CN102072956A - Nano influenza virus testing paper - Google Patents
Nano influenza virus testing paper Download PDFInfo
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- CN102072956A CN102072956A CN2010105953284A CN201010595328A CN102072956A CN 102072956 A CN102072956 A CN 102072956A CN 2010105953284 A CN2010105953284 A CN 2010105953284A CN 201010595328 A CN201010595328 A CN 201010595328A CN 102072956 A CN102072956 A CN 102072956A
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- 238000012360 testing method Methods 0.000 title claims abstract description 18
- 241000712461 unidentified influenza virus Species 0.000 title claims description 47
- 238000001514 detection method Methods 0.000 claims abstract description 65
- 239000011246 composite particle Substances 0.000 claims abstract description 47
- 239000012528 membrane Substances 0.000 claims abstract description 26
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000010931 gold Substances 0.000 claims abstract description 22
- 229910052737 gold Inorganic materials 0.000 claims abstract description 22
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 19
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 19
- 241000700605 Viruses Species 0.000 claims abstract description 18
- 239000000427 antigen Substances 0.000 claims abstract description 9
- 102000036639 antigens Human genes 0.000 claims abstract description 9
- 108091007433 antigens Proteins 0.000 claims abstract description 9
- 108010044434 Alpha-methylacyl-CoA racemase Proteins 0.000 claims abstract description 8
- 102100040410 Alpha-methylacyl-CoA racemase Human genes 0.000 claims abstract description 8
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- 102000007469 Actins Human genes 0.000 claims abstract description 7
- 108010085238 Actins Proteins 0.000 claims abstract description 7
- 239000002245 particle Substances 0.000 claims abstract description 4
- 206010022000 influenza Diseases 0.000 claims abstract 6
- 239000011859 microparticle Substances 0.000 claims abstract 3
- 238000005070 sampling Methods 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 30
- 241000712431 Influenza A virus Species 0.000 claims description 18
- 239000004005 microsphere Substances 0.000 claims description 6
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- 238000001179 sorption measurement Methods 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 150000001718 carbodiimides Chemical class 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims 1
- 239000004698 Polyethylene Substances 0.000 claims 1
- 125000003172 aldehyde group Chemical group 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 239000003446 ligand Substances 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 239000002985 plastic film Substances 0.000 claims 1
- -1 polyethylene Polymers 0.000 claims 1
- 229920000573 polyethylene Polymers 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 239000003761 preservation solution Substances 0.000 claims 1
- 238000002331 protein detection Methods 0.000 claims 1
- 239000002131 composite material Substances 0.000 abstract 1
- 239000000084 colloidal system Substances 0.000 description 20
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 12
- 230000008878 coupling Effects 0.000 description 12
- 238000010168 coupling process Methods 0.000 description 12
- 238000005859 coupling reaction Methods 0.000 description 12
- 229920000915 polyvinyl chloride Polymers 0.000 description 9
- 239000004800 polyvinyl chloride Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 208000002979 Influenza in Birds Diseases 0.000 description 5
- 206010064097 avian influenza Diseases 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 230000000274 adsorptive effect Effects 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229920001617 Vinyon Polymers 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 150000003949 imides Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
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- 238000010170 biological method Methods 0.000 description 1
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- 238000007789 sealing Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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Abstract
本发明公开了一种纳米流感病毒检测试纸，包括PVC底板，在PVC底板上表面铺有结合垫，在结合垫的左边设置加样孔，右边依次粘贴硝酸纤维膜和吸水垫，所述硝酸纤维膜上从左到右依次设有线状或带状的三条抗体检测线和β-肌动蛋白检测抗体控制线，所述结合垫由玻璃纤维膜组成，在玻璃纤维膜上均匀附着有黑色、蓝色和橙色的纳米胶体金复合微粒，所述纳米胶体金复合微粒的粒径为20-80nm，其中黑色纳米胶体金复合微粒上分别偶联三种抗目的物检测抗体，蓝色纳米胶体金复合微粒上偶联AMACR抗原,橙色纳米胶体金复合微粒上偶联抗β-肌动蛋白抗体作为内参。
The invention discloses a nano-influenza virus detection test paper, which comprises a PVC bottom plate, a binding pad is laid on the upper surface of the PVC bottom plate, a sampling hole is arranged on the left side of the binding pad, and a nitrocellulose membrane and a water-absorbing pad are pasted on the right side successively. The nitrocellulose There are three linear or strip-shaped antibody detection lines and β-actin detection antibody control lines on the membrane from left to right. The binding pad is composed of glass fiber membrane, and black, blue Color and orange nano-colloidal gold composite particles, the particle size of the nano-colloidal gold composite particles is 20-80nm, wherein the black nano-colloidal gold composite particles are respectively coupled with three kinds of anti-target detection antibodies, and the blue nano-colloidal gold composite particles The microparticles were coupled with AMACR antigen, and the orange nanocolloidal gold composite microparticles were coupled with anti-β-actin antibody as an internal reference.
Description
Technical field
The present invention relates to a kind of detection test paper, a kind of specifically nanometer influenza virus detects test paper.
Background technology
The various virus infectionses of early diagnosis are extremely important for early prevention and treatment acute infectious disease fast.At present both at home and abroad also there is not a kind of method effectively and fast for the early diagnosis of virus infections, use molecular biological method, comprise the EILSA method, imaging diagnosis or the like, all do not reach the purpose of quick diagnosis, thus now internal and international go up be badly in need of developing efficiently a kind of and fast method diagnose various virus infectionses.
In addition, present detection test paper mainly is made up of PVC base plate, well, pad, nitrocellulose membrane and adsorptive pads, only has the quality testing of 1 clauses and subclauses of band shape or wire to survey antibody on nitrocellulose membrane, can only be used to detect a kind of virus, detection efficiency is low, is not suitable for the multiple virus of fast detecting.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of nanometer influenza virus to detect test paper, can detect three kinds of influenza viruses simultaneously.
Technical scheme of the present invention is as follows: a kind of nanometer influenza virus detects test paper, comprise the PVC base plate, be covered with pad at the PVC plate upper surface, the left side at pad is provided with well, the right is pasted nitrocellulose membrane and adsorptive pads successively, from left to right be provided with wire or banded three antibody detection lines and beta-actin on the described nitrocellulose membrane successively and detect the antibody control line, described pad is made up of glass fibre membrane, on glass fibre membrane, evenly be attached with black, blue, with orange nano colloid gold composite particles, the particle diameter of described nano colloid gold composite particles is 20-80nm, wherein three kinds of anti-objects of difference coupling detect antibody on the black nano collaurum composite particles, coupling AMACR antigen on the blue nano colloid gold composite particles, the anti-beta-actin antibody of coupling is as confidential reference items on the orange nano colloid gold composite particles.
Adopt technique scheme, use the nano colloid gold composite particles to be carrier coupling different antibodies, can detect 3 kinds of viruses simultaneously, set up a multivalence biomarker molecular detection system, on sensitivity and specificity, all improve a lot, utilize nano combined particulate will locate the deposition site reinforcement that develops the color, improved be quick on the draw, point sample is easy, the test paper long shelf-life, be applicable to basic unit, laboratory and on-the-spot the detection, the sensitivity that utilizes the present invention to detect influenza virus is more than 96.8%, and specificity is more than 95.6%.
In technique scheme: described three detection antibody detection lines are that common influenza virus detects antibody detection line, avian flu virus detection antibody detection line, influenza A virus detects antibody detection line and beta-actin detects the antibody control line, and described three kinds of anti-object antibody are that common influenza virus detects antibody, avian flu virus detection antibody and influenza A virus detection antibody.Can realize like this detecting simultaneously and whether contain common influenza virus or avian influenza virus or influenza A virus.
In technique scheme: described PVC base plate for long for 10cm, wide be the rectangle vinyon sheet of 4cm, described nitrocellulose membrane long for long for 8cm, wide be that 3cm, thickness are that 0.1mm, average pore size are little porous cellulose nitrate diaphragm of 5um.
In technique scheme, dilute the polyclonal antibody of 3 kinds of influenza viruses respectively with phosphate buffer, be common influenza virus polyclonal antibody, avian influenza virus polyclonal antibody and influenza A virus polyclonal antibody, make that the polyclonal antibody weight percent concentration is 0.05-0.3mg/ml in the solution, make common influenza virus polyclonal antibody solution, avian influenza virus polyclonal antibody solution and influenza A virus polyclonal antibody solution; With some film device above-mentioned common influenza virus polyclonal antibody solution, avian influenza virus polyclonal antibody solution and influenza A virus polyclonal antibody solution are put into wire or banded common influenza virus detection antibody detection line, avian flu virus detection antibody detection line and influenza A virus detection antibody detection line respectively successively with 3 μ L/cm on nitrocellulose membrane then.
In technique scheme: must contain on the described nano colloid gold composite particles can be for the immune aglucon of coupling: carboxyl, hydroxyl, amino or aldehyde radical, earlier the nano colloid gold composite particles is carried out pre-service with anhydrous morpholino b acid, a spot of carbodiimides and N-hydroxyl amber platinum acid imide are joined in the microspheres solution concussion activation nano colloid gold composite particles on the earthquake device; The common influenza virus detection of coupling antibody, avian flu virus detection antibody and influenza A virus detect antibody respectively with black nano collaurum composite particles, blue nano colloid gold composite particles coupling AMACR antigen, with the anti-beta-actin antibody of orange nano colloid gold composite particles coupling as confidential reference items; There is not the activated group of complete reaction to seal on three kinds of immune nano collaurum composite particles surfaces then, with contingent non-specific adsorption in the test after being reduced in; Microballoon is resuspended in the protection liquid again, makes concentration and be the microspheres solution of 2mg/ml and preserve, the prescription of preserving liquid is: pH9.0 contains 0.05%Tween-20,0.1%NaN
30.005M borate buffer system with 0.1%BSA; To deceive at last, blue and three kinds of quality such as colored immune nano collaurum composite particles of orange are mixed and made into a kind of black Blue Curacao three mixture of colours immune nano collaurum composite particles; Rob the microlitre with 10-40 with moving liquid, concentration is that drying obtains pad then for the nano colloid gold composite particles of 2mg/ml drops on the glass fibre membrane.
Beneficial effect: this product has efficiently, and is fast cheap, and facility is used easily, be convenient to transportation and preserve, avirulence, can be on same test paper three kinds of advantages such as virus of quick diagnosis simultaneously, therefore have stronger competitive edge.Compared with prior art, the sensitivity of this novel nano fast diagnose test paper and specificity are more than 90%.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
Among the present invention: BSA is the abbreviation of bovine serum albumin, i.e. bovine serum albumin(BSA).
AMACR antigen is
αThe abbreviation of-methyl acyl-CoA racemase antigen.
Tween is a tween solution.
PVC is the abbreviation of Polyvinylchloride.
As shown in Figure 1, the present invention is made up of PVC base plate 1, well 2, pad 3, nitrocellulose membrane 4 and adsorptive pads 9, is covered with pad 3 on PVC base plate 1, on the left side of pad 3 well 2 is set, and the right is pasted nitrocellulose membrane 4 and adsorptive pads 9 successively; Described PVC base plate 1 for long for 10cm, wide be the rectangle vinyon sheet of 4cm, described nitrocellulose membrane 4 for long for 8cm, wide be that 3cm, thickness are that 0.1mm, average pore size are little porous cellulose nitrate diaphragm of 5um;
Dilute the polyclonal antibody of 3 kinds of influenza viruses respectively with phosphate buffer, be common influenza virus polyclonal antibody, avian influenza virus polyclonal antibody and influenza A virus polyclonal antibody, make that the polyclonal antibody weight percent concentration is 0.05-0.3mg/ml in the solution, make common influenza virus polyclonal antibody solution, avian influenza virus polyclonal antibody solution and influenza A virus polyclonal antibody solution; With some film device above-mentioned common influenza virus polyclonal antibody solution, avian influenza virus polyclonal antibody solution and influenza A virus polyclonal antibody solution are put into wire or banded common influenza virus detection antibody detection line 5, influenza virus detection antibody detection line 6 and influenza A virus detection antibody detection line 7 at nitrocellulose membrane 4 respectively successively with 3 μ L/cm then; And then point is gone up beta-actin detection antibody control line 8.
The carrier that the present invention selects is that particle diameter is that the nano colloid gold composite particles of 20-80nm is (as heteropolymer, liposome etc.), be divided into black, blueness and orange three kinds, must contain on this nano colloid gold composite particles and can supply the immune aglucon of coupling: carboxyl, hydroxyl, amino or aldehyde radical, earlier the nano colloid gold composite particles is carried out pre-service with anhydrous morpholino b acid, a spot of carbodiimides and N-hydroxyl amber platinum acid imide are joined in the microspheres solution concussion activation nano colloid gold composite particles on the earthquake device; Detect antibody, avian flu virus detection antibody and influenza A virus with the common influenza virus of black nano collaurum composite particles difference coupling and detect antibody, blue nano colloid gold composite particles coupling AMACR antigen (
α-methyl acyl-CoA racemase antigen), with the anti-beta-actin antibody of orange nano colloid gold composite particles coupling as confidential reference items; There is not the activated group of complete reaction to seal on three kinds of immune nano collaurum composite particles surfaces then, with contingent non-specific adsorption in the test after being reduced in; Microballoon is resuspended in the protection liquid again, makes concentration and be the microspheres solution of 2mg/ml and preserve, the prescription of preserving liquid is: pH9.0 contains 0.05%Tween-20, the 0.005M borate buffer system of 0.1%NaN3 and 0.1%BSA; To deceive at last, blue and three kinds of quality such as colored immune nano collaurum composite particles of orange are mixed and made into a kind of black Blue Curacao three mixture of colours immune nano collaurum composite particles; Rob the microlitre with 10-40 with moving liquid, concentration is that drying obtains pad 3 then for the nano colloid gold composite particles of 2mg/ml drops on the glass fibre membrane.
The wherein sealing of nano colloid gold composite particles: the bovine serum albumin(BSA) (BSA) that adds 1%-5%, BSA is dissolved in the borate tween buffer solution in advance, there is not the activated group of complete reaction to seal to nano colloid gold composite particles surface, and the physisorption by BSA, seal other site, space, with contingent non-specific adsorption in the experiment after reducing, capping 30min under the room temperature.
Claims (5)
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| CN2010105953284A CN102072956A (en) | 2010-12-20 | 2010-12-20 | Nano influenza virus testing paper |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707061A (en) * | 2012-06-18 | 2012-10-03 | 深圳市博卡生物技术有限公司 | Gold immunochromatographic assay quantitative detection test paper and detection method |
| CN103048467A (en) * | 2012-12-06 | 2013-04-17 | 苏州海吉亚生物科技有限公司 | Method for detecting tissue cell protein by using housekeeping protein |
| CN103105492A (en) * | 2013-01-17 | 2013-05-15 | 重庆市科学技术研究院 | Fluorescence immunochromatography flu virus detection test paper |
| CN106855571A (en) * | 2016-12-29 | 2017-06-16 | 广州华弘生物科技有限公司 | A kind of antistreptolysin O (ASO) quick diagnosis reagent kit and its application method |
| CN114689849A (en) * | 2022-05-31 | 2022-07-01 | 上海尚济生物医学工程有限责任公司 | Antigen detection product and preparation method and application thereof |
| CN115060896A (en) * | 2022-06-15 | 2022-09-16 | 张春明 | Immunoassay reagent for reducing false negative |
| GB2617621A (en) * | 2022-04-16 | 2023-10-18 | Uk Nivd Ltd | Detector |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707061A (en) * | 2012-06-18 | 2012-10-03 | 深圳市博卡生物技术有限公司 | Gold immunochromatographic assay quantitative detection test paper and detection method |
| CN103048467A (en) * | 2012-12-06 | 2013-04-17 | 苏州海吉亚生物科技有限公司 | Method for detecting tissue cell protein by using housekeeping protein |
| CN103048467B (en) * | 2012-12-06 | 2016-01-27 | 苏州海吉亚生物科技有限公司 | Utilize the method that house keeping protein detects histocyte albumen |
| CN103105492A (en) * | 2013-01-17 | 2013-05-15 | 重庆市科学技术研究院 | Fluorescence immunochromatography flu virus detection test paper |
| CN106855571A (en) * | 2016-12-29 | 2017-06-16 | 广州华弘生物科技有限公司 | A kind of antistreptolysin O (ASO) quick diagnosis reagent kit and its application method |
| GB2617621A (en) * | 2022-04-16 | 2023-10-18 | Uk Nivd Ltd | Detector |
| CN114689849A (en) * | 2022-05-31 | 2022-07-01 | 上海尚济生物医学工程有限责任公司 | Antigen detection product and preparation method and application thereof |
| CN115060896A (en) * | 2022-06-15 | 2022-09-16 | 张春明 | Immunoassay reagent for reducing false negative |
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Application publication date: 20110525 |