CN105717033A - Method for quantitatively detecting protein concentration by flow cytometer - Google Patents
Method for quantitatively detecting protein concentration by flow cytometer Download PDFInfo
- Publication number
- CN105717033A CN105717033A CN201610051003.7A CN201610051003A CN105717033A CN 105717033 A CN105717033 A CN 105717033A CN 201610051003 A CN201610051003 A CN 201610051003A CN 105717033 A CN105717033 A CN 105717033A
- Authority
- CN
- China
- Prior art keywords
- protein concentration
- fluorescent
- protein
- antibody
- magnetic microsphere
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 44
- 239000004005 microsphere Substances 0.000 claims abstract description 72
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 239000000126 substance Substances 0.000 claims description 19
- 238000003018 immunoassay Methods 0.000 claims description 14
- 239000007850 fluorescent dye Substances 0.000 claims description 10
- 238000004132 cross linking Methods 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 6
- 229920000877 Melamine resin Polymers 0.000 claims description 3
- 238000005576 amination reaction Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 239000004640 Melamine resin Substances 0.000 claims description 2
- 238000010382 chemical cross-linking Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000000684 flow cytometry Methods 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 238000005406 washing Methods 0.000 abstract description 5
- 230000001900 immune effect Effects 0.000 abstract description 4
- 238000011534 incubation Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000001917 fluorescence detection Methods 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 7
- TUFFYSFVSYUHPA-UHFFFAOYSA-M rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C(C=CC(N)=C2)C2=[O+]C2=C1C=CC(N)=C2 TUFFYSFVSYUHPA-UHFFFAOYSA-M 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000012937 correction Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000011017 operating method Methods 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229920001109 fluorescent polymer Polymers 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229960004011 methenamine Drugs 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- VCZQFJFZMMALHB-UHFFFAOYSA-N tetraethylsilane Chemical class CC[Si](CC)(CC)CC VCZQFJFZMMALHB-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610051003.7A CN105717033B (en) | 2016-01-25 | 2016-01-25 | A kind of method of flow cytometer quantitative detection of protein concentration |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610051003.7A CN105717033B (en) | 2016-01-25 | 2016-01-25 | A kind of method of flow cytometer quantitative detection of protein concentration |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105717033A true CN105717033A (en) | 2016-06-29 |
CN105717033B CN105717033B (en) | 2019-05-14 |
Family
ID=56154141
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610051003.7A Active CN105717033B (en) | 2016-01-25 | 2016-01-25 | A kind of method of flow cytometer quantitative detection of protein concentration |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105717033B (en) |
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 |
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 |
CN112946293A (en) * | 2019-12-10 | 2021-06-11 | 中国科学院大连化学物理研究所 | Quantitative detection method for target membrane protein in single cell |
CN113508299A (en) * | 2019-03-01 | 2021-10-15 | 艾瓦生命科学有限公司 | Flow cytometer measurement method and kit for carrying out the measurement method |
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 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
CN1671865A (en) * | 2002-07-24 | 2005-09-21 | 李兴祥 | Microparticle based signal amplification method and its application in the detection of analytes |
CN101939648A (en) * | 2008-01-07 | 2011-01-05 | 卢米耐克斯公司 | Immunomagnetic capture and imaging of biological targets |
CN102851369A (en) * | 2006-12-13 | 2013-01-02 | 卢米耐克斯公司 | Systems and methods for multiplex analysis of PCR in real time |
-
2016
- 2016-01-25 CN CN201610051003.7A patent/CN105717033B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1671865A (en) * | 2002-07-24 | 2005-09-21 | 李兴祥 | Microparticle based signal amplification method and its application in the detection of analytes |
CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
CN102851369A (en) * | 2006-12-13 | 2013-01-02 | 卢米耐克斯公司 | Systems and methods for multiplex analysis of PCR in real time |
CN101939648A (en) * | 2008-01-07 | 2011-01-05 | 卢米耐克斯公司 | Immunomagnetic capture and imaging of biological targets |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
US11733237B2 (en) | 2017-01-18 | 2023-08-22 | Sartorius Bioanalytical Instruments, Inc. | Methods and reagents for determining immunoglobulin gamma (IgG) antibody isotype concentration from biological samples |
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 |
CN113508299A (en) * | 2019-03-01 | 2021-10-15 | 艾瓦生命科学有限公司 | Flow cytometer measurement method and kit for carrying out the measurement method |
CN112946293A (en) * | 2019-12-10 | 2021-06-11 | 中国科学院大连化学物理研究所 | Quantitative detection method for target membrane protein in single cell |
CN112946293B (en) * | 2019-12-10 | 2022-06-24 | 中国科学院大连化学物理研究所 | Quantitative detection method for target membrane protein in single cell |
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 |
Also Published As
Publication number | Publication date |
---|---|
CN105717033B (en) | 2019-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105717033A (en) | Method for quantitatively detecting protein concentration by flow cytometer | |
US20100240150A1 (en) | Measuring reagent and turbidimetric immunoassay and sample analysis tool using the same | |
EP1801590B1 (en) | Method of assaying antigen and reagent therefor | |
CN101893619B (en) | Method for improving stability of latex suspension liquid | |
US6551788B1 (en) | Particle-based ligand assay with extended dynamic range | |
CN116539597A (en) | cTnI homogeneous phase chemiluminescence detection kit, detection method and device | |
WO2009026168A1 (en) | Anti-antibody reagent | |
JP2005510706A5 (en) | ||
CN112505322A (en) | Alzheimer disease marker p-Tau217 detection kit and manufacturing method thereof | |
CN106568967B (en) | A kind of sensitive detection method for ochratoxin A | |
CN101315379A (en) | Reagent kit for detecting Ractopamine and application thereof | |
CN110988325B (en) | Blocking agent and kit containing same | |
CN111239404B (en) | Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample | |
CN109738644B (en) | Anti-mullerian hormone immune turbidimetry quantitative detection reagent | |
Choi et al. | Rapid detection of 6×-histidine-labeled recombinant proteins by immunochromatography using dye-labeled cellulose nanobeads | |
CN114480405A (en) | Nucleic acid aptamer of colorless malachite green and application thereof | |
CN111273013A (en) | Aflatoxin B based on immunomagnetic beads1Method of measurement of | |
CN106645686A (en) | Sensitivity detection method for fumonisin B1 | |
CN112326956A (en) | Tumor specific growth factor latex enhanced immunoturbidimetry detection kit and preparation method thereof | |
WO2009042423A1 (en) | Methods and compositions for improved diagnostic assays | |
CN111650383A (en) | Chemiluminescence immunoassay method based on fluorescent dye as internal standard substance and application thereof | |
CN112858687B (en) | Serum amyloid A detection reagent and preparation method thereof | |
CN109975260B (en) | Method for detecting lysozyme based on nanogold fluorescence and application thereof | |
US20220404347A1 (en) | Methods for detecting an analyte using structure switching binding agents | |
RU2702900C1 (en) | Method for quantitative determination of antibodies to benzo[a]pyrene in human biological fluids |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |