CN103529216B - Preparation method for quantum-dot labeled immunochromatography test paper - Google Patents
Preparation method for quantum-dot labeled immunochromatography test paper Download PDFInfo
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- CN103529216B CN103529216B CN201310485259.5A CN201310485259A CN103529216B CN 103529216 B CN103529216 B CN 103529216B CN 201310485259 A CN201310485259 A CN 201310485259A CN 103529216 B CN103529216 B CN 103529216B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/12—Mumps virus; Measles virus
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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 mumps 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 mumps 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 mumps 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 mumps 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 mumps virus IgG monoclonal antibody is positioned at one end of a sampling point. The method has the advantage that the detection sensitivity is about 1000 times higher than that of the commonly-used 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 mumps virus with immunologic method.
Background technology
Adenositis virus (mumps virus) is the pathogen of mumps, belongs to Paramyxoviridae.Mumps virus is spherical, and nucleocapsid is helically symmetrical, has coating.Coating there are hemagglutinin-neuraminidase furcella (HN) and fusion factor furcella (F).Genome is single strand RNA. mumps virus can breed in chick embryo amnoitic sac or chick-embryo cell, can occur Fusion of Cells, but cytopathy is not obvious.Mumps virus only has a serotype.Resistibility is more weak, within 30 minutes 56 DEG C time, can be inactivated, to ultraviolet and fatsolvent sensitivity.People is the only host of mumps virus, and virus is through droplet transmission, and susceptible person is disease in school age children, is apt to occur in winter-spring season.In about 2 ~ 3 weeks latent period of this disease, after Virus entry airway epithelial cell and regional nodes's internal breeding, enter blood flow, then invade the parotid gland and other body of gland organ as testis, ovary, pancreas, kidney and central nervous system etc. through blood flow.Clinical manifestation is mainly side or enlargement of bilateral parotid glands, hot, weak, myalgia of occurring together etc.The course of disease 1 ~ 2 week, the easy concurrent orchitis (20%) of the infected in puberty or oaritis (5%), the infant of about 0.1% can concurrent viral meningitis.Concurrent orchitis person can cause male sterility, and mumps is also the common cause of acquired deafness cause childhood.Mumps can obtain firmly immunity after being ill.
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 with this detection paper mumps 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 mumps virus IgG 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 mumps virus IgG monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has mumps 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 mumps virus IgG 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 mumps virus IgG 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 polyclonal concentration of described mumps virus 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 mumps virus IgG 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 mumps virus IgG 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 mumps virus polyclonal antibody and rabbit against murine two, 0.5g/L mumps 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 mumps virus IgG 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 mumps virus IgG monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
With described detection paper mumps virus, comprise the steps: sample point sample one end close to mumps virus monoclonal IgG 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 mumps virus IgG 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 mumps virus IgG monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has mumps virus polyclone and rabbit against murine two to resist, and forms detection zone T and quality control band C with this;
Wherein, described quantum dot-labeled mumps virus IgG 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 mumps virus IgG 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 mumps 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 mumps virus IgG 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 mumps virus IgG 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 mumps virus polyclonal antibody and rabbit against murine two, 0.5g/L mumps 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 mumps virus IgG 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 mumps virus IgG 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 mumps 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 mumps virus blotting membranes, dry and put into quantum dot-labeled mumps virus IgG 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: with described detection paper mumps virus, comprises the steps: sample point sample one end close to mumps virus IgG 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 mumps 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 mumps 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 mumps virus IgG monoclonal antibody:
Get PBS damping fluid 100 ~ 200 μ L and the surperficial CdTe/ZnSe core-shell quanta dots being connected with carboxyl of 5 ~ 20 μ L of 0.01M;
Choose coupling reagent;
Add mumps virus IgG monoclonal antibody 150 ~ 200 μ L;
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 mumps virus polyclone and rabbit against murine two, 0.5g/L mumps virus polyclone 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 mumps virus IgG 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 mumps virus IgG 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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| 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 |
| CN101339196A (en) * | 2008-07-29 | 2009-01-07 | 上海师范大学 | Rapid checking method for bladder cancer by quantum dot mark immunity-chromatography test paper |
| CN101363848B (en) * | 2008-09-24 | 2013-05-29 | 深圳市菲鹏生物股份有限公司 | Double antigen sandwich method for detecting antibody by indirectly marked nanometer granule and kit thereof |
| CN101551398B (en) * | 2008-11-26 | 2012-09-05 | 中国计量学院 | Drug residue competition-type quantum dot-labeled immunochromatography assay test-strip and observation device thereof |
| CN102520165B (en) * | 2011-12-29 | 2014-07-30 | 北京康美天鸿生物科技有限公司 | Method for highly sensitive quantitative detection of quantum dot fluorescence immunochromatographic assay |
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