CN102128934A - Method for detecting human myocardial troponin T through cytometric bead array - Google Patents
Method for detecting human myocardial troponin T through cytometric bead array Download PDFInfo
- Publication number
- CN102128934A CN102128934A CN2010100418103A CN201010041810A CN102128934A CN 102128934 A CN102128934 A CN 102128934A CN 2010100418103 A CN2010100418103 A CN 2010100418103A CN 201010041810 A CN201010041810 A CN 201010041810A CN 102128934 A CN102128934 A CN 102128934A
- Authority
- CN
- China
- Prior art keywords
- human myocardial
- myocardial troponin
- people
- microballoon
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a method for detecting human myocardial troponin T through cytometric bead array. The method comprises the following three steps of: activating a bead, coupling a monoclonal antibody and detecting the human myocardial troponin T, specifically, firstly, activating the beads; secondly, preparing the monoclonal antibody of the carboxylation bead coupled mouse anti human myocardial troponin T; thirdly, capturing the human myocardial troponin T in a specimen; and finally, adding multiple antigenic and fluorescein isothiocyanate labeled donkey anti goat intravenous gamma globulin G of goat anti human myocardial troponin T, and detecting the fluorescence intensity of fluorescein isothiocyanate (FITC) on the bead on a flow cytometry to indirectly detect the content of the human myocardial troponin T in the specimen. The monoclonal antibody coupled bead, the polyclonal antibody of the goat anti human myocardial troponin T, and the fluorescein isothiocyanate labeled donkey anti goat intravenous gamma globulin (IgG) can be prepared into a commodity kit, which can detect the human myocardial troponin T at any time. The method is simple and feasible; and by the method, the human myocardial troponin T can be quickly detected with convenience and the basis for disease diagnosis is provided.
Description
Technical field
The invention belongs to the analytical approach of biological substance, specifically, belong to the flow microsphere analytical technique in the flow cytometry.
Background technology
The biomarker that specificity was the highest and susceptibility is higher when cardiac troponin was current diagnosis myocardial damage, necrosis, also having important use to be worth in the risk stratification of acute coronary syndrome (ACS), is to detect myocardial damage and downright bad " goldstandard ".At present, the method that detects people's myocardium calcium protein T mainly contains: 1. immune-gold labeled method, can carry out fast qualitative or sxemiquantitative; 2. electrochemiluminescence method, sensitive (lowest detectable limit 10pg/mL), precision, but reagent cost height, the electrochemiluminescence instrument of call for bids value costliness; 3. immunoturbidimetry, practical, easily and fast, cost is low, but influence factor is more, as antigen or antibody excess, soluble complex can appear, cause error, lipids contents influences turbidity in the sample, causes false increasing, and is unfavorable for the unification and the comparison of the method for inspection; Other method, as put exempt from, enzyme is exempted from etc., poor practicability.It is numerous to detect people's myocardium calcium protein T method at present clinically, some patented technology schemes are also arranged, for example No. 200720140932.1 " human muscle hemoglobin/creatine kinase isozyme/myocardium calcium protein I diagnose test paper ", 200720140929.X number " myocardium calcium protein I colored particle diagnose test paper ", No. 200780010683.7 " in Symptomatic patient, differentiating the tool and method of acute and chronic myocardial necrosis ", No. 200810036418.2 " Troponin I serum quick test kit (colloidal gold methods) " etc., but all exist standardization issue.
Flow cytometry is the unicellular or particulate that is dyeed by fluorchrome with under the high energy laser irradiation flow at high speed state, measure the intensity of the scattered light and the emitting fluorescence of its generation, thus the physics of pair cell (or particulate), biochemistry, immunity, heredity, molecular biology proterties and function is qualitative or a kind of modern cell analysis technology of detection by quantitative.The flow microsphere analytical technology is to use the monoclonal antibody bag by polystyrene microsphere, and the Acquisition Detection thing adds fluoroscopic examination antibody again, has the susceptibility height, high specificity, and lowest detectable limit can reach 10pg/ml.
Summary of the invention
The object of the present invention is to provide flow microsphere to detect the method for people's myocardium calcium protein T, be implemented in flow cytometer and detect the content that fluorescence intensity on the microballoon is come people's myocardium calcium protein T in the indirect detection sample.
The method that the flow microsphere that the inventor provides detects people's myocardium calcium protein T comprises 3 steps, promptly carries out microballoon earlier and activates, and the detection of people's myocardium calcium protein T is carried out in monoclonal antibody coupling more at last; Specific practice is: after activating microballoon, the monoclonal antibody for preparing carboxylated microballoon coupling mouse-anti people myocardium calcium protein T, catch the people's myocardium calcium protein T in the sample then, add the immunoglobulin G (IgG) of the anti-sheep of donkey of how anti-and FITC (fluorescein isothiocynate) mark of goat-anti human troponin T again, the intensity of FITC is linear relevant with the molecular number of people's myocardium calcium protein T that microballoon is caught.Therefore, by in the fluorescence intensity that detects FITC on the microballoon on the flow cytometer, get final product the content of people's myocardium calcium protein T in the indirect detection sample.
Method of the present invention can be made commercially available reagent box with the IgG of the anti-sheep of donkey of the polyclonal antibody of monoclonal antibody coupling microballoon, goat-anti people myocardium calcium protein T, marked by fluorescein isothiocyanate, can detect people's myocardium calcium protein T at any time.This method is simple and easy to do, can detect people's myocardium calcium protein T quickly, and foundation is provided for diagnosing the illness.Be applicable to all kinds of hospitals, be particularly useful for treating the hospital of cardiovascular disorder.
Embodiment
Detect people's myocardium calcium protein T according to following steps:
First step microballoon activates
1. get 100 μ L microballoon stostes (about 1.25 * 10
6Individual microballoon) put in the 1.5mL centrifuge tube, vortex oscillation device concussion microballoon 30s cleans microballoon 30s with ultrasonic cleaner.Rotating speed 14000r/min (centrifugal radius 10cm) is centrifugal 4min down, carefully abandons supernatant;
2. the microballoon lavation buffer solution [5% soil temperature-20 of the phosphate buffer of pH 7.4 (PBS) preparation] that adds 100 μ L, whirlpool concussion 10s, ultrasonic cleaning 10s, rotating speed 14000r/min (centrifugal radius 10cm) is centrifugal 4min down, carefully abandons supernatant;
3. resuspended microballoon activates damping fluid (0.1mol/LNaH in 80 μ L
2PO
4, pH=6.2), whirlpool concussion 30s, ultrasonic cleaning 30s;
4. with EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide salt) that activates the fresh 50mg/mL of damping fluid configuration and the Sul fo-NHS (sulfuration N-hydroxy-succinamide) of 50mg/mL, in the microballoon that activates, respectively add 10 μ L respectively, room temperature lucifuge shaking 20min then;
5. add 150 μ L PBS, high speed whirlpool concussion 10s, rotating speed 14000r/mi n (centrifugal radius 10cm) is centrifugal 4min down, carefully abandons supernatant;
6. resuspended activation microballoon is in 100 μ L PBS, and whirlpool shakes 30s, ultrasonic cleaning 15s.
The monoclonal antibody coupling of second step
1. the mouse-anti people myocardium calcium protein T monoclonal antibody that adds 10 μ g is regulated final volume to 500 μ L, room temperature lucifuge shaking 2h with PBS in the microballoon that activates;
2. centrifugal 4min under the rotating speed 14000r/min (centrifugal radius 10cm) carefully abandons supernatant;
3. add 500 μ L PBS, rotating speed 14000r/min (centrifugal radius 10cm) is centrifugal 4min down, carefully abandons supernatant;
4. resuspended microballoon is in 250 μ L sealing store buffer liquid (1% benzsulfamide and the 0.05%NaN of PBS preparation
3), whirlpool concussion 15s, room temperature lucifuge shaking 30min;
5. centrifugal 4min under the rotating speed 14000r/min (centrifugal radius 10cm) carefully abandons supernatant;
6. add 500 μ L sealing store buffer liquid (1% benzsulfamide and the 0.05%NaN of PBS preparation
3), rotating speed 14000r/min (centrifugal radius 10cm) is centrifugal 6min down, carefully abandons supernatant;
7. be resuspended in 200 μ L sealing store buffer liquid, calculate the concentration of microballoon with blood cell counting plate, it is standby to keep in Dark Place under 4 ℃.
This monoclonal antibody coupling microballoon can be produced in batches, at low temperature (about 4 ℃) but the lucifuge long preservation.
The detection of the 3rd step people's myocardium calcium protein T
1. get about 10000 bags by good microballoon, add 50 μ L samples, add the IgG of the anti-sheep of donkey of the polyclonal antibody of 50 μ L goat-anti people myocardium calcium protein Ts and 50 μ L FITC (fluorescein isothiocynate) marks more respectively, lucifuge room temperature shaking reaction 30min;
2. centrifugal 4min under the rotating speed 14000r/min (centrifugal radius 10cm) carefully abandons supernatant;
3. add 200 μ L damping fluids (the % benzsulfamide of PBS preparation), the whirlpool concussion is resuspended, and rotating speed 14000r/min (centrifugal radius 10cm) is centrifugal 4min down, carefully abandons supernatant;
4. add 200 μ L damping fluids (the % benzsulfamide of PBS preparation) again, whirlpool is resuspended, and the up flow type cell instrument detects, and observes the fluorescence intensity of FITC on the microballoon, gets final product the content of people's myocardium calcium protein T in the indirect detection sample.
Claims (1)
1. flow microsphere detects the method for people's myocardium calcium protein T, and its feature comprises 3 steps, promptly carries out microballoon earlier and activates, and the detection of people's myocardium calcium protein T is carried out in monoclonal antibody coupling more at last; Specific practice is: activate microballoon earlier, the monoclonal antibody that then prepares carboxylated microballoon coupling mouse-anti people myocardium calcium protein T, catch the people's myocardium calcium protein T in the sample then, add the immunoglobulin G of the anti-sheep of donkey of the how anti-and marked by fluorescein isothiocyanate of goat-anti human troponin T again; By in the fluorescence intensity that detects FITC on the microballoon on the flow cytometer, get final product the content of people's myocardium calcium protein T in the indirect detection sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100418103A CN102128934A (en) | 2010-01-13 | 2010-01-13 | Method for detecting human myocardial troponin T through cytometric bead array |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100418103A CN102128934A (en) | 2010-01-13 | 2010-01-13 | Method for detecting human myocardial troponin T through cytometric bead array |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102128934A true CN102128934A (en) | 2011-07-20 |
Family
ID=44267097
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010100418103A Pending CN102128934A (en) | 2010-01-13 | 2010-01-13 | Method for detecting human myocardial troponin T through cytometric bead array |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102128934A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507948A (en) * | 2011-11-15 | 2012-06-20 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
| CN108441950A (en) * | 2018-04-28 | 2018-08-24 | 无锡英特派金属制品有限公司 | A kind of preparation method of nano silver wires with high length-diameter ratio |
| CN108593938A (en) * | 2018-05-31 | 2018-09-28 | 吉林大学 | The magnetic macromolecular microsphere and its application in detecting serum Myocardial troponin that surface is coupled cardiac troponin antigen |
| CN109154602A (en) * | 2016-03-07 | 2019-01-04 | 奎多心血管股份有限公司 | Immunoassays control and its application method |
| CN109765358A (en) * | 2019-01-18 | 2019-05-17 | 江苏医联生物科技有限公司 | The chemical treatment method of micro-fluidic chip application protein detection |
| CN113358546A (en) * | 2021-04-21 | 2021-09-07 | 贵州安康医学检验中心有限公司 | Combined detection method for autoimmune peripheral neuropathy related antibody |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362623A (en) * | 2002-01-21 | 2002-08-07 | 陕西超英生物医学研究开发有限公司 | Multiple immunological microsphere and its prepn techn and detection method |
| CN1563990A (en) * | 2004-04-02 | 2005-01-12 | 浙江大学 | Method of detecting la grippe virus by means of immune polystyrene microballs |
| CN1588072A (en) * | 2004-09-13 | 2005-03-02 | 王占科 | Different impedance series immunological micro ball and preparing method, method and device for detecting the same |
| CN1854735A (en) * | 2005-04-19 | 2006-11-01 | 林远 | Fluid cell equipment-microcarrier clinical diagnosis chip |
| WO2006116321A2 (en) * | 2005-04-21 | 2006-11-02 | Zeta Corporation | Bead-based flow cytometry detection methods |
| CN101432626A (en) * | 2006-04-04 | 2009-05-13 | 神谷来克斯公司 | Highly sensitive system and methods for analysis of troponin |
-
2010
- 2010-01-13 CN CN2010100418103A patent/CN102128934A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362623A (en) * | 2002-01-21 | 2002-08-07 | 陕西超英生物医学研究开发有限公司 | Multiple immunological microsphere and its prepn techn and detection method |
| CN1563990A (en) * | 2004-04-02 | 2005-01-12 | 浙江大学 | Method of detecting la grippe virus by means of immune polystyrene microballs |
| CN1588072A (en) * | 2004-09-13 | 2005-03-02 | 王占科 | Different impedance series immunological micro ball and preparing method, method and device for detecting the same |
| CN1854735A (en) * | 2005-04-19 | 2006-11-01 | 林远 | Fluid cell equipment-microcarrier clinical diagnosis chip |
| WO2006116321A2 (en) * | 2005-04-21 | 2006-11-02 | Zeta Corporation | Bead-based flow cytometry detection methods |
| CN101432626A (en) * | 2006-04-04 | 2009-05-13 | 神谷来克斯公司 | Highly sensitive system and methods for analysis of troponin |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507948A (en) * | 2011-11-15 | 2012-06-20 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
| CN102507948B (en) * | 2011-11-15 | 2013-12-04 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
| CN109154602A (en) * | 2016-03-07 | 2019-01-04 | 奎多心血管股份有限公司 | Immunoassays control and its application method |
| CN109154602B (en) * | 2016-03-07 | 2022-04-15 | 奎多心血管股份有限公司 | Immunoassay controls and methods of use thereof |
| CN108441950A (en) * | 2018-04-28 | 2018-08-24 | 无锡英特派金属制品有限公司 | A kind of preparation method of nano silver wires with high length-diameter ratio |
| CN108593938A (en) * | 2018-05-31 | 2018-09-28 | 吉林大学 | The magnetic macromolecular microsphere and its application in detecting serum Myocardial troponin that surface is coupled cardiac troponin antigen |
| CN109765358A (en) * | 2019-01-18 | 2019-05-17 | 江苏医联生物科技有限公司 | The chemical treatment method of micro-fluidic chip application protein detection |
| CN113358546A (en) * | 2021-04-21 | 2021-09-07 | 贵州安康医学检验中心有限公司 | Combined detection method for autoimmune peripheral neuropathy related antibody |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018120620A1 (en) | Fluorescence immunochromatographic detection card and preparation method therefor and use thereof | |
| CN102128934A (en) | Method for detecting human myocardial troponin T through cytometric bead array | |
| CN105403693A (en) | Preparation method of magnetic particle chemiluminescence reagent | |
| CN101251540A (en) | Hepatitis B virus e antigen testing corpuscle, preparation and application thereof | |
| CN104614513A (en) | Relaxation time immunosensing analysis method based on magnetic separation | |
| CN102628867A (en) | Double antibody latex enhanced retinol binding protein detection kit | |
| CN106053826A (en) | III type procollagen N-terminal peptide quantitative measurement kit and preparation method thereof | |
| CN105651995B (en) | Detect application of CD105, CD144, CD34, KDR, Annexin V and the CD63 reagent in the reagent of the endothelium in preparing detection blood and the extracellular vesica of endothelial progenitor cells release | |
| CN103235120A (en) | Kit for compound detection of hepatitis E virus antibody profile as well as application of kit | |
| CN103235130A (en) | High-sensitivity high-linearity D-D dimer detection kit and application method thereof | |
| CN105842451A (en) | Method for detecting DNMT1 on basis of quantum dot fluorescence immunoassay | |
| CN103913444A (en) | Single-photon fluorescence excitation multi-channel quantitative determination device and detection method based on blue light optical tweezers | |
| CN102914495A (en) | Method for evaluating DNA (Deoxyribose Nucleic Acid) damages of peripheral blood lymphocytes caused by ionizing radiation | |
| JP2008539430A5 (en) | ||
| CN106324254A (en) | Anti-insulin antibody detection kit and detection method thereof | |
| CN102472749B (en) | Magnetic sensor device, method of operating such a device and sample | |
| CN106198962B (en) | Method for closing biomagnetic beads | |
| CN108061727A (en) | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof | |
| CN205484371U (en) | Tumour mark detect reagent box | |
| JP2012168012A (en) | Novel examination method and examination kit for examining vascular endothelial disorder | |
| Chugh et al. | Magnetic particle spectroscopy with one-stage lock-in implementation for magnetic bioassays with improved sensitivities | |
| CN109211866A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection CK-MB | |
| Runser et al. | An optimized and standardized rapid flow cytometry functional method for heparin-induced thrombocytopenia | |
| JP5348357B1 (en) | Method for quantifying target cells in blood and system evaluation method for quantifying the cells | |
| CN104569390A (en) | Quantitative detection method for gamma-interferon and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110720 |