CN103529212B - 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 medimmune inspection method, particularly relate to use quantum dot-labeled immunochromatographyassay assay test, detect the method for mycoplasma pneumoniae with immunologic method.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is a kind of microorganism between virus and bacterium, is that a class can at the minimum prokaryotic microorganism without growth and breeding on life nutrient culture media.MP infects and can occur at any age, especially more common with 5 ~ 20 years old.MP is propagated with the form of aerosol particles by the spittle, mycoplasma pneumoniae pneumonia (Mycoplasmal pneumonia is caused after infection, MPP), an endemic is there is every 3 ~ 5 years, 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 more than 1/3 of non-bacterial pneumonia.The outer each system of lung also can be caused in addition to change, and have death to report, cause clinical concern.
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 mycoplasma pneumoniae.
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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM monoclonal antibody is positioned at the other end of glass fibre element film B, corresponding with detection zone T and quality control band C, 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 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae polyclonal antibody and rabbit against murine two, 0.5g/L mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
With described detection paper mycoplasma pneumoniae, comprise the steps: sample point sample one end close to mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM monoclonal antibody is positioned at the other end of glass fibre element film B, corresponding with detection zone T and quality control band C, 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 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae polyclonal antibody and rabbit against murine two, 0.5g/L mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae-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 mycoplasma pneumoniae blotting membranes, dry and put into quantum dot-labeled mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody, comprises the steps: sample point sample one end close to mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae 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 mycoplasma pneumoniae
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 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, described plastic plate is pasted with from top to bottom successively glass fibre element film A, the glass fibre element film B of quantum dot-labeled mycoplasma pneumoniae IgM monoclonal antibody, nitrocellulose filter, thieving paper;
Wherein, described nitrocellulose filter one end has mycoplasma pneumoniae 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 mycoplasma pneumoniae IgM 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 mycoplasma pneumoniae IgM monoclonal antibody is positioned at sample delivery point one end;
Wherein, described quantum dot is CdTe/ZnSe core-shell quanta dots;
Described plastic plate is viscosity PVC base plate;
The concentration of described mycoplasma pneumoniae polyclonal antibody is 0.5g/L;
The concentration that described rabbit against murine two resists is 1.0g/L;
Described detection zone T and quality control band C spacing are no less than 5mm.
2. the preparation method of quantum dot-labeled immunochromatographyassay assay test as claimed in claim 1, is characterized in that, comprise the steps:
(1) coupling of quantum dot and mycoplasma pneumoniae IgM monoclonal antibody:
Get PBS damping fluid 100 ~ 200 μ L and the surperficial quantum dot being connected with carboxyl of 5 ~ 20 μ L of 0.01M;
Choose coupling reagent;
Add mycoplasma pneumoniae IgM 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;
Wherein, the coupling reagent in step (1) is selected from hydroxy succinimide, 1-(3-dimethyl aminopropyl)-3-ethyl carbon diamine hydrochloride;
(2) preparation of test paper:
Resist by the PBS damping fluid of 0.05 ~ 0.15M dilution mycoplasma pneumoniae polyclonal antibody and rabbit against murine two, 0.5g/L mycoplasma pneumoniae polyclonal antibody and 1.0g/L rabbit against murine two being resisted is sprayed on nitrocellulose filter, 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 mycoplasma pneumoniae IgM monoclonal antibody is evenly sprayed on glass fibre membrane B, 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 mycoplasma pneumoniae IgM monoclonal antibody, nitrocellulose filter, thieving paper successively;
Cut into test paper with test paper cutting knife, after drying, seal preservation.
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| CN104198703B (en) * | 2014-08-18 | 2015-12-30 | 湖北工业大学 | People's mycoplasma pneumoniae gold label silver stain immunochromatographytest test kit and its preparation method and application |
| CN105203768B (en) * | 2014-08-18 | 2017-01-18 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
| 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 |
| 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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| CN102928587A (en) * | 2012-11-16 | 2013-02-13 | 南京凯基生物科技发展有限公司 | Colloidal gold method detection test strip and reagent kit for IgM and IgG antibodies of mycoplasma pneumoniae and preparation method of reagent kit |
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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 |
| CN101051048A (en) * | 2007-05-11 | 2007-10-10 | 杨致亭 | Mycoplasma pneumiae anti-body testpaper strip |
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