CN201053965Y - Influenza virus quick detection test paper - Google Patents
Influenza virus quick detection test paper Download PDFInfo
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- CN201053965Y CN201053965Y CNU2007201494803U CN200720149480U CN201053965Y CN 201053965 Y CN201053965 Y CN 201053965Y CN U2007201494803 U CNU2007201494803 U CN U2007201494803U CN 200720149480 U CN200720149480 U CN 200720149480U CN 201053965 Y CN201053965 Y CN 201053965Y
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Abstract
The utility model relates to a flu virus fast test paper which belongs to the field of the immunity chromatographic analysis fast examination. The test paper consists of a soleplate, a water absorption board, a nitrocellulose membrane, a flu virus monoclonal antibody storage cushion and a sample liquid absorption layer; wherein, the mid part of the soleplate is provided with the nitrocellulose membrane which is provided with an experimental line and a multi-clonal antibody control line; one end of the soleplate is of the water absorption board, and the other end is of the sample liquid absorption layer; the two end of the nitrocellulose membrane is overlapped with the water absorption board and the flu virus monoclonal antibody storage cushion; the flu virus monoclonal antibody storage cushion is provided with the sample liquid absorption layer; the method of color pellet immunity chromatographic analysis is used for testing flu virus. Through the adoption of the test paper strip and the color display of the experimental line, the flu virus can be detected quickly, at the same time, the utility model has the advantages of easy operation, strong peculiarity, high sensitivity, easy reading of results, low cost and no need for professional staff, etc., thereby actually integrating the complex principle with easy operation.
Description
Technical field
The present invention relates to a kind of movably, convenient and practical influenza virus quick detection test paper, belong to immunochromatography fast detecting field.
Background technology
Influenza virus is a kind of as common Respirovirus, the antigenicity complexity is divided into first, second, the third three types according to its core protein antigenicity difference, different according to its surface antigen hemagglutinin (H) and neuraminidase (N), homologous virus divides some hypotypes again, does not have cross immunity between various.Owing to do not have cross immunity between the various virus, can repeatedly infect, repeatedly morbidity.Influenza is the most rampant in the world infectious disease, once repeatedly have swepts the globe, and brings huge disaster to the mankind.Only in the influenza large propagation of nineteen fifty-seven, the whole world has 1,500,000,000 people morbidity, and ten hundreds of old men and child are tormented deadly.
At present, detect influenza virus, at present multiple detection meanss such as viral separation, indirect immunofluorescence, enzyme linked immunosorbent assay, multiple reverse transcription polymerase chain reaction, PCR and nucleic acid hybridization that adopt both at home and abroad more.Isolation of virus is consuming time oversize and cost is higher; Indirect immunofluorescence needs expensive fluorescent microscope and sample to be difficult for preserving; PCR method can not be distinguished the virus and the dead virus of thoughts contamination power; Enzyme linked immunosorbent assay generate a reagent box need refrigerate, transportation inconvenience, and said method all needs the professional to operate simultaneously.
Exactly because therefore the backwardness of existing detection method is developed easy to usely, detects sensitively, cheap testing product is the task of top priority.
The colored particle immunochromatographiassays assays provides a new detection approach for the detection of influenza virus.Immunity particulate technology is to utilize the solid phase particle of the synthetic certain particle size size of macromolecular material as carrier, bag by on have specificity affinity various immunologic active materials (antigen or antibody), making its sensitization is immune particulate, is used for immunology and other biological and learns detection and a technology of separating.Particulate as carrier is a raw material with certain macromolecule organic monomer normally, is prepared from through high molecular polymerization methods such as emulsion polymerization, suspension polymerization and irradiation polymerizations.Because preparation material and technology difference, particulate of a great variety, inert particulate such as polystyrene latex particulate, active particles such as Carboxylated Polystyrene particulate, magnetic particle and labeled microparticles four big based fine particles such as (with isotope, fluorescein or enzyme labeling) have now been made, nearly tens kinds of quantity.The particulate and the antigen (or antibody) that prepare are formed immune particulate through sensitization methods such as the plain bridging methods of physisorption, chemical coupling and biotin affinity.Be widely used in detection, isolation and purification, cell marking and the identification etc. of various soluble large molecule materials.In recent years, the particulate technology making nucleic acid molecular hybridization, DNA and RNA separate and research field such as PCR also demonstrates wide application prospect.
Emulsion particle is a kind of as immune particulate, adopt the synthetic high polymer latex beads, the polystyrene latex that is carboxylation is as carrier, antibody or antigen are formed immune latex diagnostic reagent by physisorption is immobilized in microsphere surface, can detect corresponding antigen or antibody by the latex agglutination experiment.The latex diagnostic reagent that is prepared into is used to diagnose multiple disease, has advantages such as easy, quick, sensitive, inexpensive and processing ease.
The colloidal gold immunochromatographimethod technology is a novel vitro diagnostic techniques that has grown up on monoclonal antibody technique, immunochromatography technique and collaurum developing technology basis since the nineties in 20th century, be according to the immune response principle, utilize the large aperture miillpore filter to be carrier, detect a kind of tachysynthesis analytical technology of test substance in the sample with collaurum as the solid phase labelling thing.
The present invention improves detection method on colored particle immunochromatography technique principle basis, set up the method that adopts colloidal gold method and emulsion process to detect influenza virus.The improvement of detection method makes and detects more conveniently, and the result is more accurate, have quick, sensitive, easy and simple to handle, cost is low, need not characteristics such as professional's operation, really reaches the organic unity of complicated principle and ease of Use.
Summary of the invention
The present invention is directed to some problems of above-mentioned existence, a kind of influenza virus quick detection test paper is provided.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of influenza virus quick detection test paper is made up of base plate, water sucting plate, nitrocellulose filter, influenza virus monoclonal antibody storage pad, sample liquid-adsorption layer; The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a test wire and a polyclonal antibody control line, base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with influenza virus monoclonal antibody storage pad with water sucting plate respectively and are connected, on influenza virus monoclonal antibody storage pad, be pressed with the sample liquid-adsorption layer, utilize the colored particle immunochromatographic method to detect influenza virus.
Colored particle can be a kind of metal-sol particle, can comprise colloid gold particle, silver-colored particle, iron particle, magnetic-particle etc.; A kind of marking particle can comprise dye granule, latex particle, fluorescent grain etc.Wherein, the particle diameter of colloid gold particle size is a nanoscale, latex particle diameter 0.01～10 μ m; The adsorption mechanism of metal-sol particle is to utilize its electronegative character under alkali condition, with the positive charge group of protein molecule, by electrostatic attraction and combination; Latex particle is to utilize chemically combined mode to combine with protein molecule, forms immune diagnostic reagent.
The sample liquid-adsorption layer is made up of the trilaminate material stack, is followed successively by 10～25g/m
2Nonwoven layer, glass layer, 10～25g/m
2Nonwoven layer, above-mentioned substance all need be passed through surfactant damping fluid immersion treatment, and dry back is standby, and said apparatus also has the syphonic effect principle except that the capillarity principle is arranged, and accelerate the suction translational speed greatly.
(active component is first/New Caledonia/20/99 (HIN1)-pleiston with influenza vaccines; First/New Caledonia/20/99 recombined strain IVR-116; First/Wisconsin/67/2005 (H3N2)-pleiston; First/Hiroshima/52/2005 recombined strain IVR-142; Second/Malaysia/2506/2004-pleiston; Second/Malaysia/2506/2004) immune mouse, cell line injection mouse abdominal cavity, extract ascites and carry out purifying, in the hybridoma cell strain that filters out, obtain the influenza cell strain of monoclonal antibody Flu4G3F12 and the Flu23C7H8 of two plant height purity, pairing, one strain is used for test wire bag quilt, and a strain can be used for the colored particle mark.
Test paper sample liquid-adsorption layer is put into sample to be tested (liquid level must not surpass the MAX line), because the capillarity sample will move to water sucting plate along test strips, when moving to monoclonal antibody storage pad, influenza antigen and influenza virus labeling of monoclonal antibody probe generation specific bond in the sample, when moving to test wire, influenza antigen combines with influenza virus monoclonal antibody in the test wire again in the sample, therefore its colored particle is stranded on the test wire, and the test wire place shows red positive; If do not have influenza virus in the opposite sample to be tested, influenza virus labeling of monoclonal antibody probe just not can with the influenza virus monoclonal antibody generation specific bond on the test wire, there is not colored particle to be detained, promptly only there is a red control line negative, this test paper double antibodies sandwich method principle that Here it is adopts.According to this principle, two lines are positive, and line is negative to draw judgement.
The control line that is provided with on the nitrocellulose filter is formed by sheep anti mouse polyclonal antibody bag, when the influenza virus monoclonal antibody of sample by the colored particle mark moves to sheep anti mouse polyclonal antibody control line, no matter have or not influenza virus in the sample, the capital combines delay with the sheep anti mouse polyclonal antibody that has configured, and it is red that control line is shown.Therefore control line does not have colour band and produces that then the representative operation is wrong, during detection the sample liquid level surpass the MAX line or test paper expired.
Because adopt technique scheme, influenza virus quick detection test paper provided by the present invention has such beneficial effect, promptly make things convenient for quick, removable, as to be convenient to influenza virus field screening work, and high specificity, highly sensitive, need not the technical skill personnel operation, and readability as a result.
Description of drawings
Fig. 1 is an outside drawing of the present invention.
Fig. 2 is an exploded view of the present invention.
Fig. 3 is negative findings figure of the present invention.
Fig. 4 is positive findings figure of the present invention.
Among the figure 1, water sucting plate, 2, nitrocellulose filter, 3, control line, 4, test wire, 5, monoclonal antibody storage pad, 6, the sample liquid-adsorption layer, 7, base plate, 8, the MAX line.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is further explained:
A kind of influenza virus quick detection test paper comprises base plate (7), water sucting plate (1), nitrocellulose filter (2), influenza virus monoclonal antibody storage pad (5), sample liquid-adsorption layer (6); The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a test wire (4) and a polyclonal antibody control line (3), base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with influenza virus monoclonal antibody storage pad with water sucting plate respectively and are connected, on influenza virus monoclonal antibody storage pad, be pressed with the sample liquid-adsorption layer, utilize the colored particle immunochromatographic method to detect influenza virus.
Embodiment 1:
A kind of influenza virus colloidal gold test comprises base plate (7), water sucting plate (1), nitrocellulose filter (2), influenza virus monoclonal antibody gold mark pad (5), sample liquid-adsorption layer (6); The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a test wire (4) and a polyclonal antibody control line (3), base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with influenza virus monoclonal antibody gold mark pad with water sucting plate respectively and are connected, on influenza virus monoclonal antibody gold mark pad, be pressed with the sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect influenza virus.
The test paper method for making:
1. influenza MONOCLONAL ANTIBODIES SPECIFIC FOR: (active component is first/New Caledonia/20/99 (HIN1)-pleiston with influenza vaccines; First/New Caledonia/20/99 recombined strain IVR-116; First/Wisconsin/67/2005 (H3N2)-pleiston; First/Hiroshima/52/2005 recombined strain IVR-142; Second/Malaysia/2506/2004-pleiston; Second/Malaysia/2506/2004) immune mouse, cell line injection mouse abdominal cavity, extract ascites and carry out purifying, in the hybridoma cell strain that filters out, obtain the influenza cell strain of monoclonal antibody Flu4G3F12 and the Flu23C7H8 of two plant height purity, pairing, one strain is used for test wire bag quilt, and a strain can be used for colloid gold label.
The preparation of collaurum and with the combining of influenza monoclonal antibody Flu4G3F12
(1) get distilled water and add an amount of gold chloride magnetic agitation and be warmed to 90 ℃, add an amount of citrate three sodium and continue heated and stirred to seething with excitement 3～10 minutes, preferred 5 minutes, it was standby to keep in Dark Place after the cooling.
(2) the collaurum liquid of influenza virus monoclonal antibody Flu4G3F12 mark, on the adsorbing fiber material, dry back is standby.
3. the preparation of film: machine system film, utilize computer control transmission speed, guarantee that the antibody amount of bag quilt on the per unit film equates.
Detection line: influenza monoclonal antibody Flu23C7H8
Control line: sheep anti mouse polyclonal antibody
4. test strips is pressed the known technology combination.
5. using method and result judge: during use, test paper sample liquid-adsorption layer is put into sample to be tested (liquid level must not surpass the MAX line), observations in the time of 5 minutes.Be higher than detected level if detect influenza virus concentration, it is positive that two red lines appear in the view window place; Be lower than boundary value, it is negative a red line to occur; It is wrong that control line does not have colour band generation representative operation, and the sample liquid level is expired above MAX line or test paper during detection.
Embodiment 2:
A kind of influenza virus latex detects test paper, comprises base plate (7), water sucting plate (1), nitrocellulose filter (2), influenza virus monoclonal antibody storage pad (5), sample liquid-adsorption layer (6); The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a test wire (4) and a polyclonal antibody control line (3), base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with influenza virus monoclonal antibody storage pad with water sucting plate respectively and are connected, on influenza virus monoclonal antibody gold mark pad, be pressed with the sample liquid-adsorption layer, utilize colloidal gold immunity chromatography to detect influenza virus.
The test paper method for making:
1. influenza MONOCLONAL ANTIBODIES SPECIFIC FOR: (active component is first/New Caledonia/20/99 (HIN1)-pleiston with influenza vaccines; First/New Caledonia/20/99 recombined strain IVR-116; First/Wisconsin/67/2005 (H3N2)-pleiston; First/Hiroshima/52/2005 recombined strain IVR-142; Second/Malaysia/2506/2004-pleiston; Second/Malaysia/2506/2004) immune mouse, cell line injection mouse abdominal cavity, extract ascites and carry out purifying, in the hybridoma cell strain that filters out, obtain the influenza cell strain of monoclonal antibody Flu4G3F12 and the Flu23C7H8 of two plant height purity, pairing, one strain is used for test wire bag quilt, and a strain can be used the emulsion particle mark.
2. the preparation of emulsion particle:
1) gets 10% latex solution 100ul, add borate buffer solution 900ul, high speed centrifugation 10min;
2) abandon supernatant, add the borate buffer solution washing, once more high speed centrifugation 10min;
3) abandon supernatant, add borate buffer solution suspension precipitation, add antibody, volume is settled to 1ml;
4) in " 3 " gained solution, add 10ulEDC (15mg/ml), hatch 4h, high speed centrifugation 10min;
5) abandon supernatant, suspend with confining liquid (5% BSA) and precipitate, sealing is spent the night; Next day, line.
3. emulsion particle combines with influenza monoclonal antibody Flu4G3F12: the emulsion particle of influenza virus monoclonal antibody Flu4G3F12 mark, on the adsorbing fiber material, dry back is standby.
4. the preparation of film: machine system film, utilize computer control transmission speed, guarantee that the antibody amount of bag quilt on the per unit film equates.
Detection line: influenza monoclonal antibody Flu23C7H8
Control line: sheep anti mouse polyclonal antibody
5. test strips is pressed the known technology combination.
6. using method and result judge: during use, test paper sample liquid-adsorption layer is put into sample to be tested (liquid level must not surpass the MAX line), observations in the time of 5 minutes.Be higher than detected level if detect influenza virus concentration, it is positive that two red lines appear in the view window place; Be lower than boundary value, it is negative a red line to occur; It is wrong that control line does not have colour band generation representative operation, and the sample liquid level is expired above MAX line or test paper during detection.
Claims (4)
1. an influenza virus quick detection test paper is characterized in that being made up of base plate (7), water sucting plate (1), nitrocellulose filter (2), influenza virus monoclonal antibody storage pad (5), sample liquid-adsorption layer (6); The base plate middle part is a nitrocellulose filter, nitrocellulose filter is provided with a test wire (4) and a polyclonal antibody control line (3), base plate one end termination is a water sucting plate, other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with influenza virus monoclonal antibody storage pad with water sucting plate respectively and are connected, on influenza virus monoclonal antibody storage pad, be pressed with the sample liquid-adsorption layer, utilize the colored particle immunochromatographic method to detect influenza virus.
2. a kind of influenza virus quick detection test paper according to claim 1 is characterized in that colored particle can be a kind of metal-sol particle, can comprise colloid gold particle, silver-colored particle, iron particle, magnetic-particle; A kind of marking particle can comprise dye granule, latex particle, fluorescent grain.
3. according to claim 1 described a kind of influenza virus quick detection test paper, it is characterized in that the particle diameter size of colloid gold particle is nanoscale, latex particle diameter 0.01～10 μ m; The adsorption mechanism of metal-sol particle is to utilize its electronegative character under alkali condition, with the positive charge group of protein molecule, by electrostatic attraction and combination; Latex particle is to utilize chemically combined mode to combine with protein molecule, forms immune diagnostic reagent.
4. a kind of influenza virus colloidal gold fast detecting test paper according to claim 1 is characterized in that described sample liquid-adsorption layer is made up of trilaminate material stack, and ground floor is a nonwoven layer, and specification is 10～25g/m
2The second layer is a glass layer; The 3rd layer is nonwoven layer, and its specification is 10～25g/m
2
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701957A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Manufacturing method of fluorescent labeling test strip for rapid detection of avian influenza virus |
| CN101701959A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Immunofluorescence test strip for rapid detection of Newcastle disease virus and its application |
| CN101871938A (en) * | 2010-05-12 | 2010-10-27 | 蓝十字生物药业（北京）有限公司 | Rapid test strip with colloid gold label |
| CN102539755A (en) * | 2011-12-20 | 2012-07-04 | 广州万孚生物技术有限公司 | Test strip for detecting influenza A virus antigen in secretion and preparation method thereof |
| CN104076142A (en) * | 2014-03-05 | 2014-10-01 | 广东医学院附属医院 | Fluorescent microsphere lateral chromatographic detection strip for multiple joint inspection of trace target substances as well as preparation method and application thereof |
| CN105980038A (en) * | 2014-04-11 | 2016-09-28 | 旭化成医疗株式会社 | Virus removal membrane |
| CN111264937A (en) * | 2020-03-13 | 2020-06-12 | 北京理工大学 | Gauze mask with prevention and detection function |
| CN111387596A (en) * | 2020-03-27 | 2020-07-10 | 四川爱联科技有限公司 | Intelligent medical protective mask based on NB-IoT module and monitoring system |
-
2007
- 2007-06-06 CN CNU2007201494803U patent/CN201053965Y/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701957A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Manufacturing method of fluorescent labeling test strip for rapid detection of avian influenza virus |
| CN101701959A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Immunofluorescence test strip for rapid detection of Newcastle disease virus and its application |
| CN101871938A (en) * | 2010-05-12 | 2010-10-27 | 蓝十字生物药业（北京）有限公司 | Rapid test strip with colloid gold label |
| CN102539755A (en) * | 2011-12-20 | 2012-07-04 | 广州万孚生物技术有限公司 | Test strip for detecting influenza A virus antigen in secretion and preparation method thereof |
| CN104076142A (en) * | 2014-03-05 | 2014-10-01 | 广东医学院附属医院 | Fluorescent microsphere lateral chromatographic detection strip for multiple joint inspection of trace target substances as well as preparation method and application thereof |
| CN105980038A (en) * | 2014-04-11 | 2016-09-28 | 旭化成医疗株式会社 | Virus removal membrane |
| CN105980038B (en) * | 2014-04-11 | 2018-11-02 | 旭化成医疗株式会社 | Remove the film of virus |
| CN111264937A (en) * | 2020-03-13 | 2020-06-12 | 北京理工大学 | Gauze mask with prevention and detection function |
| CN111387596A (en) * | 2020-03-27 | 2020-07-10 | 四川爱联科技有限公司 | Intelligent medical protective mask based on NB-IoT module and monitoring system |
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