CN105116151A - Immunochromatographic test paper as well as preparation method and use method thereof - Google Patents
Immunochromatographic test paper as well as preparation method and use method thereof Download PDFInfo
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- CN105116151A CN105116151A CN201510541301.XA CN201510541301A CN105116151A CN 105116151 A CN105116151 A CN 105116151A CN 201510541301 A CN201510541301 A CN 201510541301A CN 105116151 A CN105116151 A CN 105116151A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/532—Production of labelled immunochemicals
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Abstract
The invention discloses immunochromatographic test paper which has oxidation-reduction enzyme activity and takes an alloy nano particle as a biomarker, as well as a preparation method and a use method for the test paper. The test paper comprises a sample absorption pad, a connection pad, a nitrocellulose membrane and a water absorption pad which are arrayed linearly on a base plate in sequence, wherein the connection pad is wrapped by Au-Pt alloy nano particle marking proteins and made from a glass fiber material; a quality control line and a detection line wrap the nitrocellulose membrane. A corresponding detection kit can be formed by adding an aniline, phenol or carbazole developing agent into the test paper. According to the test paper, the defects and shortcomings of the existing colloid detection technology can be avoided, and the detection sensitivity can be improved by 2-3 levels; furthermore, the experiment process is simple, convenient and high in operability; a product has great market potential, and a new and more effective measure is provided for the detection science.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper and preparation thereof, using method, particularly relate to a kind of possess oxidoreductase activity, be the immune chromatography test paper of biomarker by alloy nano particle, and the method for preparation and use of this test paper.
Background technology
Be that the chromatographic technique of representative has developed more than 20 year with collaurum, still widespread use at present, but due to qualitative or semiquantitative detection can only be used for, be difficult to the requirement meeting clinical detection indices quantification; Simultaneously testing result judges by naked eyes, particularly when testing result be the weak positive, very easily causes artificial detection leakage phenomenon, therefore there is the problems such as sensitivity is lower, needs perfect further.And some novel immunochromatography techniques do not exist collaurum need assemble the shortcoming that could develop the color in a large number, detection sensitivity can be improved with its distinctive signal amplifying system, minimizing sample background disturb, have the advantage that conventional tag thing is incomparable.
The technology that immunochromatography technique is most widely used as POCT has been deep into the various aspects such as disease, drugs, environment, and quick exactly, easy indoor and the field of being applicable to of its maximum advantage is detected.Collaurum is the main flow of application at present as mark always.It can direct visual results, without any need for equipment.Novel fluorescence, up-conversion, quantum dot are also development trends, which improve sensitivity, but need specific equipment and professional.
The main development direction of immunochromatography Fast Detection Technique has: 1. improve the sensitivity detected, reduce the gap between corresponding quantitative immunoassay.Improve the sensing range that sensitivity can widen immunochromatographiassays assays undoubtedly, and adopt signal assemble amplification system or adopt some novel markers, and may be one of most promising approach in conjunction with some corresponding easy detection instruments.2. realize sxemiquantitative and quantification.The result of qualitative detection only has the positive and feminine gender, and the content of some materials is often in certain range changing, only provides positive or negative, and result can not be satisfactory.Therefore immunochromatography technique is just towards future developments such as quantitative, high sensitivity.
Summary of the invention
The object of the present invention is to provide one can overcome prior art deficiency, not only can realize macroscopic qualitative detection, fetch equipment also can be utilized to complete the immune chromatography test paper of the quantitative detection of experimental result, and preparation method thereof and using method.
The preparation method of this immune chromatography test paper of the present invention is:
A. ultrapure water is added HAuCl successively
4, H
2ptCl
6solution and trisodium citrate, slowly drip NaBH after stirring and evenly mixing again in mixed liquor
4, make its color finally become peony from colourless purpling look, obtain Au-Pt alloy nano particle solution;
B. the pH adjusting Au-Pt alloy nano particle solution makes it be suitable for the albumen isoelectric point that will mark, then adds antibody used or antigen protein wherein, obtains the white solution of immune Au-Pt alloy nano particle mark egg;
C. Au-Pt alloy nano particle labelled protein glass fiber material is adsorbed, obtain the pad that immune Au-Pt alloy nano particle is coated with labelled antigen;
D. the antigen or the antibody that are used as quality control band and detection zone is determined respectively;
E. on the base plate of impermeable material, be linearly fixed with absorption of sample pad (absorbent material usually adopted is glass fiber material) that absorbent material makes successively, thieving paper that connection gasket, nitrocellulose membrane and absorbent material that the glass fiber material that is coated with Au-Pt alloy nano particle labelled protein is formed are made, and guarantee: between absorption of sample pad and connection gasket, connection gasket and cellulose nitrate is intermembranous and overlapped between nitrocellulose filter and adsorptive pads, formed and well connect;
F. spray as the antigen of quality control band or the nature controlling line of antibody on nitrocellulose membrane respectively and be used as the antigen of detection zone or the detection line of antibody, wherein detection line and nature controlling line line-to-line distance remain on a suitable distance, obtain test paper;
G. again test paper is cut into the strip of required width.
The method for optimizing that in preparation method of the present invention prepared by Au-Pt alloy nano particle solution is: in ultrapure water 40.0mL, add 490 μ L2.9 × 10 successively
-2mol/LHAuCl
4with 10 μ L2.9 × 10
-2mol/LH
2ptCl
6solution, and the trisodium citrate of 3.5mL10mg/mL stirring and evenly mixing, then the 0.5mg/mLNaBH slowly dripping that in mixed liquor 3.5mL newly prepares
4, be finally settled to 50mL with water, obtain Au-Pt alloy nano particle solution.
After determining concrete labelled protein and detection line, nature controlling line antigen or antibody, corresponding test paper can be prepared according to aforesaid preparation method.
Test paper using method of the present invention is: first the solution containing detected material antigen or antibody is dropped in glass fiber material region test paper being coated with Au-Pt alloy nano particle labelled protein and cellulose nitrate diaphragm area reacts, after question response terminates, phenyl amines is added again to the reaction zone on nitrocellulose membrane, phenol or carbazoles developer develop the color, can according to the yin and yang attribute whether test paper manifesting blue-black quality control band and detection zone determination test sample simultaneously, or recycling colour generation analyser or other fetch equipment are analyzed, draw quantitative or semiquantitative testing result.
Aforesaid test paper using method of the present invention can be used as a kind of detection method for non-diseases diagnosis.
Use test paper of the present invention, add the developer of phenyl amines or phenol or carbazoles, the immunochromatography detection box that a kind of alloy nano-material possessing oxidoreductase activity is label can be formed.
A kind of immune chromatography test paper that quantitatively can detect O type foot and mouth disease virus as one of test paper of the present invention concrete Test paper, it comprises: base plate, to be arranged on base plate linearly aligned absorption of sample pad successively, the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed, nitrocellulose membrane and thieving paper, nitrocellulose filter is coated with nature controlling line and detection line, between absorption of sample pad and connection gasket, connection gasket is intermembranous with cellulose nitrate and overlappedly between nitrocellulose filter and thieving paper be connected, wherein: the connection gasket albumen that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed is the O type type strain cavy antibody purification combined with Au-Pt alloy nano particle, detection line bag quilt be aftosa O type epidemic strain rabbit antibody purification, nature controlling line is goat-anti guinea pig antibodies.
Another concrete Test paper of the present invention is a kind of immune chromatography test paper that quantitatively can detect p53, it comprises: base plate, to be arranged on base plate linearly aligned absorption of sample pad successively, the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed, nitrocellulose membrane and thieving paper composition, nitrocellulose filter is coated with nature controlling line and detection line, between absorption of sample pad and connection gasket, connection gasket and cellulose nitrate is intermembranous and overlapped between nitrocellulose filter and thieving paper, wherein: the albumen on the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed is the anti-p53 antibody of rabbit combined with Au-Pt alloy nano particle, detection line bag quilt be mouse-anti p53 monoclonal antibody, nature controlling line is goat anti-rabbit antibody.
When the concrete Test paper of the present invention these two kinds uses, first by the solution containing detected material antigen or antibody, the reaction zone dropped in this test paper chromatographic film is reacted, after question response terminates, add phenyl amines, phenol or carbazoles developer to the reaction zone of nitrocellulose membrane again to develop the color, can according to the yin and yang attribute whether test paper manifesting blue-black quality control band and detection zone determination test sample simultaneously, or recycling colour generation analyser or other fetch equipment are analyzed, and draw quantitative or semiquantitative testing result.
It should be noted that test paper of the present invention is when the detection diagnosed for non-diseases, after the reaction zone reaction of detected material in test paper chromatographic film terminates, there is not corresponding colour developing in test paper, but add after phenyl amines, phenol or carbazoles developer develop the color to the reaction zone in chromatographic film again, quality control band can be made or/and detection zone manifests extremely significantly black-and-blue, make it not only can realize macroscopic qualitative detection, fetch equipment also can be utilized to complete the quantitative of experimental result or half-quantitative detection.This feature of the present invention can overcome the shortcoming and defect of existing colloid gold test paper detection technique, has again the irrealizable quantitative or half-quantitative detection function of existing colloid gold test paper detection technique institute simultaneously.
Current novel tracer material development is swift and violent, comprises rare earth element, fluorescent latex, fluorescent microsphere, quantum dot, magnetic bead etc.
Although the class enzymatic property of alloy is understood widely, its routine is applied to electrochemical field more, because it is colourless, without emission spectrum, so there is no people and selects it as the mark of immune chromatography test paper.Because can not visual inspection, equipment Inspection can not be used.
The present invention utilizes alloy to replace nm of gold as label, establishes this novel immune chromatography test paper.Can continue during this test paper application to keep colloid gold test paper to detect quick, easy, not need equipment and technician feature, can improve 100-1000 doubly than traditional gold size colour developing again, it is that following alloy material provides brand-new thinking in the application in immunochromatography technique field simultaneously.The invention has the advantages that: utilize alloy oxidation reductase activity first, its inventive application is tested in immune chromatography test paper.Testing result is made to have the enlarge-effect that significantly develops the color after being developed the color by TMB.Not only can realize macroscopic qualitative detection, fetch equipment also can be utilized to realize the quantitative of experimental result.Because spectrometer comes quantitatively, not need special design by shade, the test paper fetch equipment that therefore current any commercialization detects collaurum can be applied on it.The test paper establishment model that the foundation changing inventive method breaks traditions, has started immunochromatography mark and has selected new direction, the development of great-leap-forward that also made the application model of traditional colloid gold test paper have.Testing result is made significantly to be developed the color enlarge-effect after being developed the color by TMB.Avoid the shortcoming and defect of existing dot immune gold filtration assay, utilize the present invention can improve macroscopic qualitative analysis sensitivity, the raising of a sensitivity 2-3 order of magnitude can be realized.Due to the easy of its experimentation and operability, believe that this invention following can be used as the alternative method of alternative collaurum detection method and alternative detection kit, not only will have huge market potential, and provide a kind of more efficiently detection means newly for detection science.
Accompanying drawing explanation
Fig. 1 is test paper structure schematic diagram of the present invention, and wherein: end liner-1, nitrocellulose filter-2, thieving paper-3, is coated with the connection gasket-4 of the glass fibre membrane of alloy designation antigen, absorption of sample pad-5, quality control band-6 and detection zone-7.
Fig. 2 is Au-Pt alloy nano particle transmission electron microscope picture (left figure) and scanning electron microscope (SEM) photograph (right figure).
Fig. 3 is embodiment 1 test paper result schematic diagram.
Fig. 4 is embodiment 2 test paper result schematic diagram.
Embodiment
The present invention explains orally below in conjunction with two actual Test paper examples.
Embodiment 1_ with Au-Pt alloy nano-material for label quantitatively detects preparation and the use of the immune chromatography test paper of p53
The preparation of 1.Au-Pt alloy nano-material
Ultrapure water 40.0mL is placed in clean conical flask, under agitation adds 490 μ L2.9 × 10 successively
-2mol/LHAuCl
4with 10 μ L2.9 × 10
-2mol/LH
2ptCl
6solution, and the trisodium citrate of 3.5mL10mg/mL is in conical flask, stirs and evenly mixs, and slowly drips the 0.5mg/mLNaBH that 3.5mL newly prepares in mixed liquor
4, its color finally becomes peony from colourless purpling look, continues to stir 10min.Be settled to 50mL with water, obtain golden platinum Nanoalloy.
The preparation of 2.Au-Pt alloy nano particle mark p53 albumen
By Au-Pt alloy nano particle solution 0.1mol/LHCl or 0.02mol/LK obtained in steps A
2cO
3adjustment PH is 2-9, then in the Au-Pt alloy nano particle solution of optimum concentration 20ug/ml, the anti-p53 antibody of rabbit is added by the optimum mark amount of 6.5ug/ml, stirring at room temperature 1h, then add 10%BSA and hatch 30min, the PBS buffer solution of potpourri containing 1%BSA, be precipitated to abandon supernatant after the centrifugal 10min of 8000r/min, by precipitating the damping fluid being suspended in 1/10 initial Au-Pt alloy nano particle liquor capacity, (pH8.5 is containing 10mMPBS, 0.25%Tween-20,10% sucrose, 5%BSA) in, suspend and dissolve, for subsequent use, with coated glass fiber film 4.
3. contain the preparation of the nitrocellulose filter 2 of quality control band 6 and detection zone 7
Mouse-anti p53 monoclonal antibody is adjusted to 1mg/ml, and goat anti-rabbit antibody is adjusted to the working concentration of 0.5mg/ml, is sprayed on nitrocellulose filter 2 respectively, forms detection zone 7 and quality control band 637 DEG C of dry 2h, saves backup.
4. be coated with the preparation of the glass fibre membrane 4 of Au-Pt alloy nano particle
Get glass fibre membrane 4, the anti-p53 antibody of the rabbit marked by Au-Pt alloy nano particle is sprayed on glass fibre membrane 4, and form pad, 37 DEG C of dry 2h, save backup.
5. the preparation of absorption of sample pad 5
Absorption of sample pad 5 needs first with containing 10mMPBS, 0.1% (w/v) Tween-20, and 1% (w/v) PVP(polyvinylpyrrolidone) damping fluid of pH8.5 anticipates, and 37 DEG C of dry 2h, save backup.
6. the assembling of test paper
In end liner (1), generally adopt PVS material, on in turn mutually overlap joint ground paste absorption of sample pad 5, be coated with the glass fibre membrane 4 of Au-Pt alloy nano particle, nitrocellulose filter 2 and thieving paper 3, cut into the test paper of 4mm width as requested, namely make with Au-Pt alloy nano-material as label quantitatively detects the immune chromatography test paper of p53.
7. sample detection
Get 3 immune chromatography test papers, be respectively 1, Gold-immunochromatography assay test paper and Au-Pt alloy nano particle labeled immunochromatographyassay assay test 2.The sample solution of 50ul is dripped respectively on the absorption of sample pad of these test paper, after nitrocellulose filter completes antigen-antibody reaction, directly the reaction zone (TLINE and CLINE) of 1 Au-Pt alloy nano particle labeled immunochromatographyassay assay test adds the TMB solution through 20 × dilution wherein, after 10-20min, observations is see accompanying drawing 3.
In accompanying drawing 3 A, B, C, D respectively figure be the reaction result under different p53 protein concentration conditions, wherein: (A) 0ng/ml, (B) 10ng/ml, (C) 100ng/ml, (D) 500ng/ml.Three test paper are all had in each figure of A, B, C, D of Fig. 3, wherein one, left side is existing Gold-immunochromatography assay test paper, a middle situation being Au-Pt alloy nano particle labeled immunochromatographyassay assay test of the present invention and not adding when TMB develops the color after reacting with checking matter, right side one be Au-Pt alloy nano particle labeled immunochromatographyassay assay test of the present invention add after reacting with checking matter TMB develop the color after situation, the upper strap portion of each test paper is quality control band; Lower strip is detection zone.
8. contrast testing result:
For negative sample, no matter colloid gold test paper or test paper of the present invention all only has quality control band to develop the color, detection zone does not all develop the color, and the quality control band of colloid gold test paper is light red, and test paper of the present invention is then in black-and-blue.
For positive, when its concentration is 10ng/ml, colloid gold test paper still only has quality control band to show light red, and detection zone does not develop the color, and quality control band of the present invention is all with detection all can be significantly black-and-blue; When tested concentration is 100ng/ml, the quality control band of colloid gold test paper and detection zone can show light red, but the colour developing of its detection zone is not fairly obvious, and quality control band of the present invention and detection zone all present significantly black-and-blue; When the concentration of test sample is 250ng/ml, the quality control band of colloid gold test paper and detection zone all can show light red, but the colour developing of its quality control band is not fairly obvious, and quality control band of the present invention and detection zone all can present significantly black-and-blue.
Can be obtained by an easy colour generation analyser analysis result, with 1000 times that the sensing range of Au-Pt alloy nano particle labeled immunochromatographyassay assay test is Gold-immunochromatography assay test paper and blue latex particles labeled immunochromatographyassay assay test, it is 100 times of common magnetic nano-particle labeled immunochromatographyassay assay test.
Embodiment 2_ with Au-Pt alloy nano-material for label quantitatively detects preparation and the use of the immune chromatography test paper of O type foot and mouth disease virus
The preparation of 1.Au-Pt alloy nano-material
Ultrapure water 40.0mL is placed in clean conical flask, under agitation adds 490 μ L2.9 × 10 successively
-2mol/LHAuCl
4with 10 μ L2.9 × 10
-2mol/LH
2ptCl
6solution, and the trisodium citrate of 3.5mL10mg/mL is in conical flask, stirs and evenly mixs, and slowly drips the 0.5mg/mLNaBH that 3.5mL newly prepares in mixed liquor
4, its color finally becomes peony from colourless purpling look, continues to stir 10min.Be settled to 50mL with water, obtain golden platinum Nanoalloy.
2.Au-Pt the preparation of alloy nano particle mark O type hoof-and-mouth disease toxalbumin and antibody purification
By Au-Pt alloy nano particle solution 0.1mol/LHCl or 0.02mol/LK obtained in steps A
2cO
3adjustment PH is 2-9, then in the Au-Pt alloy nano particle solution of optimum concentration 20ug/ml, O type type strain cavy antibody purification is added by the optimum mark amount of 80ug/ml, stirring at room temperature 1h, then add 10%BSA and hatch 30min, the PBS buffer solution of potpourri containing 1%BSA, be precipitated to abandon supernatant after the centrifugal 10min of 8000r/min, by precipitating the damping fluid being suspended in 1/10 initial Au-Pt alloy nano particle liquor capacity, (pH8.5 is containing 10mMPBS, 0.25%Tween-20, 10% sucrose, 5%BSA), suspend and dissolve, for subsequent use, with coated glass fiber film 4.
3. contain the preparation of the nitrocellulose filter 2 of quality control band 6 and detection zone 7
Aftosa O type epidemic strain rabbit antibody purification is adjusted to 1.0-2.0mg/ml, and goat-anti guinea pig antibodies is adjusted to the working concentration of 0.5-1.0mg/ml, is sprayed on nitrocellulose filter 2 respectively, forms detection zone 7 and quality control band 6,37 DEG C of dry 2h, saves backup.
4. be coated with the preparation of the glass fibre membrane 4 of Au-Pt alloy nano particle
Get glass fibre membrane 4, the O type type strain cavy antibody purification marked by Au-Pt alloy nano particle is sprayed on glass fibre membrane 4, and form pad, 37 DEG C of dry 2h, save backup.
5. the preparation of absorption of sample pad 5
Absorption of sample pad 5 needs first with containing 10mMPBS, 0.1% (w/v) Tween-20, and 1% (w/v) PVP(polyvinylpyrrolidone) damping fluid of pH8.5 anticipates, and 37 DEG C of dry 2h, save backup.
6. the assembling of test paper
End liner 1 pastes absorption of sample pad 5 in turn mutually overlap joint, is coated with the glass fibre membrane 4 of Au-Pt alloy nano particle, nitrocellulose filter 2 and thieving paper 3, cut into the test paper of 4mm width as requested, namely make with Au-Pt alloy nano-material as label quantitatively detects the immune chromatography test paper of O type foot and mouth disease virus.
7. sample detection
Get 3 immune chromatography test papers, be respectively 1, Gold-immunochromatography assay test paper and Au-Pt alloy nano particle labeled immunochromatographyassay assay test 2.The sample solution of 50ul is dripped respectively on the absorption of sample pad of these test paper, after completing on antigen-antibody nitrocellulose filter reaction, directly the reaction zone (TLINE and CLINE) of 1 Au-Pt alloy nano particle labeled immunochromatographyassay assay test adds the TMB solution through 20 × dilution wherein, after 10-20min, observations is see accompanying drawing 4.In accompanying drawing 4, A, B, C, each figure are the reaction result under different O type foot and mouth disease virus concentration conditions, wherein: (A) 0ng/ml, and (B) 100ng/ml, (C) 200ng/ml.Three test paper are all had in each figure of A, B, C of Fig. 4, wherein one, left side is existing Gold-immunochromatography assay test paper, a middle situation being Au-Pt alloy nano particle labeled immunochromatographyassay assay test of the present invention and not adding when TMB develops the color after reacting with checking matter, right side one be Au-Pt alloy nano particle labeled immunochromatographyassay assay test of the present invention add after reacting with checking matter TMB develop the color after situation, the upper strap portion of each test paper is quality control band; Lower strip is detection zone.
8. result judges:
Positive: quality control band and detection zone all can develop the color; Negative: to only have quality control band to develop the color.
Contrast testing result:
For negative sample, no matter colloid gold test paper or test paper of the present invention all only has quality control band to develop the color, detection zone does not all develop the color, and the quality control band of colloid gold test paper is light red, and test paper of the present invention is then in black-and-blue.
For positive, when its concentration is 100ng/ml, colloid gold test paper still only has quality control band to show light red, and detection zone does not develop the color, and quality control band of the present invention is all with detection all can be significantly black-and-blue; When tested concentration is 200ng/ml, the quality control band of colloid gold test paper and detection zone all can show light red, but the colour developing of its quality control band is not fairly obvious, and quality control band of the present invention and detection zone all can present significantly black-and-blue.
Can be obtained by an easy colour generation analyser analysis result, with 1000 times that the sensing range of Au-Pt alloy nano particle labeled immunochromatographyassay assay test is Gold-immunochromatography assay test paper and blue latex particles labeled immunochromatographyassay assay test, it is 100 times of common magnetic nano-particle labeled immunochromatographyassay assay test.
Also can be found out by embodiment 1 and 2, when tested protein concentration increases, the colourity of its detection zone is deepened thereupon, as can be seen here, utilizes the present invention to pass through corresponding analyser, as CHR-100 colour generation analyser, can realize quantitatively or half-quantitative detection.
Claims (9)
1. the alloy nano-material possessing oxidoreductase activity is a preparation method for the immune chromatography test paper of label, it is characterized in that:
A. ultrapure water is added HAuCl successively
4, H
2ptCl
6solution and trisodium citrate, slowly drip NaBH after stirring and evenly mixing again in mixed liquor
4, make its color finally become peony from colourless purpling look, obtain Au-Pt alloy nano particle solution;
B. the pH adjusting Au-Pt alloy nano particle solution makes it be suitable for the albumen isoelectric point that will mark, then adds antibody used or antigen protein wherein, obtains the solution of immune Au-Pt alloy nano particle labelled protein;
C. Au-Pt alloy nano particle labelled protein glass fiber material is adsorbed, obtain the pad being coated with labelled antigen;
D. the antigen or the antibody that are used as quality control band and detection zone is determined respectively;
E. on the base plate of impermeable material, be linearly fixed with absorption of sample pad that absorbent material makes successively, adsorptive pads that connection gasket, nitrocellulose membrane and absorbent material that the glass fiber material that is coated with Au-Pt alloy nano particle labelled protein is formed are made, and guarantee: between absorption of sample pad and connection gasket, connection gasket and cellulose nitrate is intermembranous and overlapped between nitrocellulose filter and adsorptive pads, formed and well connect;
F. spray as the antigen of quality control band or the nature controlling line of antibody on nitrocellulose membrane respectively and be used as the antigen of detection zone or the detection line of antibody, wherein detection line and nature controlling line line-to-line distance remain on a suitable distance, obtain test paper;
G. again test paper is cut into the strip of required width.
2. preparation method according to claim 1, is characterized in that: first in ultrapure water 40.0mL, add 490 μ L2.9 × 10 successively
-2mol/LHAuCl
4with 10 μ L2.9 × 10
-2mol/LH
2ptCl
6solution, and the trisodium citrate of 3.5mL10mg/mL stirring and evenly mixing, then the 0.5mg/mLNaBH slowly dripping that in mixed liquor 3.5mL newly prepares
4, be finally settled to 50mL with water, obtain Au-Pt alloy nano particle solution.
3. the test paper prepared of claim 1 or 2 preparation method.
4. test paper using method according to claim 3, it is characterized in that first the solution containing detected material antigen or antibody being dropped on absorption of sample pad, the region being coated with the glass fiber material of Au-Pt alloy nano particle labelled protein on itself and test paper and cellulose nitrate diaphragm area are reacted, after question response terminates, phenyl amines is added again to cellulose nitrate diaphragm area, phenol or carbazoles developer develop the color, can according to the yin and yang attribute whether test paper manifesting blue-black quality control band and detection zone determination test sample simultaneously, or recycling colour generation analyser or other fetch equipment are analyzed, draw quantitative or semiquantitative testing result.
5. test paper using method according to claim 4 is used for the detection method of non-diseases diagnosis.
6. the alloy nano-material possessing oxidoreductase activity is an immunochromatography detection box for label, it is characterized in that detecting box includes test paper according to claim 3, and the developer of phenyl amines or phenol or carbazoles.
7. one kind quantitatively can be detected the immune chromatography test paper of O type foot and mouth disease virus, comprise: base plate, to be arranged on base plate linearly aligned absorption of sample pad successively, the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed, nitrocellulose membrane and thieving paper, nitrocellulose filter is coated with nature controlling line and detection line, between absorption of sample pad and connection gasket, connection gasket is intermembranous with cellulose nitrate and overlappedly between nitrocellulose filter and thieving paper be connected, it is characterized in that: the connection gasket albumen that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed is the O type type strain cavy antibody purification combined with Au-Pt alloy nano particle, detection line bag quilt be aftosa O type epidemic strain rabbit antibody purification, nature controlling line is goat-anti guinea pig antibodies.
8. one kind quantitatively can be detected the immune chromatography test paper of p53, comprise: base plate, to be arranged on base plate linearly aligned absorption of sample pad successively, the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed, nitrocellulose membrane and thieving paper composition, nitrocellulose filter is coated with nature controlling line and detection line, between absorption of sample pad and connection gasket, connection gasket is intermembranous with cellulose nitrate and overlappedly between nitrocellulose filter and thieving paper be connected, it is characterized in that: the albumen on the connection gasket that the glass fiber material being coated with Au-Pt alloy nano particle labelled protein is formed is the anti-p53 antibody of rabbit combined with Au-Pt alloy nano particle, detection line bag quilt be mouse-anti p53 monoclonal antibody, nature controlling line is goat anti-rabbit antibody.
9. the test paper using method described in claim 7 or 8, it is characterized in that the absorption of sample pad first dropped in by the solution containing detected material antigen or antibody on this test paper, the region of glass fiber material and the region of nitrocellulose filter that it is being coated with Au-Pt alloy nano particle are reacted, after question response terminates, phenyl amines is added again to the reaction zone on nitrocellulose filter, phenol or carbazoles developer develop the color, can according to the yin and yang attribute whether test paper manifesting blue-black quality control band and detection zone determination test sample simultaneously, or recycling colour generation analyser or other fetch equipment are analyzed, draw quantitative or semiquantitative testing result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510541301.XA CN105116151B (en) | 2015-08-28 | 2015-08-28 | A kind of immune chromatography test paper and its preparation, application method |
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