CN103275931A - 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.The NK cell is in the first line of defence of body nonspecific immunity, but owing to reasons such as NK cellular segregation purifying and condition of in vitro culture, its research is subjected to certain limitation.Set up at present both at home and abroad the method for multiple NK cells cell purification, but its cost height, effects limit such as complicated operation its applying at home.Yet, the quantity of NK cell in peripheral blood or splenic lymphocyte seldom, in-vitro separation and cultivate amplification and obtain relatively difficulty of a large amount of highly purified NK cells, existing method also can't directly be separated and gather trace natural killer cell in the peripheral blood.Therefore, realize the efficiently concentrating of natural killer cell in the human peripheral blood, thereby lay the foundation for inquiring into the effect of natural killer cell aspect disease treatment.
The immunity magnetic separation technique is one of important component part of peripheral blood cells sharp separation beneficiation technologies, but this technology efficient capture, concentrates target cell in the human peripheral sample, improves the target cell detection sensitivity.In recent years, based on the immunomagnetic separation (IMS) of magnetic micro-beads antibody is connected on the magnetic bead, the magnetic bead that will be connected with antibody then drop in the sample liquid to target cell catch, enrichment, magnetic separates (concrete principle is seen Fig. 2 A).Yet should have many limitation based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is less relatively, has reduced magnetic capture efficient; 2) because the particle properties of micron magnetic bead self, by heterogeneous reaction (multiphase reaction) combination, need the longer time go specificity to catch cell in the food substrate usually between itself and the cell; 3) micron magnetic bead monodispersity is relatively poor, and precipitation takes place self to assemble or form in the periphery heme easily; 4) traditional immune magnetic separation technique, directly be coupled to antibody on the immunomagnetic beads often, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change having increased space steric effect between antibody, thereby having reduced the capture rate 5 of antibody) blood viscosity is high and hemocyte concentration wherein non-natural killer cell is big, the micron magnetic bead is easy to generate non-specific adsorption, is difficult to realize the specific isolation to natural killer cell in the blood; 6) excessive concentration of micron magnetic bead can cause the breakage (magnetic field causes the cell surface magnetic bead to be attracted each other, cell is squeezed even break) of natural killer cell, causes the failure that separates.7) during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.
Summary of the invention
At the defective of prior art, the purpose of this invention is to provide a kind of capture rate height, easy disengaging time weak point, the method for natural killer cell specificity sharp separation in the peripheral blood matrix that (less than 30 T/m) are complicated under the low gradient magnetic.
The fast enriching separation method of natural killer cell in the human peripheral, may further comprise the steps: (1) whenever takes by weighing 1.0 mg branch-shape polymers, be suspended in 4 mL, 0.01 mol/L, in the pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 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, its final concentration is reached 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got branch-shape polymer-antibody complex; (2) get 30.2 mg long-chain vitamin Hs, be suspended in 4 mL, 0.01 mol/L pH, the 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-branch-shape polymer-antibody complex; (3) get the fresh peripheral blood of 1 mL people, adding 0.2 mg antibody and the co-modified branch-shape polymer of long-chain vitamin H is step (2) long-chain vitamin H-branch-shape polymer-antibody complex, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic force frame separates 3 min; (4) after the magneticseparation, after deionized water cleans gently, with the resuspended nanometer magnetic bead-Streptavidin-long-chain vitamin H-branch-shape polymer-antibody-natural killer cell mixture that is enriched with natural killer cell that namely gets of PBS damping fluid.
Described branch-shape polymer is polyamide-amide type branch-shape polymer, and its molecular weight is 42000 Da.Structure such as Fig. 1.
The described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50 nm, is preferably 30 nm.
Branch-shape polymer is realized the covalent coupling of branch-shape polymer and antibody by the carboxyl of amino and natural killer cell antibody.
Branch-shape polymer is realized the covalent coupling of branch-shape polymer and long-chain vitamin H by the carboxyl of amino and long-chain biotin molecule; Add excessive long-chain vitamin H to guarantee exposed amino sites on the sealing branch-shape polymer.
Concrete principle is seen Fig. 2 B.
Present method is specially adapted to the separation of complex sample, as human peripheral sample etc.Sample preparation gets final product according to conventional treatment method.
Adopt technical solution of the present invention to have following beneficial effect:
1, the present invention is by the cascade scale effect of branch-shape polymer, magnetic cell signal exponentially level is enlarged, under lower magneticstrength, just can realize the separation of magnetic cell, and in the identical time, compare than routine immunization magnetic bead separation method, it is stronger to be separated to the purpose cell ability, is specially adapted to the separation of complex sample, as peripheral blood sample etc.At slow, the demanding defective in magnetic field of purpose cell speed in the 20-50 nm immunomagnetic beads separate complex matrix sample behind the simple employing antibody modification, adopt branch-shape polymer to realize the amplification of nanometer magnetic bead magnetic signal, thereby improved purpose cellular segregation efficient in the complex matrices sample, realized purpose cell-specific sharp separation in (less than 30 T/m) are complicated under low gradient magnetic the food substrate.
2, this programme has been avoided in the ordinary method antibody molecule being coupled to magnetic bead surfaces and has been caused antibody activity to reduce and sterically hindered big shortcoming for antibody molecule is coupled on the branch-shape polymer.
3, the present invention adopts branch-shape polymer, can make reaction soln more stable, and difficult the precipitation increased the chance that antibody contacts with target cell, is conducive to improve capture rate; Simultaneously, be connected with a large amount of long-chain biotin molecules on the branch-shape polymer, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on the branch-shape polymer in conjunction with a large amount of nanometer magnetic beads, realize the cascade amplification of magnetic cell signal, be conducive to shorten the disengaging time of magnetic cell.
4, with behind nanometer magnetic bead (20-50 nm) the replacement micron order magnetic particle, because the nanometer magnetic bead particle diameter is little, specific surface area is big, the steric hindrance of being combined with cell-surface antigens is little, the covering efficient of cell surface magnetic bead significantly improves, and the cell of magnetic nano particle subcovering can keep normal shape, and nanometer magnetic bead also has dispersed and stable preferably in complex matrices, so the use of nanometer magnetic bead can overcome above-mentioned all because the defective of using the micron magnetic bead to cause.
5, the present invention is in sepn process, introduced tree-shaped high-polymer molecular, be connected with a large amount of long-chain biotin molecules on the branch-shape polymer, can be special and high-affinity ground be dispersed in that coupling has the identification of Streptavidin nanometer magnetic bead in the matrix solution, thereby make on the branch-shape polymer in conjunction with a large amount of nanometer magnetic beads, increase the magnetic bead quantity of target cell surface bonding greatly, realized the target cell that sharp separation is caught under magnetic field.Comparing with traditional cell magnetism separate method, is stabilized nano magnetic bead more in matrix because of what add, and this method is applicable to that more in complex matrices cell being carried out magnetic separates, and has improved purpose cellular segregation efficient in the complex matrices sample.
6, during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.Yet this experimental program is introduced branch-shape polymer in coupling process, and it has certain space size, thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the disadvantageous effect of the character of magnetic bead own and surperficial antagonist molecule.Simultaneously, the branch-shape polymer of introducing but can not influence the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
Description of drawings
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 (B) involved in the present invention.
Embodiment
In order to make the present invention clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explaining the present invention, and be not used in restriction the present invention.
The long-chain vitamin H is for buying in the carboxylated long-chain vitamin H of U.S. Thermo Fisher Scientific company (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) that is modified with Streptavidin is bought the Ocean NanoTech company in the U.S..
Amidized branch-shape polymer is amidized polyamide-amide type branch-shape polymer, and its molecular weight is 42000 Da, available from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic force frame separates magneticstrength less than 30T/m.
N-maloyl imines 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 the 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, be settled to 1000 mL again and namely get 0.01 M PBS.Volume ratio with 1/1000 (V/V) adds Tween 20 again, namely obtains 0.1%PBST.
Embodiment 1
1. branch-shape polymer-antibody complex prepares according to following steps:
(1) take by weighing 1.0 mg branch-shape polymer polyamide-amide type branch-shape polymers, be suspended in the 4 mL phosphoric acid buffers (pH 8.0 for PBS, 0.01 mol/L), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) add i.e. 3.57 mg of mouse-anti people NKp46 antibody 1 mL(to above-mentioned drips of solution), its final concentration is reached 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
2. long-chain vitamin H-branch-shape polymer-antibody complex prepares according to following steps:
(1) get 30.2 mg long-chain vitamin Hs, be suspended in the 4 mL phosphoric acid buffers (pH 8.0 for PBS, 0.01mol/L), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 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 the 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
3. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-branch-shape polymer-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min formation long-chain vitamin H-branch-shape polymer-antibody-NK cell antigen mixture of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic force frame separate 3 min;
4. after deionized water cleaned gently, mixing the resuspended mixture that is enriched with the NK cell that namely gets with the PBS damping fluid was nanometer magnetic bead-Streptavidin-vitamin H-branch-shape polymer-antibody-NK cell antigen.
The experiment of embodiment 2 concentration effects
(1) getting 1 mL concentration is 10
4The NK cell of cell/mL is in the aseptic centrifuge tube of 1.5 mL, and centrifugal 5 min of 12000 rpm abandon supernatant, and is resuspended with the aseptic PBS solution of equal-volume.
(2) enrichment is caught: the nanometer magnetic bead group of technical solution of the present invention group (the branch-shape polymer group that NK cell antibody and long-chain vitamin H are co-modified), NK cell-specific antibody modification, the micron magnetic bead group enriched target cell of NK cell-specific antibody modification are set respectively.
(3) after magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching the NK cell and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the resuspended liquid of target cell of each group enrichment is carried out gradient dilution, with flow cytometer (Flow Cytometer) amount detection, calculate the capture rate of target cell by the capture rate formula, test triplicate at every turn.Each calculation formula of organizing capture rate is as follows: (target cell sum/all total cellular score of being adsorbed by enrichment) * 100%.
The described scheme of respectively organizing enrichment acquisition target cell is as follows:
A. technical solution of the present invention group (the branch-shape polymer group that NK cell antibody and long-chain vitamin H are co-modified) enrichment acquisition target cell scheme such as embodiment 1, specific as follows:
Be that vitamin H-branch-shape polymer-antibody complex joins and contains in the target cell centrifuge tube with 0.1 mg NK cell antibody and the co-modified branch-shape polymer of vitamin H, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.Add 0.1 mg then and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
B. the nanometer magnetic bead group enrichment acquisition target cell scheme of NK cell-specific antibody modification is specific as follows:
The nanometer magnetic bead of the NK cell-specific antibody modification that 0.1 mg is prepared joins and contains in the target cell centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The nanometer magnetic bead preparation of described NK cell-specific antibody modification: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used dehydrated alcohol, 1 M NaOH successively, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g NK cell-specific antibody by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the thanomin room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
C. the micron magnetic bead group enrichment acquisition target cell scheme of NK cell-specific antibody modification is specific as follows:
The micron magnetic bead of the NK cell-specific antibody modification that 0.1 mg is prepared joins and contains in the target cell centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The micron magnetic bead preparation of described NK cell-specific antibody modification: (1) is got 10 mg micron magnetic beads (1150 nm do not have the coupling Streptavidin) and is used dehydrated alcohol, 1 M NaOH successively, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g NK cell-specific antibody by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the thanomin room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate of NK cell-specific antibody modification | The nanometer magnetic bead group capture rate of NK cell-specific antibody modification | The branch-shape polymer group capture rate that NK cell antibody and long-chain vitamin H are co-modified |
| 53.5% | 24.5% | 92.5% |
Experimental result shows, the capture rate of the micron magnetic bead group of NK cell-specific antibody modification is apparently higher than the capture rate of nanometer magnetic bead group, this explanation contrast nanometer magnetic bead group, because micron magnetic bead volume is big, magnetic is strong, at short notice just can the more target cell of separation and concentration.But, the capture rate of technical solution of the present invention group is far longer than the micron magnetic bead group of NK cell-specific antibody modification again, this shows that technical solution of the present invention can increase target cell nano surface magnetic bead fraction of coverage by branch-shape polymer, thereby magnetic is improved greatly, and then realized (3min) high efficiency separation enrichment of N K cell at short notice.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic force frame disengaging time is 30min, and all the other are with embodiment 2.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate of NK cell-specific antibody modification | The nanometer magnetic bead group capture rate of NK cell-specific antibody modification | The branch-shape polymer group capture rate that NK cell antibody and long-chain vitamin H are co-modified |
| 58.2% | 44.2% | 92.3% |
Experimental result shows, separate 3min among the comparative example 2, when disengaging time reaches 30min, three groups capture rate all is improved, particularly the capture rate of the nanometer magnetic bead group of NK cell-specific antibody modification improves the most obvious, this shows the capture rate that can improve the nanometer magnetic bead group by time expand widely, but it still is lower than the capture rate of short period of time co-modified branch-shape polymer group of NK cell antibody and long-chain vitamin H when separating (3min).This shows technical solution of the present invention (3min) high efficiency separation enrichment of N K cell at short notice.
The research of natural killer cell in the embodiment 4 nanometer magnetic bead enrichment healthy volunteer peripheral bloods
5 mL join in the aseptic centrifuge tube of 10mL with the aseptic EDTA anticoagulation cirumferential blood of healthy volunteer sample, get the 1mL peripheral blood through flow cytometer counting NK cell count wherein, and its quantity is 3.9 * 10
4It is standby that other gets the 1mL peripheral blood.
The NK cell antibody and the co-modified branch-shape polymer (0.1 mg) of long-chain vitamin H that prepare are joined respectively in the sample solution, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.Add the nanometer magnetic bead (0.1 mg) be modified with Streptavidin then, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, conventional magnetic force frame separates 3 min.After magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching the NK cell antibody and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 methods obtain, 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
| The experiment group number | The natural killer cell separation efficiency |
| Embodiment 4 | 82.1%(3.2×10 4/3.9×10 4) |
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces 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 the human peripheral, it is characterized in that may further comprise the steps: (1) whenever takes by weighing 1.0 mg branch-shape polymers, be suspended in 4 mL, 0.01 mol/L, in the pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 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, its final concentration is reached 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got branch-shape polymer-antibody complex; (2) get 30.2 mg long-chain vitamin Hs, be suspended in 4 mL, 0.01 mol/L pH, the 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 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, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-branch-shape polymer-antibody complex; (3) get the fresh peripheral blood of 1 mL people, adding 0.2 mg antibody and the co-modified branch-shape polymer of long-chain vitamin H is step (2) long-chain vitamin H-branch-shape polymer-antibody complex, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic force frame separates 3 min; (4) after the magneticseparation, after deionized water cleans gently, with the resuspended nanometer magnetic bead-Streptavidin-long-chain vitamin H-branch-shape polymer-antibody-natural killer cell mixture that is enriched with natural killer cell that namely gets of PBS damping fluid.
2. method according to claim 1 is characterized in that described branch-shape polymer is polyamide-amide type branch-shape polymer, and its molecular weight is 42000 Da.
3. method according to claim 1 is characterized in that the described nanometer magnetic bead particle diameter of having modified Streptavidin is 20-50 nm, is preferably 30 nm.
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| CN102676453A (en) * | 2011-03-17 | 2012-09-19 | 中国医学科学院肿瘤研究所 | Method for culturing natural killer (NK) and/or natural killer T (NKT) cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102676453A (en) * | 2011-03-17 | 2012-09-19 | 中国医学科学院肿瘤研究所 | Method for culturing natural killer (NK) and/or natural killer T (NKT) cells |
Non-Patent Citations (2)
| Title |
|---|
| 司宝财: ""树枝状大分子复合磁性颗粒的制备与表征"", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》 * |
| 山珊 等: ""免疫磁珠法富集沙门氏菌的优化及应用"", 《食品工业科技》 * |
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Inventor after: Song Yunjuan Inventor after: Hu Shishu Inventor after: Shen Yan Inventor before: Xu Hengyi |
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