CN103529213B - Method for preparing quantum-dot labeled immunochromatography test paper - Google Patents
Method for preparing quantum-dot labeled immunochromatography test paper Download PDFInfo
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Abstract
The invention relates to a medical immunodetection method and particularly relates to a method for using quantum-dot labeled immunochromatography test paper to detect hepatitis E virus by an immunological method. The quantum-dot labeled immunochromatography test paper is characterized in that a plastic board is stuck with a glass cellulose membrane A, a glass cellulose membrane B of a quantum-dot labeled hepatitis E virus IgG monoclonal antibody, a nitrocellulose membrane and water absorbing paper from bottom to top in sequence, wherein one end of the nitrocellulose membrane is provided with hepatitis E virus polyclonal antibody and rabbit antimouse second antibody so as to form a detecting band T and a quality control band C; the quantum-dot labeled hepatitis E virus IgG monoclonal antibody is positioned at one end of the glass cellulose membrane and corresponds to the detecting band T and the quality control band C, and the quantum-dot labeled hepatitis E virus IgG monoclonal antibody is positioned at one end of a sampling point. The method has the advantages that high specificity of immunoreactions and the fluorescence characteristic of quantum dots are combined, so that the detection sensitivity is about 1000 times higher than that of the current commonly-used detection method.
Description
Technical field
The present invention relates to medimmune inspection method, particularly relate to use quantum dot-labeled immunochromatographyassay assay test, detect the method for hepatitis E virus with immunologic method.
Background technology
Hepatitis E is the Amphixenosis of a kind of people and the many animals caused by hepatitis E virus.Mainly propagate through ight soil approach, often cause outbreak of epidemic because drinking water source is contaminated, and Sporadic cases is distribution on global.Hepatitis E accounts for 15% ~ 20% of China's acute type hepatitis.The mortality ratio suffering from the adult of viral hepatitis type E reaches 0.5% ~ 3%, and pregnant woman is then up to 15% ~ 20%.Therefore, viral hepatitis type E examination and make a definite diagnosis particularly important.
At present, conventional detection method is colloidal gold method, although this method detects fast and convenient, easily operate, accuracy rate is lower, and sensitivity is also lower.The detection method of therefore seeking a kind of low price, easy and simple to handle, sensitivity and specificity all higher is problem in the urgent need to address.
Summary of the invention
For the weak point of above-mentioned technology, the invention provides all higher Test paper of a kind of low price, easy and simple to handle, sensitivity and specificity and the method by this detection paper hepatitis E virus.
A kind of quantum dot-labeled immunochromatographyassay assay test, is provided with plastic plate, nitrocellulose filter, glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, thieving paper;
Wherein, described plastic plate is pasted with successively glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has hepatitis E virus polyclonal antibody and rabbit against murine two to resist, and forms detection zone T and quality control band C with this;
Wherein, described quantum dot-labeled E type hepatitis virus monoclonal antibody is positioned at one end of glass fibre element film B, corresponding with detection zone T and quality control band C, and quantum dot-labeled E type hepatitis virus monoclonal antibody is positioned at sample delivery point one end.
Preferably, described quantum dot is CdTe/ZnSe core-shell quanta dots.
Preferably, described plastic plate is viscosity PVC base plate.
Preferably, the concentration of described hepatitis E virus polyclonal antibody is 0.5g/L.
Preferably, the concentration that described rabbit against murine two is anti-is 1.0g/L.
Preferably, described detection zone T and quality control band C spacing are no less than 5mm.
The preparation method of test paper as above, comprises the steps:
(1) coupling of quantum dot and E type hepatitis virus monoclonal antibody:
Get PBS damping fluid 100 ~ 200uL and the surperficial quantum dot being connected with carboxyl of 5 ~ 20uL of 0.01M;
Choose coupling reagent, coupling reagent is selected from hydroxy succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochloride;
Add E type hepatitis virus monoclonal antibody 150 ~ 200uL;
Shaking table reaction 1 ~ 4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) with 1% ~ 5% is closed;
4 DEG C of preservations;
(2) preparation of test paper:
Resist by the PBS damping fluid of 0.05 ~ 0.15M dilution hepatitis E virus polyclonal antibody and rabbit against murine two, 0.5g/L hepatitis E virus polyclonal antibody and 1.0g/L rabbit against murine two are resisted and is sprayed on nitrocellulose filter one end, form detection zone T and quality control band C, T band and C are with interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, close stand-by for 37 DEG C, or dry rear 4 DEG C of preservations;
Quantum dot-labeled E type hepatitis virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, to be with and C is with corresponding, drying at room temperature, 4 DEG C of preservations with the T formed;
Plastic plate glues note glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
By described detection paper hepatitis E virus, comprise the steps: sample point sample one end close to E type hepatitis virus monoclonal antibody on the test paper assembled, after reaction 5min, observations in uv analyzer.
Compared with prior art, the present invention has the following advantages: owing to adding core-shell quanta dots in prepared test paper, particularly have employed CdTe/ZnSe water-soluble nuclear-shell quantum dot, by water miscible quantum dot and specific by covalent coupling, the photoluminescent property of double antibodies sandwich principle incorporating quantum point is then utilized whether to detect in sample containing object.Under the irradiation of uviol lamp, by observing the photoluminescence line of detection zone and quality control band on immuno-chromatographic test paper strip, judge testing result.The fluorescent characteristic of immunoreactive high specific and quantum dot combines by the present invention, utilizes quantum dot multi-wavelength excitation, high strength fluorescent emission, and emission peak is narrow, peak shape is symmetrical, the fluorescent characteristic that stability of photoluminescence is good, establishes fast, special, easy, sensitive immunochromatography detection method.The object quantitatively detected is reached by observing fluorescence signal.The detection sensitivity height about about 1000 times of the current conventional a kind of method for quick-collaurum of detection sensitivity ratio of the method.
Accompanying drawing explanation
Fig. 1 is process chart prepared by test paper.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1: a kind of quantum dot-labeled immunochromatographyassay assay test, be provided with plastic plate, nitrocellulose filter, glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, thieving paper, described glass fibre element film A is the glass fibre element film not having the market of point sample is bought;
Wherein, described plastic plate is pasted with successively glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has hepatitis E virus polyclonal antibody and rabbit against murine two to resist, and forms detection zone T and quality control band C with this;
Wherein, described quantum dot-labeled E type hepatitis virus monoclonal antibody is positioned at one end of glass fibre element film B, corresponding with detection zone T and quality control band C.
Preferably, described quantum dot is CdTe/ZnSe core-shell quanta dots.
Preferably, described plastic plate is viscosity PVC base plate.
Preferably, the concentration of described hepatitis E virus polyclonal antibody is 0.5g/L.
Preferably, the concentration that described rabbit against murine two is anti-is 1.0g/L.
Preferably, described detection zone T and quality control band C spacing are no less than 5mm.
Embodiment 2: the preparation method of test paper as above, as shown in Figure 1, comprises the steps:
(1) coupling of quantum dot and E type hepatitis virus monoclonal antibody:
Get PBS damping fluid 100 ~ 200uL and the surperficial quantum dot being connected with carboxyl of 5 ~ 20uL of 0.01M;
Choose coupling reagent, coupling reagent is selected from hydroxy succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochloride;
Add E type hepatitis virus monoclonal antibody 150 ~ 200uL;
Shaking table reaction 1 ~ 4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) with 1% ~ 5% is closed;
4 DEG C of preservations;
(2) preparation of test paper:
Resist by the PBS damping fluid of 0.05 ~ 0.15M dilution hepatitis E virus polyclonal antibody and rabbit against murine two, 0.5g/L hepatitis E virus polyclonal antibody and 1.0g/L rabbit against murine two are resisted and is sprayed on nitrocellulose filter one end, form detection zone T and quality control band C, T band and C are with interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, close stand-by for 37 DEG C, or dry rear 4 DEG C of preservations;
Quantum dot-labeled E type hepatitis virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, to be with T and C is with corresponding, drying at room temperature, 4 DEG C of preservations;
Plastic plate glues note glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
Wherein, in step (1), the detection method of coupling effect is as follows:
Gel electrophoresis: adopt 0.8 Ago-Gel, voltage is 80V, after electrophoresis 15min, in uv analyzer, observes electrophoresis result.
Dot blotting: by 0.3g/L hepatitis E virus-BSA liquid point on nitrocellulose filter, 0.01mol/L phosphate buffer (the PBS containing 5%BSA is soaked in after drying, 10nmol/L, PH=7.4, containing NaCl 8.5g/L) in, remaining protein binding site on closing membrane, hatches 1h in 37 DEG C, take out after closing, PBS washs.Prepare three identical hepatitis E virus blotting membranes, dry and put into quantum dot-labeled E type hepatitis virus monoclonal antibody respectively, quantum dot-BSA and quantum dot solution afterwards, in 37 DEG C of shaking tables reaction 30min, PBS washings.Observations in uv analyzer.
Embodiment 3: by described detection paper hepatitis E virus, comprises the steps: sample point sample one end close to E type hepatitis virus monoclonal antibody on the test paper assembled, after reaction 5min, and observations in uv analyzer.Blank is set to the blood of PBS damping fluid and normal person.
Result judges: under C band manifests the prerequisite of red fluorescence band, and the fluorescent belt intensity of visual T band, with blank, fluorescence is more weak, and the concentration represented containing checking matter in liquid to be measured is lower.
Embodiment 4: detection paper validation verification, the hepatitis E virus solution of preparation four kinds of concentration, concentration is respectively 0.5,1.0,2.0,5.0ug/L.Utilize Test paper and chemiluminescence paper box to detect sample, each Concentration Testing 6 times, measure the validity that test paper detects result.As shown in Table 1, result show the detection paper prepared with the present invention out present the positive, and the larger fluorescence intensity of solution concentration is larger.
Table one: the ELISA test strip result of various concentration hepatitis E virus
The above, be only preferred embodiment of the present invention, and professional who are familiar with this art is after understanding technological means of the present invention such as, and natural energy, according to actual needs, is changed under the teachings of the present invention.Therefore all equal changes of doing according to the present patent application the scope of the claims and modification, once should still remain within the scope of the patent.
Claims (2)
1. a preparation method for quantum dot-labeled immunochromatographyassay assay test, is characterized in that, comprises the steps:
(1) coupling of quantum dot and E type hepatitis virus monoclonal antibody:
Get PBS damping fluid 100 ~ 200uL and the surperficial CdTe/ZnSe core-shell quanta dots being connected with carboxyl of 5 ~ 20uL of 0.01M;
Choose coupling reagent;
Add E type hepatitis virus monoclonal antibody 150 ~ 200uL;
Shaking table reaction 1 ~ 4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) with 1% ~ 5% is closed;
4 DEG C of preservations;
(2) preparation of test paper:
Resist by the PBS damping fluid of 0.05 ~ 0.15M dilution hepatitis E virus polyclonal antibody and rabbit against murine two, 0.5g/L hepatitis E virus polyclonal antibody and 1.0g/L rabbit against murine two are resisted and is sprayed on nitrocellulose filter one end, form detection zone T and quality control band C, T band and C are with interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, close stand-by for 37 DEG C, or dry rear 4 DEG C of preservations;
Quantum dot-labeled E type hepatitis virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, corresponding with detection zone T and quality control band C, drying at room temperature, 4 DEG C of preservations;
Viscosity PVC base plate glues note glass fibre element film A, the glass fibre element film B of quantum dot-labeled E type hepatitis virus monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
2. the preparation method of quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, it is characterized in that, the coupling reagent in step (1) is selected from hydroxy succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochloride.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104237531A (en) * | 2014-08-25 | 2014-12-24 | 大理学院 | Viral hepatitis type E detection test strip device |
| CN105445463A (en) * | 2014-09-25 | 2016-03-30 | 苏州新波生物技术有限公司 | Hepatitis E virus IgG antibody detection kit and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1811449A (en) * | 2006-02-23 | 2006-08-02 | 上海交通大学 | Method for detecting quantum dot mark fast immune chromatographic test paper bar |
| CN1902496A (en) * | 2003-11-07 | 2007-01-24 | 赫普金尼克斯股份有限公司 | Binding assay components |
| CN101363848A (en) * | 2008-09-24 | 2009-02-11 | 深圳市菲鹏生物股份有限公司 | Sandwich method for detecting double antigen by antibody indirectly marked with nanometer granule and kit thereof |
| CN101551398A (en) * | 2008-11-26 | 2009-10-07 | 中国计量学院 | Drug residue competition-type quantum dot-labeled immunochromatography assay test-strip and observation device thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1902496A (en) * | 2003-11-07 | 2007-01-24 | 赫普金尼克斯股份有限公司 | Binding assay components |
| CN1811449A (en) * | 2006-02-23 | 2006-08-02 | 上海交通大学 | Method for detecting quantum dot mark fast immune chromatographic test paper bar |
| CN101363848A (en) * | 2008-09-24 | 2009-02-11 | 深圳市菲鹏生物股份有限公司 | Sandwich method for detecting double antigen by antibody indirectly marked with nanometer granule and kit thereof |
| CN101551398A (en) * | 2008-11-26 | 2009-10-07 | 中国计量学院 | Drug residue competition-type quantum dot-labeled immunochromatography assay test-strip and observation device thereof |
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Effective date of registration: 20160727 Address after: 102629 Beijing City, Daxing District science and Technology Park of Zhongguancun Daxing biological medicine industry base Qingfeng West Road, No. 29 Patentee after: Beijing Huawei Dieter Health Science and Technology Co Ltd Address before: 100054 Beijing city Xicheng District Baizhifang Street No. 10 Tairan Ju B block 11 layer Patentee before: Huaweiji Biological Medicines Co Ltd, Beijing |