CN103543267A - Method for detecting hepatitis A virus (HAV), quantum dot labelled immunochromatographic test strip and preparation method thereof - Google Patents
Method for detecting hepatitis A virus (HAV), quantum dot labelled immunochromatographic test strip and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a medical immunodetection method and in particular relates to a method for detecting a hepatitis A virus (HAV) by an immunological method by using a quantum dot labelled immunochromatographic test strip. The quantum dot labelled immunochromatographic test strip is characterized in that a glass cellulose membrane A, a glass cellulose membrane B of a quantum dot labelled HAV monoclonal antibody, a nitrocellulose membrane and absorbent paper are stuck to a plastic board from bottom to top in sequence, wherein one end of the nitrocellulose membrane is provided with an HAV polyclonal antibody and a rabbit anti-mouse second antibody, thereby forming a detection zone T and a quality control zone C; the quantum dot labelled HAV monoclonal antibody is arranged at the other end of the glass cellulose membrane B, corresponds to the detection zone T and the quality control zone C and is arranged at one end of a sampling point. The detection sensitivity of the method is about 1000 times higher than that of the detection method frequently used at present.
Description
Technical field
The present invention relates to medical immunology detection method, particularly relate to use quantum dot-labeled immunochromatographyassay assay test, with immunologic method, detect the method for hepatitis A virus.
Background technology
Viral hepatitis type A is called for short hepatitis A, is a kind of acute infectious disease being caused by hepatitis A virus (HAV).Show as clinically onset anxious, have chilly, heating, anorexia, nauseating, tired, hepatomegaly and dysfunction of liver.There is jaundice in some cases, symptomless infection case is more common, generally do not transfer chronic and carrier state to.The hepatitis A infection sources is acute patient and subclinical infection person normally, patient is from hiding latter stage to the latter 10 days infectiousness maximums of falling ill, fecal-oral transmission is its main route of transmission, and water, food are that fulminant master looks into mode, and daily life contact is the main route of transmission of Sporadic cases.
At present, conventional detection method is colloidal gold method, although that this method detects is fast and convenient, and easily operation, accuracy rate is lower, and sensitivity is also lower.Therefore seek a kind of low price, easy and simple to handle, sensitivity and specificity all higher detection method be 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 papers and by the method for this detection paper hepatitis A virus of a kind of low price, easy and simple to handle, sensitivity and specificity.
, be provided with plastic plate, nitrocellulose filter, glass fibre element film A, the glass fibre element film B of quantum dot-labeled hepatitis A virus monoclonal antibody, thieving paper;
Wherein, on described plastic plate, be pasted with successively glass fibre element film B, nitrocellulose filter, the thieving paper of glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
Wherein, described nitrocellulose filter one end has hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit to resist, and with this, forms and detects band T and quality control band C;
Wherein, described quantum dot-labeled hepatitis A virus monoclonal antibody is positioned at one end of glass fibre element film B, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled hepatitis A 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 A virus polyclonal antibody is 0.5g/L.
Preferably, the anti-concentration of the anti-mouse two of described rabbit is 1.0g/L.
Preferably, described detection band 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 hepatitis A virus monoclonal antibody:
Get PBS damping fluid 100～200uL of 0.01M and the quantum dot that 5～20uL surface is connected with carboxyl;
Choose coupling reagent, coupling reagent is selected from hydroxyl sulfo-succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochlorides;
Add hepatitis A virus monoclonal antibody 150～200uL;
Shaking table reaction 1～4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) sealing with 1%～5%;
4 ℃ of preservations;
(2) preparation of test paper:
Anti-with PBS damping fluid dilution hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L hepatitis A virus polyclonal antibody and the anti-nitrocellulose filter one end that is sprayed on of the anti-mouse two of 1.0g/L rabbit, form and detect band T and quality control band C, T band and C band interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, and 37 ℃ of sealings are stand-by, or dry rear 4 ℃ of preservations;
Quantum dot-labeled hepatitis A virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, with the T band forming and C be with corresponding, drying at room temperature, 4 ℃ of preservations;
On plastic plate, glue successively glass fibre element film B, nitrocellulose filter, the thieving paper of note glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
With test paper cutting knife, cut into test paper, dry rear sealing is preserved.
With described detection paper hepatitis A virus, comprise the steps: sample point sample to approach one end of hepatitis A virus monoclonal antibody on the test paper assembling, after reaction 5min, observations in uv analyzer.
Compared with prior art, the present invention has the following advantages: owing to having added core-shell quanta dots in prepared test paper, particularly adopted CdTe/ZnSe water-soluble nuclear-shell quantum dot, by water miscible quantum dot and specific by covalent coupling, then utilize double antibodies sandwich principle to detect in sample whether contain object in conjunction with the photoluminescent property of quantum dot.Under the irradiation of uviol lamp, by observing the photoluminescence line that detects band and quality control band on immuno-chromatographic test paper strip, judgement testing result.The present invention combines the fluorescent characteristic of immunoreactive high specific and quantum dot, utilizes quantum dot multi-wavelength excitation, high strength fluorescent emission, and emission peak is narrow, peak shape is symmetrical, and the fluorescent characteristic that stability of photoluminescence is good has been set up fast, special, easy, sensitive immunochromatography detection method.By observing fluorescence signal, reached the object of quantitative detection.The detection sensitivity of the method is than the high approximately 1000 times of left and right of the detection sensitivity of conventional at present a kind of method for quick-collaurum.
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 hepatitis A virus monoclonal antibody, thieving paper, described glass fibre element film A does not have the glass fibre element film bought on the market of point sample;
Wherein, on described plastic plate, be pasted with successively glass fibre element film B, nitrocellulose filter, the thieving paper of glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
Wherein, described nitrocellulose filter one end has hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit to resist, and with this, forms and detects band T and quality control band C;
Wherein, described quantum dot-labeled hepatitis A virus monoclonal antibody is positioned at one end of glass fibre element film B, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled hepatitis A 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 A virus polyclonal antibody is 0.5g/L.
Preferably, the anti-concentration of the anti-mouse two of described rabbit is 1.0g/L.
Preferably, described detection band 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 hepatitis A virus monoclonal antibody:
Get PBS damping fluid 100～200uL of 0.01M and the quantum dot that 5～20uL surface is connected with carboxyl;
Choose coupling reagent, coupling reagent is selected from hydroxyl sulfo-succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochlorides;
Add hepatitis A virus monoclonal antibody 150～200uL;
Shaking table reaction 1～4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) sealing with 1%～5%;
4 ℃ of preservations;
(2) preparation of test paper:
Anti-with PBS damping fluid dilution hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L hepatitis A virus polyclonal antibody and the anti-nitrocellulose filter one end that is sprayed on of the anti-mouse two of 1.0g/L rabbit, form and detect band T and quality control band C, T band and C band interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, and 37 ℃ of sealings are stand-by, or dry rear 4 ℃ of preservations;
Quantum dot-labeled hepatitis A virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, with T band and C be with corresponding, drying at room temperature, 4 ℃ of preservations;
On plastic plate, glue successively glass fibre element film B, nitrocellulose filter, the thieving paper of note glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
With test paper cutting knife, cut into test paper, dry rear sealing is preserved.
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, observes electrophoresis result in uv analyzer.
Dot blotting: by 0.3g/L hepatitis A virus-BSA liquid point on nitrocellulose filter, after drying, be soaked in the 0.01mol/L phosphate buffer (PBS containing 5%BSA, 10nmol/L, PH=7.4, containing NaCl8.5g/L) in, remaining protein binding site on closing membrane, hatches 1h in 37 ℃, after sealing, take out PBS washing.Prepare three identical hepatitis A virus blotting membranes, dry the rear quantum dot-labeled hepatitis A virus monoclonal antibody of putting into respectively, quantum dot-BSA and quantum dot solution, in 37 ℃ of shaking table reaction 30min, PBS washing.Observations in uv analyzer.
Embodiment 3: with described detection paper hepatitis A virus, comprise the steps: sample point sample to approach one end of hepatitis A virus monoclonal antibody on the test paper assembling, and after reaction 5min, observations in uv analyzer.With PBS damping fluid and normal person's blood, be made as blank.
Result is judged: at C band, manifests under the prerequisite of red fluorescence band, and the fluorescent belt intensity of visual T band, with blank, fluorescence is more weak, represents that in liquid to be measured, the concentration containing checking matter is lower.
Embodiment 4: detection paper validation verification, prepare the hepatitis A virus solution of four kinds of concentration, and concentration is respectively 0.5,1.0,2.0,5.0ug/L.Utilize Test paper and chemiluminescence paper box to detect sample, each concentration detects 6 times, measures 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 A 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 instruction of the present invention.Therefore all equal variation and modifications of doing according to the present patent application the scope of the claims, once should still remain within the scope of the patent.
Claims (9)
1. a quantum dot-labeled immunochromatographyassay assay test, is characterized in that, is provided with plastic plate, nitrocellulose filter, glass fibre element film A, the glass fibre element film B of quantum dot-labeled hepatitis A virus monoclonal antibody, thieving paper;
Wherein, on described plastic plate, be pasted with successively from top to bottom glass fibre element film B, nitrocellulose filter, the thieving paper of glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
Wherein, described nitrocellulose filter one end has hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit to resist, and with this, forms and detects band T and quality control band C;
Wherein, described quantum dot-labeled hepatitis A virus monoclonal antibody is positioned at one end of glass fibre element film B, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled hepatitis A virus monoclonal antibody is positioned at sample delivery point one end.
2. quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, described quantum dot is CdTe/ZnSe core-shell quanta dots.
3. quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, described plastic plate is viscosity PVC base plate.
4. quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, the concentration of described hepatitis A virus polyclonal antibody is 0.5g/L.
5. quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, the anti-concentration of the anti-mouse two of described rabbit is 1.0g/L.
6. quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, described detection band T and quality control band C spacing are no less than 5mm.
7. the as above preparation method of the quantum dot-labeled immunochromatographyassay assay test described in any one claim, is characterized in that, comprises the steps:
(1) coupling of quantum dot and hepatitis A virus monoclonal antibody:
Get PBS damping fluid 100～200uL of 0.01M and the quantum dot that 5～20uL surface is connected with carboxyl;
Choose coupling reagent;
Add hepatitis A virus monoclonal antibody 150～200uL;
Shaking table reaction 1～4 hour;
Chromatographic column filters, centrifugal purification;
Bovine serum albumin(BSA) sealing with 1%～5%;
4 ℃ of preservations;
(2) preparation of test paper:
Anti-with PBS damping fluid dilution hepatitis A virus polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L hepatitis A virus polyclonal antibody and the anti-nitrocellulose filter one end that is sprayed on of the anti-mouse two of 1.0g/L rabbit, form and detect band T and quality control band C, T band and C band interval 5mm, room temperature is dried, nitrocellulose filter is put into PBS damping fluid, and 37 ℃ of sealings are stand-by, or dry rear 4 ℃ of preservations;
Quantum dot-labeled hepatitis A virus monoclonal antibody is evenly sprayed in glass fibre membrane B one end, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled hepatitis A virus monoclonal antibody is positioned at sample delivery point one end, drying at room temperature, 4 ℃ of preservations;
On plastic plate, glue successively glass fibre element film B, nitrocellulose filter, the thieving paper of note glass fibre element film A, quantum dot-labeled hepatitis A virus monoclonal antibody;
With test paper cutting knife, cut into test paper, dry rear sealing is preserved.
8. the preparation method of quantum dot-labeled immunochromatographyassay assay test as claimed in claim 7, is characterized in that, the coupling reagent in step (1) is selected from hydroxyl sulfo-succinimide, 1-(3-dimethyl aminopropyl)-3 ethyl carbon diamine hydrochlorides.
9. with the detection paper hepatitis A virus described in claim 1-6 any one, it is characterized in that, comprise the steps: sample point sample to approach one end of hepatitis A virus monoclonal antibody on the test paper assembling, after reaction 5min, observations in uv analyzer.
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Cited By (1)
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| CN104502590A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Hepatitis A virus chromatography test paper strip based on low-noise excitation fluorescent label |
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Application publication date: 20140129 |