CN105738620A - Test paper strip and method for detecting canine distemper virus through immunofluorescence chromatography technique - Google Patents
Test paper strip and method for detecting canine distemper virus through immunofluorescence chromatography technique Download PDFInfo
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- CN105738620A CN105738620A CN201410753886.7A CN201410753886A CN105738620A CN 105738620 A CN105738620 A CN 105738620A CN 201410753886 A CN201410753886 A CN 201410753886A CN 105738620 A CN105738620 A CN 105738620A
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- canine distemper
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Abstract
The invention relates to a test paper strip and method for detecting a canine distemper virus through immunofluorescence chromatography technique, and specifically discloses the test paper strip for detecting a canine distemper virus through immunofluorescence chromatography technique. The test paper strip includes a marker pad containing a fluorescent-microsphere-labeled canine distemper virus antibody, and a capture membrane containing another canine distemper virus antibody. The invention further discloses the method for detecting canine distemper virus through immunofluorescence chromatography technique. The method comprises: dropwise adding a canine-distemper-virus -containing solution to the sample pad of the test paper strip, and detecting a signal through a strip reader after reaction for a while.
Description
Technical field
The present invention relates to the fields such as nano material technology, immunological technique, Sidestream chromatography technology.More specifically, the present invention relates to the test strips of a kind of fluorescence immune chromatography technology for detection canine distemper virus；Moreover, it relates to a kind of method that fluorescence immune chromatography technology detects canine distemper virus.
Background technology
Canine distemper virus main infection Canis animals, after infection, symptom is mainly limited to upper respiratory tract, so can cough, rhinorrhea, be likely to that serious symptoms occurs without the pup of immunity, such as pneumonia, diarrhoea, dehydration, anorexia etc., it is easy to dead.
If dog has infected canine distemper virus, it is necessary to detection as early as possible and early treatment.The traditional method of detection canine distemper virus is colloidal gold immunity chromatography, but colloidal gold immunochromatographimethod sensitivity is relatively low, and stability is also good not, it is easy to false cloudy false sun occur.The test kit sensitivity of the fluorescence immune chromatography method detection canine distemper virus of my company's exploitation is significantly high, can detect that the canine distemper virus antigen of 10ng/ml, the dog that canine distemper virus is infected can early detect and early isolate early treatment, improve the survival rate of pup, simultaneously as sensitivity improves, considerably reduce the appearance of false cloudy false sun, improve the stability of product, therefore there is important application prospect.
Summary of the invention
Present invention seek to address that the above-mentioned various problems in this area and realize following target.Specifically, present invention aim to provide a kind of easily and fast and also detect test strips and the method for canine distemper virus highly sensitively.
Concrete, the invention provides the test strips of a kind of fluorescence immune chromatography technology for detection canine distemper virus.
In one embodiment, the invention provides the test strips of a kind of fluorescence immune chromatography technology for detection canine distemper virus, including labeling pad and catch film, it is characterised in that, described labeling pad contains the canine distemper virus detection antibody of fluorescent microsphere labelling, described in catch film and contain capture probe.
In further embodiment, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
In further embodiment, it is characterised in that described time-resolved fluorescence microsphere is the fluorescent microsphere containing europium complex.
In another embodiment, a kind of method that the invention provides fluorescence immune chromatography technology for detection canine distemper virus, it is characterised in that drip the sample containing canine distemper virus in described test strips sample pad, detects fluorescence signal after reaction.
In another embodiment, the invention provides the test kit of a kind of fluorescence immune chromatography technology for detection canine distemper virus, comprise reagent bottle and test strips, wherein test strips comprises again labeling pad and catches film, it is characterized in that, described reagent bottle contains the canine distemper virus detection antibody of fluorescent microsphere labelling, described in catch film and contain capture probe.
In further embodiment, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
In another embodiment, a kind of method that the invention provides fluorescence immune chromatography technology for detection canine distemper virus, it is characterised in that add the sample containing canine distemper virus in described reagent bottle, it is added drop-wise in test strips sample pad after mixing, after reaction, detects fluorescence signal.
Describe in detail
Show with test strips such as Fig. 1 of fluorescence immune chromatography technology for detection canine distemper virus, including sample pad 1, labeling pad 2, NC film 3, absorbent paper 4, PVC base plate 5.Containing C line 6 and T line 7 on NC film 3.
Wherein sample pad is generally glass fibre, filter paper or hemofiltration film.Labeling pad can be glass fibre, filter paper or polyester film.Can combine when sample pad is identical with labeling pad material, complete the function of sample pad and labeling pad with a glass fibre, filter paper or polyester film.
Canine distemper virus detection antibody in described labeling pad or containing fluorescent microsphere labelling, or the canine distemper virus detection antibody and the biotin labeled canine distemper virus that contain fluorescent microsphere labelling catch antibody simultaneously, canine distemper virus or the antibody of other fluorescent microsphere labellings being used as positive control can also be contained, such as streptavidin or rabbit antibody.If there being reagent bottle in test kit, then labeling pad can be only glass fibre, filter paper or polyester film and without fluorescent microsphere or antibody.
Catching film can be nitrocellulose filter (NC film), nylon membrane etc., the described T line catching film or catch antibody (double antibody sandwich method) containing canine distemper virus, or containing streptavidin (labeling pad simultaneously canine distemper virus detection antibody containing fluorescent microsphere labelling and biotin labeled canine distemper virus catch antibody), C line or containing can in conjunction with canine distemper virus detect antibody two resist, or containing the pure canine distemper virus of biotin labeled Sanguis Bovis seu Bubali or egg white canine distemper virus (labeling pad is containing streptavidin), or containing goat anti-rabbit antibody (labeling pad is containing rabbit antibody).
nullThe method detecting canine distemper virus with immunochromatography technique，As Fig. 1 shows，After sample solution is added drop-wise in the sample pad 1 of test strips，Liquid will move along the horizontal direction of arrow，Arrive first and reach labeling pad 2，Canine distemper virus in sample and the canine distemper virus detection antibodies of fluorescent microsphere labelling in labeling pad 2 form ' canine distemper virus-canine distemper virus detection antibody-fluorescent microsphere ' complex，When solution continues to move to arrive the T line catching film，' canine distemper virus-canine distemper virus detection antibody～fluorescent microsphere ' in solution forms ' canine distemper virus catches antibody-canine distemper virus-canine distemper virus detection antibody～fluorescent microsphere ' complex by catching antibody with canine distemper virus herein and is fixed on T line，The fluorescence signal on T line is detected subsequently with detector，The height of canine distemper virus concentration is may determine that according to signal intensity，The strong then canine distemper virus concentration of fluorescence signal is high.
Catch the fluorescent microsphere of antibody capture due to T line place canine distemper virus and include a lot of fluorescence molecule, so T line place can detect very strong fluorescence signal, so that the method for this immunochromatography technique detection canine distemper virus has significantly high sensitivity.When fluorescence molecule is time-resolved fluorescence complex, the fluorescence lifetime of hundreds of microsecond to several milliseconds is had owing to launching light, optical signal can be launched to the detection of hundreds of microseconds by tens of microseconds after exciting light removes, now exciting light and background fluorescence all disappear, the fluorescence signal that only fluorescence molecule is special, so the specificity of detection and sensitivity are all greatly improved.
' microsphere ' in the present invention can be silicon dioxide microsphere, polystyrene microsphere or other polymer microspheres, and microsphere diameter is at 10nm to 10μBetween m, its surface can with functional groups such as carboxyl, amino or ketone groups.
' fluorescent microsphere ' in the present invention refers to embed in spheroid the microsphere of fluorescence molecule.Described ' fluorescence molecule ' includes lanthanide series complex fluorescence molecule and the phosphorescent molecules such as platinum complexes, palladium complex such as the fluorescence molecule or derivatives thereof such as fluorescein, rhodamine, europium complex, samarium complex, terbium complex, wherein europium complex is the complex formed with other organic molecules again after europium and ligand sequestration, platinum complexes is the complex that platinum is formed with part (including porphyrin, porphin, pyridine and its derivatives) chelating, wherein europium complex is again time-resolved fluorescence complex, and platinum complexes is again phosphorescence complex.
' the canine distemper virus detection antibody of fluorescent microsphere labelling ' in the present invention refers to the canine distemper virus detection antibody that fluorescent microsphere couples.More specifically, canine distemper virus detection antibody is can with the specific binding antibody of canine distemper virus with what occur in animal blood after the protein immune animal of canine distemper virus.
' capture probe ' in the present invention refers to be combined in the canine distemper virus that test strips catches on film and catches antibody (double antibody sandwich method), streptavidin (labeling pad canine distemper virus detection antibody containing fluorescent microsphere labelling simultaneously and biotin labeled canine distemper virus catch antibody).More specifically, it is can with the specific binding antibody of canine distemper virus with what occur in animal blood after canine distemper virus protein immune animal that canine distemper virus catches antibody, but canine distemper virus catches the different parts of antibody and canine distemper virus detection antibody recognition canine distemper disease toxalbumin, two antibody identify and respectively in conjunction with leaning on double antibody sandwich method identification canine distemper virus after canine distemper disease toxalbumin.
In the present invention, test strips is caught ' the T line ' on film and is referred to the region at capture probe place, the canine distemper virus that namely the canine distemper virus detection antibody of fluorescent microsphere labelling is fixed after being combined with canine distemper virus again catches the region of antibody capture (double antibody sandwich method), or streptavidin and biotin labeled canine distemper virus catch the region (the canine distemper virus detection antibody of fluorescent microsphere labelling and biotin labeled canine distemper virus catch antibody sandwich identification canine distemper virus) of antibodies.
In the present invention, test strips catches the region that ' the C line ' on film refers to canine distemper virus detection antibody and its two anti-binding of fluorescent microsphere labelling, or refers to containing other antigen or antibody (goat anti-rabbit antibody) and the region that the positive control of fluorescent microsphere labelling is combined.
Because what be in a bad way after dog infected dogs distemper virus even can cause death, so needing quickly to detect as early as possible in initial infection, early diagnosing, early knowing result, in order to adopt remedial measures in time or treatment means.And the present invention can convenient by fluorescence immune chromatography method, detect the canine distemper virus of animal simply, quickly and in high sensitivity and early treat, it is to avoid its further development animal bodies is produced grievous injury.
Accompanying drawing explanation
According to one or more different embodiments, with reference to drawings below to the present invention have been described in detail.The merely illustrative purpose of accompanying drawing and provide, and the embodiment that the present invention is typical or exemplary is only described.These accompanying drawings are provided to promote reader's the understanding of the present invention, are not to be regarded as the restriction width of the present invention, scope or application.
Fig. 1 is with the test strips schematic diagram of fluorescence immune chromatography technology for detection canine distemper virus in the present invention.In Fig. 1, accompanying drawing labelling 1-7 illustrates: 1-sample pad；2-labeling pad；3-catches film；4-adsorptive pads；5-support baseboard；6-C line；7-T line.
Fig. 2 is the comparison detecting canine distemper virus result respectively with fluorescence immune chromatography and colloidal gold immunochromatographimethod
Detailed description of the invention
Embodiment 1, with the preparation of the test strips of fluorescence immune chromatography technology for detection canine distemper virus antigen
The test strips preparation method of the use fluorescence immune chromatography technology for detection canine distemper virus shown in Fig. 1 is as follows:
1, the SiO containing europium complex of carboxyl modified2Microsphere is provided by Zhi Ang bio tech ltd, Changchun, and canine distemper virus antibody and antigen are purchased from antibody line company (antibodies-online) company.
2, fluorescent microsphere couples with canine distemper virus antibody
By 200μL20mMPBS, pH7.4, the fluorescent microsphere of solid content 1% and 6 milligrams of carbodiimides mixing, shaken at room temperature 30 minutes, 12,000rpm are centrifuged 10 minutes, clean 2 times with 0.1M borate buffer solution, the fluorescent microsphere of activation is resuspended in 300μIn l borate buffer solution.Microsphere suspensions adds 200μThe canine distemper virus antibody of l1mg/ml, at room temperature oscillating reactions is overnight.Reacted solution is centrifuged 10 minutes in 12,000rpm, adds 400μL0.25M ethanolamine vibrates 30 minutes in room temperature again.2 times will be washed with PBS again after reacted solution centrifugal.300 are added after centrifugationμThe BSA of l2mg/ml is resuspended, and room temperature is washed 3 times with PBS after vibrating 30 minutes, then with 200μThe canine distemper virus antibody of l50mMPBS, pH7.4,0.1%Tween20,5% sucrose further fluorescent microsphere labelling.
3, the canine distemper virus detection antibody treatment labeling pad of fluorescent microsphere labelling, canine distemper virus catches antibody and sheep anti-mouse antibody (Sigma Co., USA) processes and catches film (here for NC film)
The canine distemper virus of fluorescent microsphere labelling detecting antibody-solutions BioDot spray film instrument and is sprayed onto in labeling pad (Millipore company), spray speed is 10μl/cm.1mg/ml canine distemper virus is caught antibody and 1mg/ml sheep anti-mouse antibody BioDot company's spray film instrument line NC film (Millipore company), and line speed is 3μl/cm.Then the labeling pad sprayed and NC film are placed in 37 degree of calorstats dry 1 hour, then the part of the canine distemper virus antibody containing fluorescent microsphere labelling in labeling pad is cut down, obtain 5mm width, the band of 30cm length.
4, the assembling of test strips kilocalorie and cutting
According in Fig. 1 shown in the structure of test strips, first NC film is pasted onto on support baseboard (Millipore company), by T line side successively binding mark thing pad and sample pad (Millipore company), adsorptive pads (Millipore company) is being pasted by C line side, then cutting into the wide test strips of 5mm with BioDot company cutting machine, in aluminium foil bag, Seal and preservation is standby.
Embodiment 2, detects canine distemper virus antigen with fluorescence immune chromatography test paper bar
By 100μL is containing 0ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml, the solution of 10000ng/ml canine distemper virus antigen is added drop-wise in embodiment 1 in the fluorescence immune chromatography test paper bar sample pad of preparation respectively, fluorescence signal is detected with the fluorescence detector (Shang Haihai jump mold company limited) launching 395nm exciting light, simultaneously by 100 after reacting 15 minutesμL is added drop-wise on colloidal gold immuno-chromatography test paper strip containing the solution of 0ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml, 10000ng/ml canine distemper virus antigen, observed result after reacting 5 minutes.The testing result of two kinds of methods compares such as Fig. 2
As seen from Figure 2, when canine distemper virus antigen concentration is 0ng/ml, two kinds of detection method T lines (below that line position), all without signal, illustrate that both of which does not have false positive.And add 10ng/ml, 100ng/ml, 103Ng/ml and 104During ng/ml canine distemper virus antigen, only fluorescence immune chromatography method T line can detect signal, and strengthens gradually along with antigen concentration raises fluorescence signal.Colloidal gold immuno-chromatography test paper strip is even at adding 104During ng/ml canine distemper virus antigen, T line is all without going out red line.Visible, fluorescence immune chromatography than colloidal gold immunochromatographimethod sensitive at least 1000 times herein, and do not have false positive.
Embodiment 3, detects canine distemper virus antigen with fluorescence immune chromatography test kit
Fluorescence immune chromatography test kit includes reagent bottle and test strips.The preparation method of test strips, with embodiment 1, simply need not spray the canine distemper virus detection antibody of fluorescent microsphere labelling in the labeling pad of this test strips.But, containing fluorescently-labeled canine distemper virus antibody in the reagent bottle of test kit.
During detection, first the sample solution containing canine distemper virus is added in reagent bottle, be added drop-wise in above-mentioned test strips after mixing, after reacting 5 minutes, detect fluorescence signal.Fig. 2 same with colloidal gold immunochromatographimethod result of the comparison, distinguishes not quite with embodiment 2.
Although the application describes specific embodiment in detail, but the application is not limited to these embodiments, on the contrary, its intention contains the various amendments and equivalent that include in the spirit and scope of embodiment, in some particular cases, can lacking and increase certain or some technical characteristics without deviating from the spirit and scope of the present invention, scope of the present application is limited only by the appended claims.
Claims (7)
1. by a test strips for fluorescence immune chromatography technology for detection canine distemper virus, including labeling pad and catch film, it is characterised in that described labeling pad contains the canine distemper virus detection antibody of fluorescent microsphere labelling, described in catch film and contain capture probe.
2. test strips as claimed in claim 1, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
3. test strips as claimed in claim 2, it is characterised in that described time-resolved fluorescence microsphere is the fluorescent microsphere containing europium complex.
4. the method with fluorescence immune chromatography technology for detection canine distemper virus, it is characterised in that drip the sample containing canine distemper virus in the test strips sample pad described in claim 1, detects fluorescence signal after reaction.
5. the test kit with fluorescence immune chromatography technology for detection canine distemper virus, comprising reagent bottle and test strips, wherein test strips comprises again labeling pad and catches film, it is characterised in that, described reagent bottle contains the canine distemper virus detection antibody of fluorescent microsphere labelling, described in catch film and contain capture probe.
6. test kit as claimed in claim 5, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
7. the method with fluorescence immune chromatography technology for detection canine distemper virus, it is characterised in that add the sample containing canine distemper virus in the reagent bottle described in claim 5, is added drop-wise in test strips sample pad after mixing, detect fluorescence signal after reaction.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107589268A (en) * | 2017-11-01 | 2018-01-16 | 杭州微瑞科技有限公司 | CDV antibody Quantitative detection card and application method |
| CN108181476A (en) * | 2018-01-08 | 2018-06-19 | 中国农业科学院兰州畜牧与兽药研究所 | Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof |
| CN108362877A (en) * | 2018-01-24 | 2018-08-03 | 中国农业科学院兰州畜牧与兽药研究所 | Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof |
| CN109975541A (en) * | 2018-12-26 | 2019-07-05 | 山东绿都生物科技有限公司 | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen |
| CN112129936A (en) * | 2020-09-21 | 2020-12-25 | 嘉兴朝云帆生物科技有限公司 | Parvovirus detection test paper and method based on fluorescence immunochromatography technology |
| CN113474657A (en) * | 2019-02-21 | 2021-10-01 | 丁勤学 | Method for removing non-specific binding signals using microparticles |
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2014
- 2014-12-11 CN CN201410753886.7A patent/CN105738620A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589268A (en) * | 2017-11-01 | 2018-01-16 | 杭州微瑞科技有限公司 | CDV antibody Quantitative detection card and application method |
| CN108181476A (en) * | 2018-01-08 | 2018-06-19 | 中国农业科学院兰州畜牧与兽药研究所 | Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof |
| CN108362877A (en) * | 2018-01-24 | 2018-08-03 | 中国农业科学院兰州畜牧与兽药研究所 | Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof |
| CN109975541A (en) * | 2018-12-26 | 2019-07-05 | 山东绿都生物科技有限公司 | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen |
| CN109975541B (en) * | 2018-12-26 | 2022-06-21 | 山东绿都生物科技有限公司 | Detection card for rapidly detecting canine distemper virus antigen and preparation method thereof |
| CN113474657A (en) * | 2019-02-21 | 2021-10-01 | 丁勤学 | Method for removing non-specific binding signals using microparticles |
| CN112129936A (en) * | 2020-09-21 | 2020-12-25 | 嘉兴朝云帆生物科技有限公司 | Parvovirus detection test paper and method based on fluorescence immunochromatography technology |
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Application publication date: 20160706 |