CN103275931B - Quick enriching and separating method of natural killer cells in peripheral blood of human - Google Patents
Quick enriching and separating method of natural killer cells in peripheral blood of human Download PDFInfo
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Abstract
The invention discloses a method for separating and enriching natural killer cells (natural killer cells, NK) in peripheral blood for further providing a basis for subsequent study of natural killer cells, and relates to the biomedical field. The method comprises the following steps of: covalent coupling of dendritic polymers and mouse anti-human NKp46 antibodies; further coating long-chain biotin molecules by dendritic polymers modified by mouse anti-human NKp46 antibodies; capturing NK in a peripheral blood sample by dendritic polymers co-modified by mouse anti-human NKp46 antibodies and long-chain biotin; identifying and coupling long-chain dendritic polymers in the peripheral blood by streptavidin modified nanomagnetic beads; and separating and resuspending the captured NK. The resuspension can be directly used for subsequent analysis. Compared with conventional cell separation methods, the method is more suitable for magnetic separation of NK in complex peripheral blood samples, so that the magnetic separation time is shortened, and the NK separation efficiency in the peripheral blood sample is improved.
Description
Technical field
The present invention relates to biomedical sector, specifically relate to the natural killer cell separation method based on nanometer magnetic bead.
Background technology
Natural killer cell (natural killer cell, NK) is the important immunocyte of body, not only with antitumor, anti-virus infection is relevant with immunomodulatory, and participates in the generation of allergy and autoimmune disorder in some cases.NK cell is in the first line of defence of body nonspecific immunity, but due to reasons such as NK cellular segregation purifying and condition of in vitro culture, is subject to a definite limitation to its research.Established at present both at home and abroad the method for multiple NK cells cell purification, but its cost is high, the factors such as complicated operation limit its applying at home.But the quantity of NK cell in peripheral blood or splenic lymphocyte is little, in-vitro separation and cultivate amplification to obtain a large amount of highly purified NK cell more difficult, existing method also cannot directly be separated and gather by trace natural killer cell in human peripheral blood.Therefore, realize the efficiently concentrating of natural killer cell in human peripheral blood, thus lay the foundation for inquiring into the effect of natural killer cell in disease treatment.
Immunity magnetic separation technique is one of important component part of peripheral blood cells fast separating concentration technology, this technology can efficient capture, target cell in concentrated human peripheral sample, improve target cell detection sensitivity.In recent years, antibody is connected on magnetic bead by the immunomagnetic separation (IMS) based on magnetic micro-beads, then the magnetic bead being connected with antibody is dropped in sample liquid target cell is caught, enrichment, Magneto separate (concrete principle is shown in Fig. 2 A).But, many limitation should be there is based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is relatively little, reduces magnetic capture efficiency; 2) due to the particle properties of micron magnetic bead self, combined by heterogeneous reaction (multiphase reaction) between itself and cell, usually need the time more grown to go specificity to catch cell in food substrate; 3) micron magnetic bead monodispersity is poor, self assemble easily occurs in the heme of periphery or forms precipitation; 4) traditional immune magnetic separation technique, often antibody is directly coupled on immunomagnetic beads, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change the space steric effect increased between antibody, thus reduce the capture rate 5 of antibody) blood viscosity is high and hematocrite concentration that is wherein non-natural killer cell is large, micron magnetic bead easily produces non-specific adsorption, is difficult to realize the specific isolation to natural killer cell in blood; 6) excessive concentration of micron magnetic bead can cause the breakage of natural killer cell (magnetic field causes cell surface magnetic bead to be attracted each other, and cell is squeezed and even breaks), causes the failure be separated.7), during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.
Summary of the invention
For the defect of prior art, the object of this invention is to provide high, the easy disengaging time of a kind of capture rate short, the method for natural killer cell specificity sharp separation in the peripheral blood matrix that (to be less than 30 T/m) under low gradient magnetic complicated.
The fast enriching separation method of natural killer cell in human peripheral, comprise the following steps: (1) often takes 1.0 mg branch-shape polymers, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration of glutaraldehyde is made to be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 1 mL 3.57 mg/ mL mouse-anti people NKp46 antibody, make its final concentration reach about 3 mg/mL, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain branch-shape polymer-antibody complex; (2) 30.2 mg long-chain biological elements are got, be suspended in 4 mL 0.01 mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 1.0 mg branch-shape polymer-antibody complexes, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-branch-shape polymer-antibody complex; (3) get 1 mL people's fresh peripheral blood, add 0.2 mg antibody and long-chain biological element co-modified branch-shape polymer and step (2) long-chain biological element-branch-shape polymer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; (4), after magneticseparation, after deionized water cleans gently, namely obtain with PBS damping fluid is resuspended nanometer magnetic bead-Streptavidin-long-chain biological element-branch-shape polymer-antibody-natural killer cell the mixture being enriched with natural killer cell.
Described branch-shape polymer is polyamide-amine type branch-shape polymer, and its molecular weight is 42000 Da.Structure is as Fig. 1.
The described nanometer magnetic bead particle diameter having modified Streptavidin is 20-50 nm, is preferably 30 nm.
Branch-shape polymer realizes the covalent coupling of branch-shape polymer and antibody by carboxyl that is amino and natural killer cell in human body.
Branch-shape polymer, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of branch-shape polymer and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed branch-shape polymer.
Concrete principle is shown in Fig. 2 B.
Present method is specially adapted to the separation of complex sample, as human peripheral sample etc.Sample preparation is treatment process conveniently.
Technical solution of the present invention is adopted to have following beneficial effect:
1, the present invention by means of the Cascaded amplification effect of branch-shape polymer, magnetic cell signal exponentially level is expanded, the separation of magnetic cell just can be realized under lower magneticstrength, and within the identical time, comparatively routine immunization Beads enrichment method is compared, be separated to object cell ability stronger, be specially adapted to the separation of complex sample, as peripheral blood sample etc.For the defect that object cell speed is slow, magnetic field requirements is high in the simple 20-50 nm immuno magnetic cell separation complex matrices sample adopted after antibody modification, branch-shape polymer is adopted to realize the amplification of nanometer magnetic bead magnetic signal, thus improve object cellular segregation efficiency in complex matrices sample, achieve object cell-specific sharp separation in the food substrate that (to be less than 30 T/m) under low gradient magnetic complicated.
2, this programme is for be coupled on branch-shape polymer by antibody molecule, avoid in ordinary method antibody molecule is coupled to magnetic bead surfaces cause antibody activity reduce and sterically hindered large shortcoming.
3, the present invention adopts branch-shape polymer, reaction soln can be made more stable, not easily precipitate, add the chance that antibody contacts with target cell, is conducive to improving capture rate; Simultaneously, branch-shape polymer is connected with a large amount of long-chain biological element molecules, the nanometer magnetic bead can modified in conjunction with Streptavidin, thus makes on branch-shape polymer in conjunction with a large amount of nanometer magnetic beads, achieve the Cascaded amplification of magnetic cell signal, be conducive to the disengaging time shortening magnetic cell.
4, after replacing micron order magnetic particle with nanometer magnetic bead (20-50 nm), because nanometer magnetic bead particle diameter is little, specific surface area is large, the steric hindrance be combined with cell-surface antigens is little, the covering efficiency of cell surface magnetic bead significantly improves, and the cell of magnetic nano particle subcovering can keep normal shape, nanometer magnetic bead also has dispersed and stability preferably in complex matrices, and therefore the use of nanometer magnetic bead can overcome above-mentioned all defects owing to using micron magnetic bead to cause.
5, the present invention is in sepn process, introduce tree-shaped high-polymer molecular, branch-shape polymer is connected with a large amount of long-chain biological element molecules, can special and high-affinity ground be dispersed in coupling in matrix solution and have the identification of Streptavidin nanometer magnetic bead, thus make on branch-shape polymer in conjunction with a large amount of nanometer magnetic beads, considerably increase the magnetic bead quantity that target cells combines, achieve the target cell that sharp separation is caught under magnetic field.Compared with traditional Cell magnetic separation method, be nanometer magnetic bead more stable in matrix because of what add, the method is more suitable for carries out Magneto separate to cell in complex matrices, improves object cellular segregation efficiency in complex matrices sample.
6, during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.But this experimental program introduces branch-shape polymer in coupling process, it has certain space size, thus makes antibody molecule away from magnetic bead and magnetic bead surfaces, avoids the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Meanwhile, the branch-shape polymer of introducing but can not affect antibody space conformation, thus serves the bioactive effect of protection antibody molecule.
Accompanying drawing explanation
The structural representation of Fig. 1 branch-shape polymer.
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique involved in the present invention (B).
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Long-chain biological element is for buying in the carboxylated long-chain biological element of Thermo Fisher Scientific company of the U.S. (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) being modified with Streptavidin is bought in Ocean NanoTech company of the U.S..
Amidized branch-shape polymer is amidized polyamide-amine type branch-shape polymer, and its molecular weight is 42000 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magneticstrength and is less than 30T/m.
N-hydroxysuccinimide NHSS, ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC etc. is conventional reagent, repeats no more.
0.1%PBST compound method: 8.0 g NaCl, 0.2 g KCl, 0.24 g KH
2pO
4, 1.44 g Na
2hPO
4be dissolved in 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, then be settled to 1000 mL and namely obtain 0.01 M PBS.Add Tween 20 with the volume ratio of 1/1000 (V/V) again, namely obtain 0.1%PBST.
Embodiment 1
1. branch-shape polymer-antibody complex, prepare in accordance with the following steps:
(1) take 1.0 mg branch-shape polymer polyamide-amine type branch-shape polymers, be suspended in 4 mL phosphoric acid buffers (PBS, 0.01 mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) i.e. 3.57 mg of mouse-anti people NKp46 antibody 1 mL(are dripped to above-mentioned solution), make its final concentration reach about 3 mg/mL.Room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
2. long-chain biological element-branch-shape polymer-antibody complex is prepared in accordance with the following steps:
(1) get 30.2 mg long-chain biological elements, be suspended in 4 mL phosphoric acid buffers (PBS, 0.01mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) 1.0 mg branch-shape polymer-antibody complexes are joined in above-mentioned solution, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
3. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain biological element-branch-shape polymer-antibody complex, be placed on blending instrument, form long-chain biological element-branch-shape polymer-antibody-NK cell antigen mixture with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame and be separated 3 min;
4., after deionized water cleans gently, mix with PBS damping fluid and resuspendedly namely obtain the mixture and nanometer magnetic bead-Streptavidin-vitamin H-branch-shape polymer-antibody-NK cell antigen that are enriched with NK cell.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4the NK cell of cell/mL is in 1.5 mL sterile centrifugation tube, and centrifugal 5 min of 12000 rpm, abandon supernatant, resuspended by the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange technical solution of the present invention group (NK cell antibody and the plain co-modified branch-shape polymer group of long-chain biological), the nanometer magnetic bead group of NK cell-specific antibodies modification, the micron magnetic bead group enrich target cells of NK cell-specific antibodies modification respectively.
(3), after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch NK cell immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the target cell re-suspension liquid of each group of enrichment is carried out gradient dilution, with flow cytometer (Flow Cytometer) amount detection, by the capture rate of capture rate formulae discovery target cell, and each experiment in triplicate.The calculation formula of each group of capture rate is as follows: (the target cell sum adsorbed by enrichment/all total cellular score) × 100%.
The scheme of described each group of enrichment acquisition target cell is as follows:
A. enrichment acquisition target cell protocol is as embodiment 1, specific as follows for technical solution of the present invention group (NK cell antibody and the plain co-modified branch-shape polymer group of long-chain biological):
0.1 mg NK cell antibody and the co-modified branch-shape polymer of vitamin H and vitamin H-branch-shape polymer-antibody complex are joined containing in target cell centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
B. the nanometer magnetic bead group enrichment acquisition target cell protocol of NK cell-specific antibodies modification is specific as follows:
The nanometer magnetic bead that the NK cell-specific antibodies prepared by 0.1 mg is modified joins containing in target cell centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
The nanometer magnetic bead preparation that described NK cell-specific antibodies is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g NK cell-specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
C. the micron magnetic bead group enrichment acquisition target cell protocol of NK cell-specific antibodies modification is specific as follows:
The micron magnetic bead that the NK cell-specific antibodies prepared by 0.1 mg is modified joins containing in target cell centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
The micron magnetic bead preparation that described NK cell-specific antibodies is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g NK cell-specific antibodies by every mg magnetic bead, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
Each group of capture rate is as follows:
| The micron magnetic bead group capture rate that NK cell-specific antibodies is modified | The nanometer magnetic bead group capture rate that NK cell-specific antibodies is modified | NK cell antibody and the plain co-modified branch-shape polymer group capture rate of long-chain biological |
| 53.5% | 24.5% | 92.5% |
Experimental result shows, the capture rate of the micron magnetic bead group that NK cell-specific antibodies is modified is apparently higher than the capture rate of nanometer magnetic bead group, this illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic strong, and the target cell that just energy separation and concentration is more at short notice.But, the capture rate of technical solution of the present invention group is far longer than again the micron magnetic bead group of NK cell-specific antibodies modification, this shows that technical solution of the present invention can increase target cells nanometer magnetic bead fraction of coverage by branch-shape polymer, thus magnetic is improved greatly, and then achieve (3min) high efficiency separation enrichment of N K cell at short notice.
Experiment is caught in embodiment 3 enrichment
Conventional magnetic frame disengaging time is 30min, and all the other are with embodiment 2.
Each group of capture rate is as follows:
| The micron magnetic bead group capture rate that NK cell-specific antibodies is modified | The nanometer magnetic bead group capture rate that NK cell-specific antibodies is modified | NK cell antibody and the plain co-modified branch-shape polymer group capture rate of long-chain biological |
| 58.2% | 44.2% | 92.3% |
Experimental result shows, 3min is separated in comparative example 2, when reaching 30min when disengaged, the capture rate of three groups is obtained for raising, particularly the capture rate of the nanometer magnetic bead group of NK cell-specific antibodies modification improves the most obvious, this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but the capture rate of NK cell antibody and long-chain biological element co-modified branch-shape polymer group when it is still separated (3min) lower than the short period of time.This shows that technical solution of the present invention can (3min) high efficiency separation enrichment of N K cell at short notice.
The research of natural killer cell in embodiment 4 nanometer magnetic bead enrichment healthy volunteer peripheral blood
Joined in 10mL sterile centrifugation tube by aseptic for healthy volunteer EDTA anticoagulation cirumferential blood sample 5 mL, get 1mL peripheral blood through Flow cytometry NK cell count wherein, its quantity is 3.9 × 10
4.Separately get 1mL peripheral blood for subsequent use.
The NK cell antibody prepared and the co-modified branch-shape polymer (0.1 mg) of long-chain biological element are joined in sample solution respectively, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add the nanometer magnetic bead (0.1 mg) being modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, conventional magnetic frame is separated 3 min.After Magneto separate, supernatant liquor is poured in sterile centrifugation tube, separate catch NK cell antibody immunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 method obtains, and all the other are with embodiment 2.The results are shown in Table 1, show the NK cell antibody in this programme energy efficiently concentrating sample separation.
Natural killer cell separating effect in table 1 peripheral blood
| Experiment group number | Natural killer cell separation efficiency |
| Embodiment 4 | 82.1%(3.2×10 4/3.9×10 4) |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. the fast enriching separation method of natural killer cell in human peripheral, it is characterized in that comprising the following steps: (1) often takes 1.0 mg branch-shape polymers, be suspended in 4 mL 0.01 mol/L, in pH 8.0 PBS phosphoric acid buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration of glutaraldehyde is made to be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 1 mL 3.57 mg/ mL mouse-anti people NKp46 antibody, make its final concentration reach about 3 mg/mL, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain branch-shape polymer-antibody complex; (2) 30.2 mg long-chain biological elements are got, be suspended in 4 mL 0.01 mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 1.0 mg branch-shape polymer-antibody complexes, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-branch-shape polymer-antibody complex; (3) get 1 mL people's fresh peripheral blood, add 0.2 mg antibody and long-chain biological element co-modified branch-shape polymer and step (2) long-chain biological element-branch-shape polymer-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; The described nanometer magnetic bead particle diameter being modified with Streptavidin is 20-50 nm(4) after magneticseparation, after deionized water cleans gently, namely obtain with PBS damping fluid is resuspended nanometer magnetic bead-Streptavidin-long-chain biological element-branch-shape polymer-antibody-natural killer cell the mixture being enriched with natural killer cell.
2. method according to claim 1, it is characterized in that described branch-shape polymer is polyamide-amine type branch-shape polymer, its molecular weight is 42000 Da.
3. method according to claim 1, is characterized in that the described nanometer magnetic bead particle diameter having modified Streptavidin is 30 nm.
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