CN103305464A - Method for directly separating CD<4+> and CD<8+> lymphocytes - Google Patents
Method for directly separating CD<4+> and CD<8+> lymphocytes Download PDFInfo
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- CN103305464A CN103305464A CN2013102194596A CN201310219459A CN103305464A CN 103305464 A CN103305464 A CN 103305464A CN 2013102194596 A CN2013102194596 A CN 2013102194596A CN 201310219459 A CN201310219459 A CN 201310219459A CN 103305464 A CN103305464 A CN 103305464A
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Abstract
The invention discloses a method for directly separating CD<4+> and CD<8+> lymphocytes, lays a better foundation for the subsequent research on the CD<4+> and CD<8+> lymphocytes, and relates to the field of biomedicines. The method comprises steps of: multi-arm well and star-shaped polymer and mouse anti-human CD<4+> or CD<8+> monoclonal antibody covalent coupling, long-chain biotin molecule coating through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody-modified multi-arm well and star-shaped polymer, peripheral blood sample CD<4+> and CD<8+> lymphocyte acquiring through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody and long-chain biotin co-modified multi-arm well and star-shaped polymer, peripheral blood long-chain biotinylation multi-arm well and star-shaped polymer identifying and coupling through utilizing streptavidin-modified nano magnetic beads, captured CD<4+> and CD<8+> lymphocyte separating and suspending and the like. A suspension can be directly used for subsequent analysis; and compared with a conventional cell separating method, the method is suitable for magnetically separating complicated peripheral blood sample CD<4+> and CD<8+> lymphocytes, so that the peripheral blood sample CD<4+> and CD<8+> lymphocyte separation efficiency is increased.
Description
Technical field
The present invention relates to biomedical sector, specifically relate to the CD4 based on nanometer magnetic bead
+And CD8
+The separation of lymphocytes method.
Background technology
The T lymphocyte is the most important a group cell of function in the body immune system.The T lymphocyte derives from the lymphocyte of marrow, carry out cellular immune function, be not only the main body of direct immunization effect, and produce cytokine profiles and express adhesion molecule, by with direct or indirect the contacting of other immunocytes, the performance immunoregulation effect.Each lymphocyte subgroup interacts in normal body, is keeping the normal immunologic function of body.When the quantity of different lymphocyte subgroups and function generation are unusual, can cause body's immunity disorderly and a series of pathological changes occur.Peripheral blood CD4
+And CD8
+Lymphocyte is as most important cell in the lymphocyte subgroup, between the genesis of itself and tumour and the immunologic function of body close relationship arranged.Therefore, to CD4
+And CD8
+The Mechanism Study of the lymphocytic growth of T, differentiation and activation seems more and more important.Yet, CD4 in normal people's peripheral blood
+And CD8
+The lymphocytic content of T is very low, existing method also can't human peripheral blood in trace CD4
+And CD8
+The T lymphocyte directly separates and gathers.Therefore, set up a kind of high efficiency separation and Purification of Human peripheral blood CD4
+And CD8
+The lymphocytic method of T, and it is carried out phenotype and biological function identify is further research CD4
+And CD8
+The effect of T lymphocyte in immunne response provides higher degree CD4
+And CD8
+The T lymphocyte.
The immunity magnetic separation technique is one of important component part of peripheral blood cells fast separating concentration technology, but target cell in this technology efficient capture, concentrated human peripheral sample is improved 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 then will be connected with antibody drop in the sample liquid to target cell catch, enrichment, magnetic separates (concrete principle is seen Fig. 2 A).Yet, should there be many limitation based on the isolation technique of micron order immunomagnetic beads at present: 1) the specific surface area less of micron magnetic bead, reduced magnetic capture efficient; 2) because the particle properties of micron magnetic bead self, by heterogeneous reaction (multiphase reaction) combination, usually need the longer time go specificity to catch cell in the food substrate between itself and the cell; 3) micron magnetic bead monodispersity is relatively poor, and precipitation easily occurs self to assemble or form in the periphery heme; 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 has reduced the capture rate 5 of antibody) the high and non-CD4 wherein of blood viscosity
+And CD8
+Lymphocytic hemocyte concentration is large, and the micron magnetic bead easily produces non-specific adsorption, is difficult to realize CD4 in the blood
+And CD8
+Lymphocytic specific isolation; 6) excessive concentration of micron magnetic bead can cause CD4
+And CD8
+Lymphocytic breakage (magnetic field causes the cell surface magnetic bead to be attracted each other, cell is squeezed even break) causes the failure that separates; 7) during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode that the activated antibody of tool is connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of magnetic bead nature 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
For the defective of prior art, the purpose of this invention is to provide high, the easy disengaging time of a kind of capture rate short, CD4 in the peripheral blood matrix that (less than 30 T/m) are complicated under the low gradient magnetic
+And CD8
+The method of lymphocyte specific sharp separation.
Direct separation of C D4
+And CD8
+Lymphocytic method may further comprise the steps:
(1) whenever gets the amidized multi-arm well of 1.0 mg star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD4
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument; The 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 is got multi-arm well star polymkeric substance-CD4
+Antibody complex; (2) whenever get the amidized multi-arm well of 1.0 mg star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD8
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument; The mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize multi-arm well star polymkeric substance-CD8 that obtains
+Antibody complex; (3) whenever get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids; With 0.1 mg multi-arm well star polymkeric substance-CD4
+Or CD8
+Antibody complex joins in the mentioned solution, and room temperature places and stirs 30 min on the blending instrument; The 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 is got long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex; (4) enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD4
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame fully separates; Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell; With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal 300rpm/min, 20 ℃ of 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell; Add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD8
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame fully separates.Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell; (5) after deionized water cleans gently, be enriched with CD4 with mixed resuspended namely the getting of PBS damping fluid
+Or CD8
+The mixture of cell is nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymkeric substance-antibody-CD4
+Or CD8
+Cell antigen.
Described multi-arm well star polymkeric substance is the end modified amino multi-arm well star polymkeric substance that has, and its molecular weight is 70000 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.
Multi-arm well star polymkeric substance is by amino and CD4
+Or CD8
+The carboxyl of lymphocyte antibody is realized the covalent coupling of multi-arm well star polymkeric substance and antibody.
Multi-arm well star polymkeric substance is realized the covalent coupling of multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance.
Concrete principle is seen Fig. 2 B.
Present method is specially adapted to the separation of complex sample, such 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 multi-arm well star polymkeric substance, magnetic cell signal exponentially level is enlarged, under lower magneticstrength, just can realize the separation of magnetic cell, and within the identical time, compare than routine immunization magnetic bead separation method, be separated to the purpose cell ability stronger, be specially adapted to the separation of complex sample, such as peripheral blood sample etc., magnetic field demanding defective slow for the purpose cell speed in the 20-50 nm immunomagnetic beads separate complex matrix sample behind the simple employing antibody modification, adopt multi-arm well star polymkeric substance 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 large shortcoming for antibody molecule is coupled on the multi-arm well star polymkeric substance.
3, the present invention adopts multi-arm well star polymkeric substance, 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 multi-arm well star polymkeric substance, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on the multi-arm well star polymkeric substance 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 large, 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 multi-arm well star polymkeric substance, be connected with a large amount of long-chain biotin molecules on the multi-arm well star polymkeric substance, 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 multi-arm well star polymkeric substance in conjunction with a large amount of nanometer magnetic beads, greatly increase the magnetic bead quantity of target cell surface bonding, realized the target cell that sharp separation is caught under magnetic field.Comparing with traditional cell magnetism separate method, is nanometer magnetic bead more stable in matrix because of what add, and the method more is applicable in complex matrices cell to be 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 that the activated antibody of tool is connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of magnetic bead nature 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 multi-arm well star polymkeric substance in coupling process, it has certain space size, thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Simultaneously, the multi-arm well star polymkeric substance of introducing but can not affect the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
Description of drawings
The structural representation of Fig. 1 multi-arm well star polymkeric substance.
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 explain the present invention, is not intended to limit 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..
Multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 Da, available from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic 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 again 1000 mL 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. multi-arm well star polymkeric substance-CD4
+Antibody complex, in accordance with the following steps preparation:
(1) whenever gets 1.0 mg multi-arm well star polymkeric substance multi-arm well star polyamide-amides and be dissolved in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min;
(2) get 2.2 mg mouse-anti people CD4
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument;
(3) the 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. multi-arm well star polymkeric substance-CD8
+Antibody complex, in accordance with the following steps preparation:
(1) whenever gets the amidized multi-arm well of 1.0 mg star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS, add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min;
(2) get 2.2 mg mouse-anti people CD8
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument;
(3) the 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. long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex prepares in accordance with the following steps:
(1) whenever get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids;
(2) with 0.1 mg multi-arm well star polymkeric substance-CD4
+Or CD8
+Antibody complex joins in the mentioned solution, and room temperature places and stirs 30 min on the blending instrument;
(3) the 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.
4. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD4
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame fully separate.Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell.With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal (300rpm/min, 20 ℃) 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD8
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame fully separate.Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell.
5. after deionized water cleans gently, be enriched with CD4 with mixed resuspended namely the getting of PBS damping fluid
+Or CD8
+The mixture of cell is nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymkeric substance-antibody-CD4
+Or CD8
+Cell antigen.
The experiment of embodiment 2 concentration effects
(1) getting 1 mL concentration is 10
4The CD4 of cell/mL
+Or CD8
+Cell 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: technical solution of the present invention group (CD4 is set respectively
+Or CD8
+The multi-arm well star polymkeric substance group that cell antibody and long-chain vitamin H are co-modified), CD4
+Or CD8
+The nanometer magnetic bead group of cell-specific antibody modification, CD4
+Or CD8
+The micron magnetic bead group enriched target cell of cell-specific antibody modification.
(3) after magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, caught CD4 and separate
+Or CD8
+The immunomagnetic beads of cell then cleans twice with PBST, mixes, 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 (CD4
+Or CD8
+The multi-arm well star polymkeric substance group that cell antibody and long-chain vitamin H are co-modified) enrichment acquisition target cell scheme such as embodiment 1, specific as follows:
With 0.1 mg CD4
+The co-modified multi-arm well star polymkeric substance of cell antibody and vitamin H is that vitamin H-multi-arm well star polymkeric substance-antibody complex joins and contains in the target cell centrifuge tube, places on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, centrifuge tube is inserted conventional magnetic frame and separate fully separation.Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell.With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal (300rpm/min, 20 ℃) 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD8
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame fully separate.Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell.
B. CD4
+Or CD8
+The nanometer magnetic bead group enrichment acquisition target cell scheme of cell-specific antibody modification is specific as follows:
The CD4 that 0.1 mg is prepared
+The nanometer magnetic bead of cell-specific antibody modification joins and contains in the target cell centrifuge tube, places on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic frame and separate 3 min.Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell.With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal (300rpm/min, 20 ℃) 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell.Add 0.1 mg CD8
+The nanometer magnetic bead of cell-specific antibody modification places on the blending instrument, and rotating speed incubated at room 15 min with 30 rpm insert conventional magnetic frame with centrifuge tube and fully separate.Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell.
Described CD4
+Or CD8
+The nanometer magnetic bead preparation of cell-specific antibody modification: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the blending instrument 37 ℃ of activation 2 h.(2) magnetic 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 CD4 by every mg magnetic bead
+Or CD8
+Cell-specific antibody places 37 ℃ of coupling 2 h on the blending 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 for subsequent use in 4 ℃ of Refrigerator stores.
C. CD4
+Or CD8
+The micron magnetic bead group enrichment acquisition target cell scheme of cell-specific antibody modification is specific as follows:
The CD4 that 0.1 mg is prepared
+The micron magnetic bead of cell-specific antibody modification joins and contains in the target cell centrifuge tube, places on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic frame and separate 3 min.Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell.With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal (300rpm/min, 20 ℃) 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell.Add 0.1 mg CD8
+The micron magnetic bead of cell-specific antibody modification places on the blending instrument, and rotating speed incubated at room 15 min with 30 rpm insert conventional magnetic frame with centrifuge tube and fully separate.Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell.
Described CD4
+Or CD8
+The micron magnetic bead preparation of cell-specific antibody modification: (1) is got 10 mg micron magnetic beads (1150 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the blending instrument 37 ℃ of activation 2 h.(2) magnetic 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 CD4 by every mg magnetic bead
+Or CD8
+Cell-specific antibody places 37 ℃ of coupling 2 h on the blending 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 for subsequent use in 4 ℃ of Refrigerator stores.
It is as follows that each organizes capture rate:
| CD4 +The micron magnetic bead group capture rate of cell-specific antibody modification | CD4 +The nanometer magnetic bead group capture rate of cell-specific antibody modification | CD4 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified |
| 52.1% | 20.8% | 87.6% |
| CD8 +The micron magnetic bead group capture rate of cell-specific antibody modification | CD8 +The nanometer magnetic bead group capture rate of cell-specific antibody modification | CD8 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified |
| 49.8% | 19.5% | 88.3% |
Experimental result shows, CD4
+Or CD8
+The capture rate of the micron magnetic bead group of cell-specific antibody modification is apparently higher than the capture rate of nanometer magnetic bead group, and this explanation contrasts nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic is strong, and 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 again CD4
+Or CD8
+The micron magnetic bead group of cell-specific antibody modification, this shows that technical solution of the present invention can increase target cell nano surface magnetic bead fraction of coverage by multi-arm well star polymkeric substance, thereby magnetic is improved greatly, and then realized at short notice (3min) high efficiency separation enrichment CD4
+Or CD8
+Cell.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic frame disengaging time is 30min, and all the other are with embodiment 2.
It is as follows that each organizes capture rate:
| CD4 +The micron magnetic bead group capture rate of cell-specific antibody modification | CD4 +The nanometer magnetic bead group capture rate of cell-specific antibody modification | CD4 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified |
| 51.7% | 38.1% | 89.8% |
| CD8 +The micron magnetic bead group capture rate of cell-specific antibody modification | CD8 +The nanometer magnetic bead group capture rate of cell-specific antibody modification | CD8 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified |
| 50.9% | 36.9% | 88.7% |
Experimental result shows, separates 3min among the comparative example 2, and when disengaging time reached 30min, three groups capture rate all was improved, particularly CD4
+Or CD8
+The capture rate of the nanometer magnetic bead group of cell-specific antibody modification improves the most obvious, and this shows the capture rate that can improve widely the nanometer magnetic bead group by time expand, but CD4 when it still is lower than short period of time separation (3min)
+Or CD8
+The capture rate of the multi-arm well star polymkeric substance group that cell antibody and long-chain vitamin H are co-modified.This shows at short notice (3min) high efficiency separation enrichment CD4 of technical solution of the present invention
+Or CD8
+Cell.
CD4 in the embodiment 4 nanometer magnetic bead enrichment healthy volunteer peripheral bloods
+And CD8
+Lymphocytic research
Get aseptic EDTA anticoagulation cirumferential blood 20 mL of healthy volunteer, utilize the density gradient centrifugation separating peripheral blood mononuclear cells, with 2 mL PBS solution re-suspended cells.Get the resuspended liquid of above-mentioned PBS cell in centrifuge tube, centrifugal (300 rpm/min, 20 ℃) 10 min, supernatant discarded, re-suspended cell, per 80 μ L PBS contain cell count 10
7Individual, per 10
7Individual cell adds 0.1mg long-chain vitamin H-multi-arm well star polymkeric substance-mouse-anti people CD4
+Antibody complex, fully mixing is hatched 15 min at 4-8 ℃.With the cell suspension after separating with isopyknic PBS solution washing, centrifugal (300rpm/min, 20 ℃) 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD8
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame fully separate.Magnetic is poured supernatant liquor in the aseptic centrifuge tube into after separating, and has caught CD4 and separate
+Or CD8
+The immunomagnetic beads of cell then cleans twice with PBST, mixes, 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 CD4 in this programme energy efficiently concentrating sample separation
+Or CD8
+Cell.
CD4 in table 1 peripheral blood
+And CD8
+The separation of lymphocytes effect
| The experiment group number | CD4 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified | CD8 +The multi-arm well star polymkeric substance group capture rate that cell antibody and long-chain vitamin H are co-modified |
| Embodiment 4 | 83.6% | 82.3% |
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. directly separation of C D4+ and the lymphocytic method of CD8+ is characterized in that may further comprise the steps:
(1) whenever gets 1.0 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD4
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument; The 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 is got multi-arm well star polymkeric substance-CD4
+Antibody complex; (2) whenever get 1.0 mg arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD8
+Monoclonal antibody adds in the above-mentioned reaction solution, and room temperature places and stirs 30 min on the blending instrument; The mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize multi-arm well star polymkeric substance-CD8 that obtains
+Antibody complex; (3) whenever get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids; With 0.1 mg multi-arm well star polymkeric substance-CD4
+Or CD8
+Antibody complex joins in the mentioned solution, and room temperature places and stirs 30 min on the blending instrument; The 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 is got long-chain vitamin H-multi-arm well star polymkeric substance-antibody complex; (4) enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD4
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame fully separates; Magnetic with isopyknic PBS solution re-suspended cell, is CD4 after separating
+Cell; With the supernatant liquor after separating with isopyknic PBS solution washing, centrifugal 300 rpm/min, 20 ℃ of 10 min, supernatant discarded is with isopyknic PBS solution re-suspended cell; Add 0.1 mg long-chain vitamin H-multi-arm well star polymkeric substance-CD8
+Antibody complex places on the blending instrument, forms long-chain vitamin H-multi-arm well star polymkeric substance-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm
+The cell antigen mixture; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame fully separates; Magnetic with isopyknic PBS solution re-suspended cell, is CD8 after separating
+Cell; (5) after deionized water cleans gently, be enriched with CD4 with mixed resuspended namely the getting of PBS damping fluid
+Or CD8
+The mixture of cell is nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymkeric substance-antibody-CD4
+Or CD8
+Cell antigen.
2. method according to claim 1 is characterized in that described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 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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| CN108118029A (en) * | 2016-11-30 | 2018-06-05 | 深圳宾德生物技术有限公司 | The screening technique of high anti-cancer activity T cell and application |
| CN108753716A (en) * | 2018-06-14 | 2018-11-06 | 杭州启澜生物医学技术有限公司 | A kind of amplification in vitro human peripheral CD3+The method of T cell |
| CN109239323A (en) * | 2018-10-12 | 2019-01-18 | 东南大学 | A kind of preparation method and applications of the immunomagnetic beads for whole blood T lymphocyte |
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| US11866465B2 (en) | 2017-04-27 | 2024-01-09 | Juno Therapeutics Gmbh | Oligomeric particle reagents and methods of use thereof |
| US11913024B2 (en) | 2015-10-22 | 2024-02-27 | Juno Therapeutics Gmbh | Methods for culturing cells and kits and apparatus for same |
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| CN106459917A (en) * | 2014-04-23 | 2017-02-22 | 朱诺治疗学股份有限公司 | Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy |
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| CN108118029A (en) * | 2016-11-30 | 2018-06-05 | 深圳宾德生物技术有限公司 | The screening technique of high anti-cancer activity T cell and application |
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