CN103529212A - Method for detecting pneumonic mycoplasma, quantum dot-labeled immunochromatographic test paper and preparation method thereof - Google Patents
Method for detecting pneumonic mycoplasma, quantum dot-labeled immunochromatographic test paper and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a medimmune inspection method, and particularly relates to quantum dot-labeled immunochromatographic test paper, and a method for detecting pneumonic mycoplasma by adopting an immunological method. According to the quantum dot-labeled immunochromatographic test paper, a glass cellulose membrane A, a quantum dot-labeled pneumonic mycoplasma IgM monoclonal antibody glass cellulose membrane B, a cellulose nitrate membrane and absorbent paper are sequentially are bonded on a plastic board from bottom to top, wherein a pneumonic mycoplasma polyclonal antibody and a rabbit-anti-mouse secondary antibody are on one end of the cellulose nitrate membrane so as to form an inspection strip T and a quality control strip C; a quantum dot-labeled pneumonic mycoplasma IgM monoclonal antibody is located at the other end of the glass cellulose membrane B and is corresponding to the inspection strip T and the quality control strip C, and the quantum dot-labeled pneumonic mycoplasma IgM monoclonal antibody is located at one end of a sample feeding point. The inspection sensitivity of the method is higher than of the currently used inspection method by about 1000 times.
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 mycoplasma pneumoniae.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is a kind of microorganism between virus and bacterium, be a class can be on without life nutrient culture media the minimum prokaryotic microorganism of growth and breeding.MP infects and can occur at any age, especially more common with 5～20 years old.MP propagates with the form of aerosol particles by the spittle, after infection, cause mycoplasma pneumoniae pneumonia (Mycoplasmal pneumonia, MPP), every 3～5 years, there is an endemic, account for 10%～20% of each parapneumonia sum, the bezonian person of suffering from an inflammation of the lungs 30%～50% is caused by mycoplasma pneumoniae; Account for the more than 1/3 of non-bacterial pneumonia.Also can cause that in addition outer each system of lung changes, and have death report, cause clinical concern.
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 mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM monoclonal antibody is positioned at the other end of glass fibre element film B, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody;
With test paper cutting knife, cut into test paper, dry rear sealing is preserved.
With described detection paper mycoplasma pneumoniae, comprise the steps: sample point sample to approach one end of mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM monoclonal antibody is positioned at the other end of glass fibre element film B, and band T and quality control band C are corresponding with detecting, and quantum dot-labeled mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae-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 mycoplasma pneumoniae blotting membranes, dry the rear quantum dot-labeled mycoplasma pneumoniae IgM 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: by described detection paper mycoplasma pneumoniae IgM monoclonal antibody, comprise the steps: sample point sample to approach one end of mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae 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 mycoplasma pneumoniaes
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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae polyclonal antibody and the anti-mouse two of rabbit of 0.05～0.15M, by 0.5g/L mycoplasma pneumoniae polyclonal antibody and anti-being sprayed on nitrocellulose filter 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 mycoplasma pneumoniae IgM monoclonal antibody is evenly sprayed in glass fibre membrane B above to 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM described in claim 1-6 any one, it is characterized in that, comprise the steps: sample point sample to approach one end of mycoplasma pneumoniae IgM monoclonal antibody on the test paper assembling, after reaction 5min, observations in uv analyzer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104198703A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof |
| CN105116150A (en) * | 2015-08-12 | 2015-12-02 | 杭州创新生物检控技术有限公司 | Mycoplasma pneumoniae antigen detection kit and detection method thereof |
| CN105203768A (en) * | 2014-08-18 | 2015-12-30 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
| CN105424933A (en) * | 2016-01-26 | 2016-03-23 | 姜竹泉 | Collaurum immunochromatography test strip detecting mycoplasma pneumoniae and preparing method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104198703A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof |
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| CN105424933A (en) * | 2016-01-26 | 2016-03-23 | 姜竹泉 | Collaurum immunochromatography test strip detecting mycoplasma pneumoniae and preparing method thereof |
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