CN105717033A - Method for quantitatively detecting protein concentration by flow cytometer - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a method for quantitatively detecting protein concentration by a flow cytometer, and specific protein concentration is detected by the combination of fluorescence immunological magnetic microspheres and flow cytometric detection. The method comprises the specific steps: preparing a fluorescence detection antibody; preparing fluorescence immunological magnetic microspheres and calibrating magnetic microspheres; making a standard article working curve, and acquiring actual protein concentration through unknown sample average fluorescence strength. The flow cytometry and the fluorescence immunological magnetic microspheres are combined for detecting protein concentration; specific protein molecular concentration is detected by using the method, a reaction system is small, measuring is more accurate, a detection lower limit is pg/ml level, specificity and sensitivity are high, operating is more convenient and quick, operating steps are simpler, incubation is direct without stepwise incubation and washing so that reaction time is shortened, detection results are obtained within a shorter time, and quantitating is more accurate.
Description
Technical field
The present invention relates to the quantitative detecting method of a kind of protein concentration, a kind of method being specifically related to flow cytometer quantitative detection of protein concentration.
Background technology
Biomacromolecule is by the simple in construction relative molecular weight relatively low organic compound polymolecular system by being polymerized, including protein, nucleic acid, lipid and saccharide.Biomacromolecule has various biological activity and plays an important role in biological metabolism, therefore is the basic substance constituting life.Particularly protein, it is composition tissue basic function unit, it is the main undertaker of vital movement, plays biological function by complicated architectural characteristic simultaneously, as provided energy and material, the enhancing development of life needs and producing immunologic function etc..
Albumen Quality Research is qualitatively and quantitatively studied firstly the need of to protein.Current quantification of protein method for measuring is broadly divided into physical method and chemical method, and wherein physical method is mainly ultraviolet absorption method, and chemical method is mainly dye method, biuret is sent out and phenol reagent process.But what these methods all detected is the concentration of gross protein, therefore exist can not the drawback of specific detection a certain kind protein concentration.Along with molecular biological development, the mankind illustrate the pathogenesis of disease molecular mechanism, so the detection for these specific proteins molecules becomes particularly important.Being now widely used in the index of Clinical Laboratory major part is the mensuration to specific protein molecule, including various enzymes, autoantigen, autoantibody and tumor markers etc..
Detection method for specific protein molecular concentration carries out the quantitative of protein mainly by the enzyme-linked immunosorbent assay (ELISA) in immunological method.The method is in soluble antigen or antibodies to the solid phase carriers such as polystyrene, antigen-antibody binding specificity will to be utilized to carry out immunoreactive principle, then use chemical colour reaction reaction that it is carried out qualitative and quantitative detecting method.The method has quick, sensitive, easy and carrier and is prone to the advantages such as standardization, but concrete operation step is loaded down with trivial details, and each reaction system can only detect an index, and the Monitoring lower-cut of the method is ng/ml level simultaneously.
Flow cytometry (FCM) has the feature of detection and evaluation of markers different kinds of molecules in simple sample simultaneously in scientific research, be a kind of high-throughout on functional level, single-row cell or other particle are carried out one by one, multiparameter, quickly, the detection technique of accurate quantitative analysis and sorting, the characteristics such as this technology has that detection speed is fast, detection parameter is many, it is big to gather data message amount, analyzes comprehensively, separating purity is high and method is flexible.Developing rapidly of this technology, depends primarily on the powerful characteristic of fluorescent dye, particularly different fluorescent labelinies or the fluorescence of varying strength and can differentiate in flow cytometer and open.The character of this uniqueness, prompting can use flow cytometer to detect the index of multiple not isolabeling in same sample simultaneously, thus reaching quickly and conveniently testing goal.Flow cytometer is mainly used in the surface markers detection of cell and hives off with cell, and to detect certain a part then it is necessary to have other particles are as carrier, and this carrier size must be nanoscale and have good size uniformity.Assisting at present Application comparison maturation in isolation technics, particularly biological magnetic separation technique in magnetic field is magnetic microsphere, and magnetic microsphere material just possesses that size is homogeneous, magnetic responsiveness strong and the characteristic such as good dispersion in water.By analyzing FCM technology, there is compared to ECLIA higher accuracy and high throughput testing characteristic, therefore can by FCM quickly, the characteristic such as high flux, multiparameter and accurate quantitative analysis be applied to the detection of protein concentration, select to be sized to simultaneously nanoscale, uniform particle sizes magnetic microsphere material as the carrier of flow cytometer, the method setting up fluorescence immunoassay magnetic microsphere associating flow cytomery protein concentration.
Summary of the invention
For above-mentioned prior art Problems existing, a kind of method that the invention provides flow cytometer quantitative detection of protein concentration, combine with fluorescence immunoassay magnetic microsphere detection protein concentration by flow cytometry, the method is adopted to carry out specific protein molecular concentration detection, reaction system is little, measure more accurate, Monitoring lower-cut is pg/ml level, there is higher specificity and sensitivity, more convenient operation is quick, operating procedure simplifies, directly hatch and hatch washing without substep, shorten the response time, only need 2.5h just can complete detection, the testing result time is shorter, quantitatively more accurate.
To achieve these goals, the technical solution used in the present invention is:
A kind of method of flow cytometer quantitative detection of protein concentration, associating fluorescence immunoassay magnetic microsphere and flow cytomery specific protein concentration, specifically include following steps:
Step (1): fluorescent dye is modified on magnetic microsphere and prepares fluorescent magnetic microspheres；
Step (2): fluoroscopic examination antibody is prepared in fluorophor crosslinking in the monoclonal antibody that protein is special；
Step (3): the fluorescent magnetic microspheres surface that step (1) obtains is carried out amination and amide-carboxylated modification, again respectively by antibody linked to the monoclonal antibody specific of this protein difference fluoroscopic examination antibody correspondence epi-position and the fluoroscopic examination of the specific fluorescent labelled amount fluorescent magnetic microspheres surface in activation, make fluorescence immunoassay magnetic microsphere and calibration magnetic microsphere；
Step (4): make standard substance working curve and obtain actual protein concentration by unknown sample average fluorescent strength.
Preferably, fluorescent dye is modified on magnetic microsphere any of the above-described scheme by the method that described step (1) is polymerized by melamine resin.
Any of the above-described scheme preferably, uses the method for chemical crosslinking that fluorophor crosslinking is prepared fluoroscopic examination antibody in the monoclonal antibody that protein is special in described step (2).
Any of the above-described scheme is preferably, the manufacture method making standard substance working curve in step (4) is use the calibration magnetic microsphere that step (3) obtains, the average fluorescent strength that fluorescence immunoassay magnetic microsphere is corresponding with the fluoroscopic examination antibody combined flow cytomery protein variable concentrations standard substance that step (2) obtains, and makes standard substance working curve.
The invention has the beneficial effects as follows: a kind of method of flow cytometer quantitative detection of protein concentration and preparation method thereof is provided, associating fluorescence immunoassay magnetic microsphere and flow cytomery specific protein concentration, specifically include following steps: step (1): is modified at by fluorescent dye on magnetic microsphere and prepares fluorescent magnetic microspheres；Step (2): fluoroscopic examination antibody is prepared in fluorophor crosslinking in the monoclonal antibody that protein is special；Step (3): the fluorescent magnetic microspheres surface that step (1) obtains is carried out amination and amide-carboxylated modification, again respectively by antibody linked to the monoclonal antibody specific of this protein difference fluoroscopic examination antibody correspondence epi-position and the fluoroscopic examination of the specific fluorescent labelled amount fluorescent magnetic microspheres surface in activation, make fluorescence immunoassay magnetic microsphere and calibration magnetic microsphere；Step (4): make standard substance working curve and obtain actual protein concentration by unknown sample average fluorescent strength, flow cytometry is combined detection protein concentration by the inventive method with fluorescence immunoassay magnetic microsphere, the method is adopted to carry out specific protein molecular concentration detection, reaction system is little, measure more accurate, Monitoring lower-cut is pg/ml level, there is higher specificity and sensitivity, more convenient operation is quick, operating procedure simplifies, directly hatch and hatch washing without substep, shorten the response time, only need 2.5h just can complete detection, the testing result time is shorter, quantitatively more accurate.
Accompanying drawing explanation
Fig. 1 is the fluorescent microscopy images of difference mark fluorescent magnetic microsphere of the present invention.
Fig. 2 is the laser scanning co-focusing microscope photo of labelling Rhodamine 123 fluorescent magnetic microspheres of the present invention.
Fig. 3 is the flow cytometry figure that the present invention corrects microsphere correction.
Fig. 4 is the flow cytometry figure of flow cytomery variable concentrations BSA standard sample of the present invention.
Fig. 5 is the standard curve (0～8000pg/mL) of fluorescent magnetic microspheres of the present invention associating flow cytomery BSA.
Fig. 6 is the flow cytometry figure of flow cytomery the unknown BSA sample concentration of the present invention.
Detailed description of the invention
In order to be more fully understood that and implement the present invention, below in conjunction with the example being embodied as, the present invention is done further detailed description.
A kind of method of flow cytometer quantitative detection of protein concentration, associating fluorescence immunoassay magnetic microsphere and flow cytomery specific protein concentration, choose Rhodamine 123 labelling magnetic microsphere, and PE-cy7 marker detection resists, and concrete grammar is as follows:
1. preparation fluorescent magnetic microspheres: being modified at by fluorescent dye on magnetic microsphere and prepare fluorescent magnetic microspheres, concrete preparation method is as follows:
(1) weigh 3mg Rhodamine 123 (Rh123) dyestuff, be dissolved in 10mL deionized water prepare 1mmol/L dye mother solution.Rhodamine 123 or PE-cy7 are for illustrating herein, and the method can choose the fluorescent dye of any two flow cytometer difference passage.Adding the powder of 300mg melamine and 750mg hexamethylenamine in 10mL deionized water, stirring makes it fully dispersed, is placed under 65 DEG C of water bath condition heated and stirred and reacts 2 hours.Add the Rhodamine 123 mother solution of 10 μ L1mM after filter paper, be sufficiently mixed, and its room temperature standing is placed in 4 DEG C of preservations for 1 hour again；
(2) weigh 20mg magnetic microsphere, be scattered in 10mL dust technology (pH=3.5), and add in the fluorescent polymer solution of the above-mentioned preparation of 2mL.After supersound process makes the fully dispersed Homogeneous phase mixing of reactant, it is placed in 95 DEG C of oil bath heating systems and reacts 30 minutes, add 20mL mixture of ice and water and terminate reaction.The fluorescent magnetic microspheres of centrifugation gained, repeatedly 3 centrifugations heavily disperseing with washed product, vacuum drying with water；
(3) weighing the fluorescent magnetic microspheres of the above-mentioned preparation of 10mg, be scattered in 2mL water with the mixed solvent of 8mL ethanol, supersound process makes microsphere fully dispersed.In this microsphere suspension liquid, add 0.1mL ammonia (25%, wt) and 30 μ L tetraethyl silanes, react 24 hours under stirring condition.Reaction is 3 centrifugations heavily disperseing with washed product, vacuum drying with water repeatedly after terminating.
2. preparing fluorescence immunoassay magnetic microsphere and fluoroscopic examination antibody, concrete preparation method is as follows:
(1) weigh the fluorescent magnetic microspheres of the above-mentioned preparation of 10mg, be scattered in 10mL dust technology (pH=3.5) and in the mixed solvent of 40mL ethanol, add 0.05mL aminopropyltriethoxywerene werene after fully dispersed, react 24 hours under stirring condition.Reaction is 3 centrifugations heavily disperseing with washed product, vacuum drying with water repeatedly after terminating.
(2) weigh the dried magnetic microsphere of above-mentioned 10mg, be scattered in 5mL oxolane, add 5mg succinic anhydride after fully dispersed, react 6 hours under stirring condition.The fluorescent magnetic microspheres of activation being scattered in 5mL again and cross-links in buffer solution (morpholino b acid solution, pH=6.0), add BSA monoclonal antibody, after mixing, room temperature lucifuge hatches 2 hours.After crosslinking terminates, add 1mg glycine and terminate reaction, centrifugal, abandon supernatant, the surface-crosslinked microsphere having antibody is resuspended in preservation liquid, 4 DEG C of preservations.
(3) with above-mentioned cross-linking method crosslinkable fluorescent dye PE-cy7 in the BSA monoclonal antibody of different epi-positions, 4 DEG C of preservations.
(4) method cross-linked with above-mentioned activation microsphere and antibody surface, the BSA monoclonal antibody of crosslinkable fluorescent dye PE-cy7 cross-links surface in activation fluorescent magnetic microspheres according to a certain percentage, and 4 DEG C preserve the fluorescence for flow cytometer FSC, SSC parameter and PE-cy7 and regulate.
3. associating flow cytomery protein concentration, concrete operational approach is as follows:
(1) weighing commercialization BSA, be dissolved in deionized water making its concentration is 5ng/mL, and room temperature stands 30min.These BSA standard substance resuspended, are diluted to 5ng/mL standard substance according to the method for gradient dilution the standard substance of 9 pipe variable concentrations.
(2) from the standard substance of 9 pipe variable concentrations, take 50 μ L respectively add in new EP pipe, in 1 negative control, add 50 μ L deionized waters simultaneously.It is sufficiently mixed BSA fluorescence immunoassay magnetic microsphere and fluoroscopic examination antibody, in each pipe, adds 50 μ L, room temperature lucifuge stationary incubation 2 hours respectively.Using the resuspended washing microsphere of 1mL washing liquid after hatching end, room temperature 200g is centrifuged 5min.After discarding supernatant, with the 300 μ resuspended microspheres of L re-suspension liquid, for lower step flow cytomery.
(3) abundant vortex fluorescent calibration microsphere, flow cytometer detects.Regulating the parameter of FSC and SSC, arrange P door, the fluorescence intensity of fluorescent calibration microsphere PE-cy7 in detection P door, the average fluorescent strength of microsphere is controlled in 6000～8000 scopes by the voltage regulating PE-cy7.
Under the parameter of above-mentioned adjustment, abundant vortex microsphere to be detected detects, and starts from low concentration to high concentration examination criteria product successively from negative control pipe, collects the fluorescence intensity of about 500 microsphere PE-cy7, record its average fluorescent strength.
(4) by statistics software, concentration corresponding for standard substance and average fluorescent strength are carried out four parameter fittings, generate quadruplex parameters, the concentration of unknown sample can be calculated by the equation.
Concrete operations and testing result are as described below, owing to the fluorescent decoration of magnetic microsphere has the motility of height, by the labelling of multiple fluorophor, thus obtaining the fluorescent microsphere with different fluorescence emitting characteristics, as shown in Figure 1；The uniform labelling fluorescent magnetic microspheres of Rhodamine 123, as shown in Figure 2.
Using antibody linked for BSA on fluorescent magnetic microspheres surface as catching microsphere, again fluorescence PE-cy7 is cross-linked on different BSA antibody as detection antibody, associating flow cytometer make BSA standard substance working curve, its concentration range is 0～8000pg/mL, least concentration be 31pg/mL as shown in Figure 5；The concrete correction with correction microsphere flow cytometer, as shown in Figure 3；Use the flow cytometry figure of blind associating flow cytomery variable concentrations BSA standard sample, as shown in Figure 4；Final detection makes standard curve according to the average fluorescent strength of variable concentrations standard sample, as shown in Figure 5, it is determined that its unknown sample concentration, as shown in Figure 6.
The technical scheme above embodiment of the present invention provided is described in detail, principle and the embodiment of the embodiment of the present invention are set forth by specific case used herein, and the explanation of above example is only applicable to help to understand the principle of the embodiment of the present invention；Simultaneously for one of ordinary skill in the art, according to the embodiment of the present invention, all will change in detailed description of the invention and range of application, in sum, this specification content should not be construed as limitation of the present invention.
Claims (4)
1. the method for a flow cytometer quantitative detection of protein concentration, it is characterised in that: associating fluorescence immunoassay magnetic microsphere and flow cytomery specific protein concentration, specifically include following steps:
Step (1): fluorescent dye is modified on magnetic microsphere and prepares fluorescent magnetic microspheres；
Step (2): fluoroscopic examination antibody is prepared in fluorophor crosslinking in the monoclonal antibody that protein is special；
Step (3): the fluorescent magnetic microspheres surface that step (1) obtains is carried out amination and amide-carboxylated modification, again respectively by antibody linked to the monoclonal antibody specific of this protein difference fluoroscopic examination antibody correspondence epi-position and the fluoroscopic examination of the specific fluorescent labelled amount fluorescent magnetic microspheres surface in activation, make fluorescence immunoassay magnetic microsphere and calibration magnetic microsphere；
Step (4): make standard substance working curve and obtain actual protein concentration by unknown sample average fluorescent strength.
2. the method for a kind of flow cytometer quantitative detection of protein concentration according to claim 1, it is characterised in that: fluorescent dye is modified on magnetic microsphere by the method that described step (1) is polymerized by melamine resin.
3. the method for a kind of flow cytometer quantitative detection of protein concentration according to claim 1, it is characterised in that: described step (2) is used the method for chemical crosslinking fluorophor crosslinking is prepared fluoroscopic examination antibody in the monoclonal antibody that protein is special.
4. the method for a kind of flow cytometer quantitative detection of protein concentration according to claim 1, it is characterized in that, the manufacture method making standard substance working curve in described step (4) is use the calibration magnetic microsphere that step (3) obtains, the average fluorescent strength that fluorescence immunoassay magnetic microsphere is corresponding with the fluoroscopic examination antibody combined flow cytomery protein variable concentrations standard substance that step (2) obtains, and makes standard substance working curve.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030322A (en) * | 2018-08-17 | 2018-12-18 | 成都赋智健康科技有限公司 | A kind of flow cytometry assays of save the cost |
| CN110178034A (en) * | 2017-01-18 | 2019-08-27 | 埃森仪器公司Dba埃森生物科学公司 | For measuring the method and reagent of immunoglobulin γ (IgG) antibody isotype concentration from biological sample |
| CN112161913A (en) * | 2020-09-25 | 2021-01-01 | 深圳唯公生物科技有限公司 | Analysis method and equipment for flow type fluorescence analysis system |
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| CN110178034A (en) * | 2017-01-18 | 2019-08-27 | 埃森仪器公司Dba埃森生物科学公司 | For measuring the method and reagent of immunoglobulin γ (IgG) antibody isotype concentration from biological sample |
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| CN112161913A (en) * | 2020-09-25 | 2021-01-01 | 深圳唯公生物科技有限公司 | Analysis method and equipment for flow type fluorescence analysis system |
| CN112710596A (en) * | 2020-11-30 | 2021-04-27 | 浙江正熙生物医药有限公司 | Method for qualitative/quantitative detection of target antibody concentration using flow cytometer |
| CN117607423A (en) * | 2023-10-14 | 2024-02-27 | 武汉中生毓晋生物医药有限责任公司 | Method for detecting concentration of anti-human lymphocyte immunoglobulin active antibody |
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