcohol and ultrapure water washing several times are adopted in the acetone sealing, can obtain the nano SiO 2 particle suspension.(2) preparation fluorescent silicon dioxide nano particle: get above-mentioned 1mL nano SiO 2 particle suspension and adopt the 3-aminopropyl trimethoxysilane to carry out amination; With adding an amount of BHHCT (chlorosulfonyl-adjacent diphenyl benzene) reaction 2h behind the absolute ethanol washing again; With being suspended in 50mmol/L Tris7.8 behind the absolute ethanol washing, add EuCl
3(europium chloride) makes its chelating on the BHHCT of particle surface, promptly obtains the fluorescent silicon dioxide nano particle through absolute ethanol washing again.(3) preparation of fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent: adopting the 0.5mol/L sodium metaperiodate is aldehyde radical with the hydroxyl oxidize of the anti-isoenzymes of creatine kinase MB of 0.4mg monoclonal antibody sugar chain molecule; Again with the fluorescent silicon dioxide nano particle mixing of this antibody and 2mg surface amination, covalent reaction takes place and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent.(that is, the Fc of antibody holds oxidized generation aldehyde radical, can be with the fluorescent silicon dioxide nano grain surface amino crosslinked, be fixed to particle surface thereby be directed.) (4) be sprayed on an amount of fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent on the spun glass; After the freeze dryer freeze drying as the cross-linking agent pad; With Biodot point film appearance 2mg/mL anti-isoenzymes of creatine kinase MM monoclonal antibody and 2mg/mL sheep anti-mouse igg are sprayed onto on the test strip and control band of nitrocellulose filter, fully dry.(5) cross-linking agent pad, nitrocellulose filter and undressed sample pad, bonding pad, thieving paper are affixed on the PVC base plate.(6) the above-mentioned PVC base plate that pastes is placed on the cutting cutter, cut into single part of test strips.(7) pack single part test strips into kit bar card supporting in.(8) kit bar card and drying agent are put into aluminium foil bag, seal, normal temperature is preserved.(9) test test strips to be checked is investigated its sensitivity, specificity and stability.The final fluorescent silicon dioxide nano particle test strips finished product that obtains to detect isoenzymes of creatine kinase.
Of the present invention another preferred embodiment in, said fluorescent silicon dioxide nano particle is modified the rare earth chelate (like BHHCT-Eu through the mode of covalent cross-linking after adopting the synthetic blank nano SiO 2 particle of water-in-oil microemulsion system more in its surface
3+), under uviol lamp, can observe the rare-earth fluorescent nano particle of intense fluorescence.
Said anti-isoenzymes of creatine kinase CK-MB antibody is purchased the monoclonal antibody (lot number M9910207) in U.S. Fitzgerald company, and said anti-isoenzymes of creatine kinase CK-MM antibody refers to purchase the monoclonal antibody (lot number X10032404) in U.S. Fitzgerald company.Isoenzymes of creatine kinase CK-MB purchases the standard antigen (lot number A10051201) in U.S. Fitzgerald company in the sensitivity experiment.
The method and the result that adopt kit of the present invention to detect judge as follows, referring to accompanying drawing 2.From aluminium foil bag, take out and detect test card, lie against on the operator's console, 70 microlitres sample to be checked is added in the well, add 50 microlitre damping fluids again, insert fluorescence detector behind the 15min and measure fluorescent value.If there is the isoenzymes of creatine kinase that will detect to exist in the sample, then the fluorescent value at test strip place can be greater than 10, and the fluorescent value of controlling band simultaneously also can be greater than 10, and this moment, the result was positive; If the isoenzymes of creatine kinase that will not detect in the sample exists, then the fluorescent value at test strip place is less than 10, but the fluorescent value at control band place is greater than 10, and this moment, the result was negative; If control band place fluorescent value is less than 10, then product is invalid.
In another embodiment of detection method of the present invention, adopt the mode judged result of fluorescence imaging under the ultraviolet light.Can test strips be placed under the uviol lamp, adopt the digital camera that has optical filter to gather fluoroscopic image.On the fluoroscopic image, if test strip all has the fluorescence band with the control band, then the result is positive; If only existing fluorescence band is taken in control out of, then the result is negative; If test strip the fluorescence band all do not occur with the control band, then product is invalid.
Fluorescence imaging is to be under the uviol lamp of 312nm and 360nm in centre wavelength under the said ultraviolet light, and the fluorescence that digital camera carried out that adopts camera lens to have optical filter (filter range 584-664nm) is taken.
The excitation wavelength of said fluorescence detector is 365nm, and emission wavelength is the quantitative instrument that can stick into capable fluorescent scanning to the kit bar of 625nm.
In the embodiment of another preparation kit of the present invention; Under magnetic agitation, add 30mL cyclohexane, 10mL Triton X-100,10mL n-hexyl alcohol to the triangular flask that has magnetite; Add 500 μ L ultrapure waters, 60 μ L ammoniacal liquor and 100 μ LTEOS down in stirring successively again; Add equal-volume acetone capping after stirring 24h; With absolute ethyl alcohol and ultrapure water washing several times, be suspended at last in the 1mL absolute ethyl alcohol, obtaining the milky turbid solution, to be synthetic particle diameter be the nano SiO 2 particle suspension about 80nm again.Again this lmL nano SiO 2 particle suspension is transferred in the round bottom bottle that has magnetite; In add under the magnetic agitation 3-aminopropyl trimethoxysilane with the nano SiO 2 particle finishing on amido functional group; Reaction is spent the night the back with adding 120 μ L BHHCT (4mg/mL) reaction 2h behind the absolute ethanol washing again; Behind absolute ethanol washing, be suspended in 50mmol/L Tris7.8 again, add 30 μ L EuCl
3(0.04mol/L) make its chelating on the BHHCT of particle surface, promptly obtain the fluorescent silicon dioxide nano particle with washing with alcohol again.After this again thereby this fluorescent silicon dioxide nano particle microballoon is carried out amination further with antibody linked, make its biologically active.Is aldehyde radical at first adopting the 0.5mol/L sodium metaperiodate when antibody linked with the hydroxyl oxidize of the anti-isoenzymes of creatine kinase MB of 0.4mg monoclonal antibody sugar chain molecule; Again with the fluorescent silicon dioxide nano particle mixing of this antibody and the above-mentioned surface amination of 2mg; Reaction is spent the night, fluorescent silicon dioxide nano particle and antibody generation covalent reaction and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent.Must be sprayed on the spun glass this cross-linking agent is even, every milliliter is sprayed 16 square centimeters, encloses aluminium foil bag after the freeze drying, preserves subsequent use under the room temperature.
On nitrocellulose filter, draw two parallel bands with machine, two bands are 8mm at interval, wherein sprays anti-isoenzymes of creatine kinase MM monoclonal antibody (being diluted to 2mg/mL with 10mmol/L pH 7.4PBS) near the band of spun glass one end and forms the detection band; Another band is sprayed sheep anti mouse two and is resisted formation control band, dries 6h down for 37 ℃, and inclosure aluminium foil bag room temperature preservation under dry environment is subsequent use.
On the single face plastic plate, post the nitrocellulose filter (long 30cm, wide 2.5cm) of parallel coated antibody prior to central authorities; Stick application of sample pad spun glass (long 30cm, wide 1.4cm) more successively, be sprayed with cross-linking agent pad glass fibre membrane (long 30cm, wide 0.8cm), bonding pad spun glass (the long 30cm of the antibody linked thing of fluorescent silicon dioxide nano particle; Wide 0.8cm); Stick thieving paper (long 30cm, wide 3.4cm) at last, all overlapping 0.2cm between film and the film; So that it closely links to each other each other, process the immunity-chromatography test cardboard.With cutting cutter the plastic plate that pastes vertically is cut into the wide immuno-chromatographic test paper strip of 4mm again, in the paper slip draw-in groove of packing into, is encapsulated into aluminium foil bag again, preserve subsequent use under the room temperature.
Use in the embodiment of kit detection at of the present invention another, take out the paper slip card of packed encapsulation, add 70 μ L serum samples in well; Add 50 μ L damping fluid (10mmol/L Tris-HCl 9.1,0.5%BSA, 0.4%NaCl again; 0.25%, 0.6%Triton-X 100), only need chromatography 15min; Then test card is inserted fluorescence paper slip detector and carry out the fluorescence signal collection, realize quantitative test.If the fluorescence value of reading that detects band and control band place on the film then is illustrated as positive findings all greater than 10, the content of the high more then myocardium mark isoenzymes of creatine kinase of the fluorescence value of reading of detection band is high more; If fluorescence value of reading of having only control band place is greater than 10, then explaining does not have isoenzymes of creatine kinase, negative result in the sample to be tested.If the fluorescence signal value of reading that detects band and control band place explains that all less than 10 detection is invalid.The sensitivity of this detection method is 2ng/mL, in batch with batch between repeatability better.
Claims (10)
1. kit that detects isoenzymes of creatine kinase; Said kit comprises base plate; Be provided with sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, the thieving paper that connects successively from an end to the other end on the said base plate; It is characterized in that the cross-linking agent pad has encapsulated the cross-linking agent of fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody, said nitrocellulose filter is provided with test strip and Quality Control band; Said test strip is arranged near a side of bonding pad and contains anti-isoenzymes of creatine kinase MM monoclonal antibody, and said Quality Control band is arranged at away from a side of bonding pad and contains sheep anti-mouse igg.
2. kit according to claim 1 is characterized in that, said sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter, thieving paper are respectively in that the junction is overlapped separately.
3. kit according to claim 2 is characterized in that, said cross-linking agent pad covers on said sample pad and the bonding pad.
4. kit according to claim 3 is characterized in that said sample pad and bonding pad are docking together, and said cross-linking agent pad covers on said sample pad and the bonding pad.
5. a method for preparing each said kit of claim 1-4 is characterized in that, comprises the steps:
(1) preparation nano SiO 2 particle: the water-in-oil microemulsion system with containing cyclohexane, n-hexyl alcohol, Triton X-100 prepares nano SiO 2 particle;
(2) preparation fluorescent silicon dioxide nano particle: with said nano SiO 2 particle surface amination, pass through mode rare-earth fluorescent chelate on it is surface-crosslinked of covalent cross-linking again with the 3-aminopropyl trimethoxysilane;
(3) preparation fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent: will resist the oxidation of isoenzymes of creatine kinase MB monoclonal antibody with sodium metaperiodate; Again with the fluorescent silicon dioxide nano particle mixing of said antibody and surface amination, covalent reaction takes place and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent;
(4) preparation cross-linking agent pad: said fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent is encapsulated and on spun glass, processes the cross-linking agent pad;
(5) preparation nitrocellulose filter: will resist isoenzymes of creatine kinase MM monoclonal antibody and sheep anti-mouse igg setting respectively and encapsulate on the test strip of nitrocellulose filter and control band with spray film appearance, 37 ℃ of abundant dryings down;
(6) successively sample pad, cross-linking agent pad, bonding pad, nitrocellulose filter and thieving paper are affixed on the PVC base plate with interconnecting.
6. method according to claim 5 is characterized in that,
In said step (1), be 3: 1: 1 cyclohexane, Triton X-100, n-hexyl alcohol mixing with ratio, under agitation add ultrapure water, ammoniacal liquor and tetraethyl orthosilicate successively; Stir 24h; With the acetone sealing, adopt the washing of absolute ethyl alcohol and ultrapure water, promptly make nano SiO 2 particle;
In said step (2); Adopt the 3-aminopropyl trimethoxysilane to carry out amination the nano SiO 2 particle of step (1) gained; With adding BHHCT reaction 2h behind the absolute ethanol washing again; Using and being suspended in 50mmol/L, pH value behind the absolute ethanol washing is 7.8 Tris, and adding EuCl3 makes its chelating on the BHHCT of particle surface, promptly obtains the fluorescent silicon dioxide nano particle through absolute ethanol washing again;
In said step (3); Adopting sodium metaperiodate will resist the hydroxyl oxidize of isoenzymes of creatine kinase MB monoclonal antibody sugar chain molecule is aldehyde radical; Again with the said fluorescent silicon dioxide nano particle mixing of this antibody and surface amination, covalent reaction takes place and form fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent.
7. method according to claim 6; It is characterized in that; In said step (4) and (5), after said fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody cross-linking agent is sprayed on the spun glass, with after the freeze dryer freeze drying as the cross-linking agent pad; To resist isoenzymes of creatine kinase MM monoclonal antibody and sheep anti-mouse igg to be sprayed onto on the test strip and control band of nitrocellulose filter with Biodot point film appearance, fully dry.
8. method according to claim 5; It is characterized in that; Further said PVC base plate is cut into single part test strips, pack single part of test strips into kit bar card supporting in, kit bar card and drying agent are put into aluminium foil bag; Seal the final fluorescent silicon dioxide nano particle kit that obtains to detect isoenzymes of creatine kinase.
9. a method that adopts the described kit measurement isoenzymes of creatine kinase of claim 1-4 is characterized in that, may further comprise the steps:
(1) adds to sample drop on the said sample pad and drip dilution;
(2) isoenzymes of creatine kinase in the said sample is through combining the formation compound in the process of said cross-linking agent pad with fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody specificity;
(3) said compound combines with the anti-isoenzymes of creatine kinase MM monoclonal antibody and the sheep anti-mouse igg opposite sex in shortage with respect to said compound in the process process of said nitrocellulose filter successively;
(4) measure fluorescent value with fluorescence detector;
(5) if the fluorescent value of Quality Control band, shows then that testing result is effective greater than preset value; If the fluorescent value of Quality Control band, shows then that sample fails or experimental implementation are wrong less than preset value, testing result is invalid;
(6) if the fluorescent value of test strip and Quality Control band all greater than said preset value, show to have isoenzymes of creatine kinase in the sample, positive; If the fluorescent value of test strip is less than said preset value, the fluorescent value of Quality Control band shows not have isoenzymes of creatine kinase in the sample greater than preset value, and is negative.
10. a method that adopts the described kit measurement isoenzymes of creatine kinase of claim 1-4 is characterized in that, may further comprise the steps:
(1) adds to sample drop on the said sample pad and drip dilution;
(2) isoenzymes of creatine kinase in the said sample is through combining the formation compound in the process of said cross-linking agent pad with fluorescent silicon dioxide nano particle-anti-isoenzymes of creatine kinase MB monoclonal antibody specificity;
(3) said compound combines with the anti-isoenzymes of creatine kinase MM monoclonal antibody and the sheep anti-mouse igg opposite sex in shortage with respect to said compound in the process process of said nitrocellulose filter successively;
(4) gather fluoroscopic image with uviol lamp;
(5), show that then testing result is effective if fluorescence appears in the Quality Control band; If fluorescence does not appear in the Quality Control band, show that then sample fails or experimental implementation are wrong, testing result is invalid;
(6) if the equal Quality Control band of test strip and Quality Control band show to have isoenzymes of creatine kinase in the sample, positive; Fluorescence appears in the Quality Control band if fluorescence does not appear in test strip, shows not have isoenzymes of creatine kinase in the sample, and is negative.
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| CN104374925A (en) * | 2014-11-28 | 2015-02-25 | 山东博科生物产业有限公司 | Creatine kinase isoenzyme detection reagent |
| CN105486859A (en) * | 2015-11-20 | 2016-04-13 | 润和生物医药科技(汕头)有限公司 | Novel improved immunochromatographic test strip, and preparation and application thereof |
| CN105974117A (en) * | 2016-05-04 | 2016-09-28 | 同昕生物技术（北京）有限公司 | CK-MB calibrating substance dilution solution and application thereof in clinic CK detection |
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| 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 |
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| CN106841633A (en) * | 2016-12-27 | 2017-06-13 | 郑乐民 | In the method and its purposes in saccharification Apolipoprotein A1 detection of fluorescent microsphere surface orientation coated antibody |
| CN112630420A (en) * | 2020-12-07 | 2021-04-09 | 北京科技大学 | Method for realizing directional coupling by using glycosyl of antibody and solid phase carrier material |
| CN114236119A (en) * | 2021-11-08 | 2022-03-25 | 润和生物医药科技(汕头)有限公司 | Colloidal gold rapid detection test paper for new crown total antibody and neutralizing antibody and preparation method thereof |
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