CN103217535A - Immunochromatography detection card for detecting troponin I - Google Patents
Immunochromatography detection card for detecting troponin I Download PDFInfo
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- CN103217535A CN103217535A CN2013100871239A CN201310087123A CN103217535A CN 103217535 A CN103217535 A CN 103217535A CN 2013100871239 A CN2013100871239 A CN 2013100871239A CN 201310087123 A CN201310087123 A CN 201310087123A CN 103217535 A CN103217535 A CN 103217535A
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Abstract
The invention relates to a detection tool in the technical field of biological engineering, especially an immunochromatography detection card for detecting troponin I. The detection card comprises a sample pad, a nitrocellulose membrane coated membrane and an absorbent paper, which are sequentially pasted on the bottom plate in overlap joint, and a sample diluent and a freeze-drying probe preservation tube. The probe preservation tube contains fluorescent latex particle labeled troponin I monoclonal antibody; the nitrocellulose membrane coated membrane comprises a detection area and a quality control area; the detection area is coated with another troponin I monoclonal antibody, which is in different epitopes with the fluorescent latex particle labeled troponin I monoclonal antibody; and the quality control zone is coated with goat anti mouse IgG. The above structure has beneficial effect of high precision and good repeatability.
Description
Technical field
The present invention relates to a kind of testing tool of technical field of bioengineering, especially a kind of immunochromatographydetection detection card that detects Troponin I.
Background technology
Cardiovascular disease incidence rate height, disability rate height, mortality ratio height, complication are many, and be very serious to health hazard.Cardiovascular check is " bottleneck " subject in the whole cardiovascular field, only correctly diagnoses the illness in the time of section as far as possible, just might timely and effectively advanced treatment means be used for clinically, and the patient is benefited.Deep day by day along with to cardiovascular diagnosis mark research, clearer and more definite correlating markings thing be for clinical meaning, and clinical diagnosis, risk stratification, the therapeutic scheme that the detection of cardiac marker can directly influence cardiovascular patient selects and prognosis is judged.Therefore shorten the turnaround time (turn-aroundtime that cardiac marker detects, TAT) have great importance for clinical early diagnosis and early treatment angiocardiopathy, in the clinical diagnosis and examination process of heart disease, being applied as of various detection techniques finds timely morning, diagnoses and treatment provides strict scientific basis.Heart disease at present commonly used detects diagnostic method, mainly comprises: non-laboratory diagnosis as detecting of family's HDH investigation, Electrocardioscopy, X ray Chest X-rays inspection, the experiment of cardiogram load, cardiac catheterization, heart ultrasonic inspection etc. and laboratory inspection etc. as heart mark creatine kinase (CK) and creatine kinase isozyme (CK-MB), Troponin I (cTnl) or TnT (cTnT), myoglobins (Myoglobin), BNP or NT-proBNP.Wherein cardiac troponin (cTn) is again clinical susceptibility of present angiocardiopathy and the best myocardial damage mark of specificity, has become the most important diagnosis basis of myocardial tissue damage (as myocardium infarct).
The cardiovascular and cerebrovascular disease urgency of feeling nervous, prognosis is critical.So the Fast Detection Technique of cardiac marker and product are parts with fastest developing speed.Main in the market detection system comprises: the Biosite Triage detection system of U.S. Biosite, the RAMP detection system of Canadian Response, the Pathfast detection system of the i-SATA detection system of U.S. Abbott and Japanese Mitsubishi Kagaku Iatron etc.
The fluorescent chromatographic immune analysis method is that development in recent years plays a kind of microanalysis method not, according to the spectrum and the fluorescence intensity of fluorescence, material is carried out qualitative or quantitative test, has advantages such as high sensitivity, selectivity is strong, need sample amount is few and easy and simple to handle.But most at present fluorescence immune chromatography technical products are continued to use the qualitative immunochromatography technique of traditional collaurum, use the immobilization carrier of glass fibre as fluorescence probe, utilize sample to add the release of back to the infiltration realization probe of glass fibre, reagent detects the homogeneity less stable.
Summary of the invention
The objective of the invention is to, a kind of immunochromatographydetection detection card that detects the detection Troponin I of homogeneity stability is provided.
The technical solution adopted for the present invention to solve the technical problems is: a kind of immunochromatographydetection detection card that detects Troponin I, by sample pad, the nitrocellulose membrane coated film, thieving paper overlaps in turn and sticks on the base plate, and be furnished with sample diluting liquid and freeze-drying probe in addition and preserve pipe and constitute, described probe is preserved the Troponin I monoclonal antibody that contains fluorescent latex particulate mark in the pipe, described nitrocellulose membrane coated film comprises detection zone and Quality Control district, described detection zone is coated with the another kind of Troponin I monoclonal antibody that is in different epi-positions with the Troponin I monoclonal antibody of described fluorescent latex particulate mark, and wrap by sheep anti-mouse igg in described Quality Control district.
The further setting of the present invention is: the concentration of the Troponin I monoclonal antibody of described fluorescent latex particulate mark is 0.5-l.5mg/ml, presses the dilutability of 1:20-1:500, and the consumption of 5u l/ pipe joins probe and preserves in the pipe (6), and freeze-drying.
The further setting of the present invention is: the concentration of the Troponin I monoclonal antibody of described detection zone bag quilt is 0.5～2mg/ml, and spray film consumption is 100U l/20-100cm.
The detection zone bag was sprayed film exactly during this was provided with on detection zone, therefore related to spray film consumption.
The further setting of the present invention is: the concentration of described sheep anti-mouse igg is 1.0 1 2mg/ml, and spray film consumption is 100u1/20-100cm.
Quality Control district bag was sprayed film exactly during this was provided with in the Quality Control district, therefore related to spray film consumption.
The further setting of the present invention is: the diameter of described fluorescent latex particulate is 200-500nm.
The further setting of the present invention is: the wavelength of emission was 500nm-600nm after described fluorescent latex particulate was stimulated.
The beneficial effect of said structure is: precision height, good reproducibility.
Description of drawings
In order to be illustrated more clearly in the embodiment of the invention or technical scheme of the prior art, to do to introduce simply to the accompanying drawing of required use in embodiment or the description of the Prior Art below, apparently, accompanying drawing in describing below only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the structural representation of present embodiment;
Embodiment
With reference to figure 1 as can be known, overlapped in turn by sample pad 1, nitrocellulose membrane coated film 2, thieving paper 3 and to stick on the base plate 4, and be furnished with sample diluting liquid and freeze-drying probe in addition and preserve pipe and constitute, nitrocellulose membrane coated film 2 comprises detection zone 5 and Quality Control district 6.
In embodiments of the present invention, the Troponin I antibody that is adopted is the monoclonal antibody of conventional monoclonal antibody technique preparation, and the principle of utilizing double antibody sandwich method to detect Troponin I antigen detects sample.
The present invention detects the immunochromatographydetection detection card of Troponin I, and sample pad 1 contains a hydrophilic poroid barrier film, and sample pad l is a sample application zone, is used to draw Troponin I to be detected and detects sample.Sample pad 1 is overlapped with nitrocellulose filter coated film 2 and thieving paper 3 successively, and sample pad 1, nitrocellulose filter coated film 2 and thieving paper 3 all are pasted on the base plate (4).Use fluorescent latex (the about 300nm of diameter) the mark l strain Troponin I monoclonal anti (0.5-7mg/ml) of specific exciting light (580nm)/emission light (615nm) wavelength; Detection zone 5 places of cellulose nitrate coated film 2 adopt another strain monoclonal antibody (0.5-7mg/ml) of Troponin I to wrap quilt.At the Quality Control district of coated film 26 place's working concentrations is that the sheep anti-mouse igg of 1mg/ml wraps quilt, plays the effect of filtration, prevents that non-specific mouse lgG in the serum is to the influence of Quality Control district, the reaction of detection zone place.
The preparation that detects the immunochromatographydetection detection card of Troponin I may further comprise the steps:
1. fluorescent latex is covalent activated
Ultrasonic Treatment latex microsphere body is after 30 seconds, and regulating the latex microsphere bulk concentration is 1%(w/v), centrifugal 10 minutes of 10000～16000rpm, centrifugal back collecting precipitation thing dissolves with lOOmM pH6.O Hepes damping fluid, and ultrasound wave 200W handled 30 seconds; Add the lOOmg/ml EDC of 100ul earlier, shake mixing, add the 50mg/mlN-hydroxy thiosuccinimide (Sulfo-NHS) of 50ul again, the concussion mixing; Incubated at room centrifugal 5～15 minutes of 10000～15000rpm after 30 minutes, precipitation is placed under 2～8 ℃ of conditions standby with the MES damping fluid dissolving of lOOmM, pH5.0～6.0;
2. the preparation of fluorescent latex particulate labelled protein
After fluorescent latex ultrasound wave 200W after the activation handled 30 seconds, ratio according to 50u g labelled antibody/100ul fluorescent latex adds the Troponin I monoclonal antibody, the stirring at room reaction is 2 hours behind the mixing, centrifuge washing 3 times, centrifugal 10 minutes of each 10000～15000rpm rotating speed, precipitation was handled 30 seconds with PBS-TBN dissolving and ultrasound wave 2OOW, recovered centrifugal front volume with the PBS-TBN damping fluid.Press the dilutability of 1:20-1:500, the consumption of 5ul/ pipe joins probe and preserves in the pipe 6, and freeze-drying.
3. prepare the cellulose nitrate coated film
To be positioned at another strain Troponin I monoclonal antibody (coated antibody) of different epi-positions and sheep anti-mouse igg respectively with the Troponin I monoclonal antibody on the glass fibre membrane label pad is cushioned liquid and is adjusted to 0.5mg/ml and 1mg/ml respectively with bag, to be 1u1/cm. be sprayed onto detection zone 5 and Quality Control district 6 corresponding on the cellulose nitrate coated film with Troponin I monoclonal antibody and sheep anti-mouse igg wraps quilt by film coating buffer amount, detection zone 5 and Quality Control district 6 be 3-8mm at interval, dried 2-3 hour for 37 ℃ under humidity＜30% condition, envelope is standby;
4. on base plate 4, paste sample pad 1, cellulose nitrate coated film 2 and thieving paper 3 mutually successively obtain test paper plate to overlap joint, cut into the test card of proper width as requested.
Test card structure among this embodiment is all identical with EXAMPLE l.The concentration of the Troponin I monoclonal antibody of described fluorescent latex particulate mark is 0.1mg/ml, and the concentration of the Troponin I monoclonal antibody of described detection zone bag quilt is 1mg/ml, and spray film consumption is 1.5ul/cm.The concentration of described sheep anti-mouse igg is 1.5mg/ml, and spray film consumption is 1.5ul/cm.
[preparation method of this embodiment is except Troponin I monoclonal antibody in the step 2: fluorescent latex is 1% (w/v).All the other are all identical with EXAMPLE l, and using method is also identical with EXAMPLE l.
In the present invention, in use, be assembled in by plastics upper casing and plastics lower casing and fasten in the plastic casing that forms, the plastics upper casing is provided with well and detection window, well is corresponding to described Troponin I immunochromatographydetection detection card sample pad l, and detection window is corresponding to the detection zone 5 and the Quality Control district 6 of described detection Troponin I immunity-chromatography test test card.
Aspect precision, the reagent strip that utilizes embodiment 1-2 is respectively the sample of high value, low value and intermediate value to content, and continuous detecting at least 10 times is calculated the coefficient of variation (CV).For the high value of Troponin I content (21.4ng/ML), intermediate value (3.39ng/ML), low value (0.34ng/ML) blood sample is a case each measures respectively 10 times, according to its determination data, utilize SPSS statistical study assay CV value all less than 10%, so precision is very high.
The fluorescent microsphere that the present invention is built up by detection zone 5 on the light source activation coated film and 6 places, Quality Control district when in use, the fluorescence signal of launching after fluorescent microsphere is excited is received by corresponding detecting system, and photosignal is changed into electric signal by processes such as light-to-current inversion, photoelectricity conversions, and by the automatic control system that is provided with in the system signal is exported, demonstrate final quantitative result.
Obviously, the foregoing description only be for explanation clearly done for example, and be not qualification to embodiment.To those of ordinary skill in the art, can also make other changes in different forms on the basis of the above description.Here need not also can't give exhaustive to all embodiments.And conspicuous variation of being extended out thus or change still are in protection scope of the present invention.
Claims (6)
1. immunochromatographydetection detection card that detects Troponin I, it is characterized in that: by sample pad, the nitrocellulose membrane coated film, thieving paper overlaps in turn and sticks on the base plate, and be furnished with sample diluting liquid and freeze-drying probe in addition and preserve pipe and constitute, described probe is preserved the Troponin I monoclonal antibody that contains fluorescent latex particulate mark in the pipe, described nitrocellulose membrane coated film comprises detection zone and Quality Control district, described detection zone is coated with the another kind of Troponin I monoclonal antibody that is in different epi-positions with the Troponin I monoclonal antibody of described fluorescent latex particulate mark, and wrap by sheep anti-mouse igg in described Quality Control district.
2. according to the immunochromatographydetection detection card of the described detection Troponin I of claim 1, it is characterized in that: the concentration of the Troponin I monoclonal antibody of described fluorescent latex particulate mark is 0.5-l.5mg/ml, press the dilutability of 1:20-1:500, the consumption of 5ul/ pipe joins probe and preserves in the pipe (6), and freeze-drying.
3. according to the immunochromatographydetection detection card of the described detection Troponin I of claim 1, it is characterized in that: the concentration of the Troponin I monoclonal antibody of described detection zone bag quilt is 0.5～2mg/ml, and spray film consumption is 100Ul/20-100cm.
4. according to the immunochromatographydetection detection card of the described detection Troponin I of claim 1, it is characterized in that: the concentration of described sheep anti-mouse igg is 1.0 1 2mg/ml, and spray film consumption is 100u1/20-100cm.
5. according to the immunochromatographydetection detection card of the described detection Troponin I of claim 1, it is characterized in that: the diameter of described fluorescent latex particulate is 200-500nm.
6. according to the immunochromatographydetection detection card of the described detection Troponin I of claim 1, it is characterized in that: the wavelength of emission was 500nm-600nm after described fluorescent latex particulate was stimulated.
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| CN2013100871239A CN103217535A (en) | 2013-03-18 | 2013-03-18 | Immunochromatography detection card for detecting troponin I |
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| CN2013100871239A CN103217535A (en) | 2013-03-18 | 2013-03-18 | Immunochromatography detection card for detecting troponin I |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015176589A1 (en) * | 2014-05-22 | 2015-11-26 | 江苏金标世纪生物科技有限公司 | Kit for rapidly detecting myocardial infarction, preparation method therefor, and application thereof |
| CN106370859A (en) * | 2016-08-22 | 2017-02-01 | 北京华科泰生物技术有限公司 | Test strip used for detecting canine cTnI, and production method of test strip |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015176589A1 (en) * | 2014-05-22 | 2015-11-26 | 江苏金标世纪生物科技有限公司 | Kit for rapidly detecting myocardial infarction, preparation method therefor, and application thereof |
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| CN106370859A (en) * | 2016-08-22 | 2017-02-01 | 北京华科泰生物技术有限公司 | Test strip used for detecting canine cTnI, and production method of test strip |
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Application publication date: 20130724 |