CN103305463B - Method for separating CD4<+> and CD25<+> lymphocytes in human peripheral blood by use of magnetic beads - Google Patents
Method for separating CD4<+> and CD25<+> lymphocytes in human peripheral blood by use of magnetic beads Download PDFInfo
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- CN103305463B CN103305463B CN201310219433.1A CN201310219433A CN103305463B CN 103305463 B CN103305463 B CN 103305463B CN 201310219433 A CN201310219433 A CN 201310219433A CN 103305463 B CN103305463 B CN 103305463B
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Abstract
The invention discloses a method for separating CD4<+> and CD25<+> regulatory lymphocytes in peripheral blood through separation and enrichment, and better provides a basis to the follow-up study of the CD4<+> and CD25<+> regulatory lymphocytes, relating to the field of biological medicine. The method comprises the following steps of: performing covalent coupling of a multi-arm well star polymer and a mouse anti-human CD4<+> or CD25<+> monoclonal antibody; coating a long-chain biotin molecule with the multi-arm well star polymer modified by the mouse anti-human CD4<+> or CD25<+> monoclonal antibody; capturing the CD4<+> and CD25<+> lymphocytes in a peripheral blood sample by use of the multi-arm well star polymer modified by the mouse anti-human CD4<+> or CD25<+> monoclonal antibody and the long-chain biotin; identifying streptavidin-modified nano magnetic beads and coupling with the long-chain biotinylation multi-arm well star polymer in the peripheral blood; separating and re-suspending the captured CD4<+> and CD25<+> lymphocytes, and the like, wherein the re-suspension liquid can be directly subjected to follow-up analysis. Compared with a traditional cell separation method, the method disclosed by the invention is more suitable for the magnetic separation of the CD4<+> and CD25<+> lymphocytes in a complicated peripheral blood sample, and improves the separation efficiency of CD4<+> and CD25<+> lymphocytes in a peripheral blood sample.
Description
Technical field
The present invention relates to biomedical sector, specifically relate to the CD4 based on nanometer magnetic bead
+and CD25
+regulator lymphocytes separation method.
Background technology
T lymphocyte is the most important a group cell of function in body immune system.T lymphocyte derives from the lymphocyte of marrow, perform cellular immune function, be not only the main body of direct immunization effect, and produce cytokine profiles and express adhesion molecule, by the contact direct or indirect with other immunocytes, play immunoregulation effect.In normal body, each lymphocyte subgroup interacts, and maintains body normal immunological function.When quantity and the function generation exception of different lymphocyte subgroup, body's immunity can be caused disorderly and a series of pathological change occurs.Recent years abroad scholar's research finds the T cell subgroup having unique immune regulatory function a: CD4
+and CD25
+autoimmune disease, can not only suppress autoimmune disorder to occur, also may participate in the adjustment of tumour immunity.This group of cells have Immune anergy and immunosuppressive properties, play a role by directly contacting with cell, and do not rely on the cytokine of its secretion.CD4 in peripheral blood
+and CD25
+autoimmune disease ratio increases, and causes tumour immunity lack of proper care, removes that this group of cells can effectively induced tumor be immune, for oncotherapy provides a kind of new method.But, CD4 in normal human peripheral blood
+and CD25
+the lymphocytic content of T is very low, and existing method also cannot trace CD4 in human peripheral blood
+and CD25
+autoimmune disease is directly separated and gathers.Therefore, a kind of high efficiency separation and Purification of Human peripheral blood CD4 is set up
+and CD25
+the method of Autoimmune disease, and phenotype and biological function qualification are carried out, for better studying CD4 to it
+and CD25
+the function of Autoimmune disease provides basis.
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 reduces the capture rate 5 of antibody) the high and wherein non-CD4 of blood viscosity
+and CD25
+lymphocytic hematocrite concentration is large, and micron magnetic bead easily produces non-specific adsorption, is difficult to realize CD4 in blood
+and CD25
+lymphocytic specific isolation; 6) excessive concentration of micron magnetic bead can cause CD4
+and CD25
+lymphocytic breakage (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, CD4 in the peripheral blood matrix that (to be less than 30 T/m) under low gradient magnetic complicated
+and CD25
+the method of lymphocyte specific sharp separation.
Utilize CD4 in Beads enrichment human peripheral
+and CD25
+lymphocytic method, comprises the following steps:
(1) often get 1.0 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.1 mg mouse-anti people CD4
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; 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 obtained to obtain multi-arm well star polymkeric substance-mouse-anti people CD4
+antibody complex; (2) often get 1.0 mg amidized multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.1 mg mouse-anti people CD25
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; 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 obtained to obtain multi-arm well star polymkeric substance-mouse-anti people CD25
+antibody complex; (3) often get 15 mg long-chain biological elements, 5.4 mg NHSS, 3.6 mg EDC are dissolved in 3 mL 0.02 M pH 6.5 PBS damping fluids; By 0.1 mg multi-arm well star polymkeric substance-mouse-anti people CD4
+or CD25
+antibody complex joins in above-mentioned solution, and room temperature is placed on blending instrument and stirs 30 min; 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 long-chain biological element-multi-arm well star polymer-antibody complex that will obtain; (4) enrichment is caught: get testing sample solution 1mL, adds 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD4
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell; Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal 300rpm/min, 20 DEG C of 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD25
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD25 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell; (5), after deionized water cleans gently, mix resuspended namely obtaining with PBS damping fluid and be enriched with CD4
+or CD25
+the mixture of cell and nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymer-antibody-CD4
+cD25
+cell.
Described multi-arm well star polymkeric substance is the end modified multi-arm well star polymkeric substance having amino, and its molecular weight is 70000 Da.Structure is as Fig. 1.
The described nanometer magnetic bead particle diameter having modified Streptavidin is 20-50 nm, is preferably 30 nm.
Multi-arm well star polymkeric substance is by amino and CD4
+or CD25
+the carboxyl of lymphocyte antibody realizes the covalent coupling of multi-arm well star polymkeric substance and antibody.
Multi-arm well star polymkeric substance, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of multi-arm well star polymkeric substance and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed multi-arm well star polymkeric substance.
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 multi-arm well star polymkeric substance, 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, multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance, 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, multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance 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 high-polymer molecular, multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance 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 multi-arm well star polymkeric substance.
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..
Multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, and its molecular weight is 70000 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: 8.0g NaCl, 0.2g KCl, 0.24g KH
2pO
4, 1.44g Na
2hPO
4be dissolved in 800mL distilled water, adjust pH to 7.4 with 5M NaOH, then be settled to 1000mL and namely obtain 0.01M PBS.Add Tween 20 with the volume ratio of 1/1000 (V/V) again, namely obtain 0.1%PBST.
Embodiment 1
1. multi-arm well star polymkeric substance-mouse-anti people CD4
+antibody complex, prepare in accordance with the following steps:
(1) often get 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 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min;
(2) 2.1 mg mouse-anti people CD4 are got
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 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. multi-arm well star polymkeric substance-mouse-anti people CD25
+antibody complex, prepare in accordance with the following steps:
(1) often get 1.0 mg amidized multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min;
(2) 2.1 mg mouse-anti people CD25 are got
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 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. long-chain biological element-multi-arm well star polymer-antibody complex is prepared in accordance with the following steps:
(1) often get 15 mg long-chain biological elements, 5.4 mg NHSS, 3.6 mg EDC are dissolved in 3 mL 0.02 M pH 6.5 PBS damping fluids;
(2) by 0.1 mg multi-arm well star polymkeric substance-mouse-anti people CD4
+or CD25
+antibody complex joins in above-mentioned solution, and room temperature is placed on blending instrument and stirs 30 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.
4. enrichment is caught: get testing sample solution 1mL, adds 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD4
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell.Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal (300rpm/min, 20 DEG C) 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD25
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD25 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell.
5., after deionized water cleans gently, mix resuspended namely obtaining with PBS damping fluid and be enriched with CD4
+or CD25
+the mixture of cell and nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymer-antibody-CD4
+cD25
+cell.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4the CD4 of cell/mL
+or CD25
+cell 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 (CD4 respectively
+or CD25
+cell antibody and the co-modified multi-arm well star polymkeric substance group of long-chain biological element), CD4
+or CD25
+nanometer magnetic bead group, CD4 that cell-specific antibodies is modified
+or CD25
+the micron magnetic bead group enrich target cells that cell-specific antibodies is modified.
(3) after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, and separate and caught CD4
+or CD25
+the immunomagnetic beads of cell then with PBST cleaning twice, mixes, 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 target cell sum) × 100%.
The scheme of described each group of enrichment acquisition target cell is as follows:
A. technical solution of the present invention group (CD4
+or CD25
+cell antibody and the plain co-modified multi-arm well star polymkeric substance group of long-chain biological) enrichment acquisition target cell protocol is as embodiment 1, specific as follows:
By 0.1 mg CD4
+cell antibody and the co-modified multi-arm well star polymkeric substance of vitamin H and vitamin H-multi-arm well star polymer-antibody complex join containing in target cell centrifuge tube, are 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 fully separation.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell.Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal (300rpm/min, 20 DEG C) 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD25
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD25 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell.
B. CD4
+or CD25
+the nanometer magnetic bead group enrichment acquisition target cell protocol that cell-specific antibodies is modified is specific as follows:
By the CD4 that 0.1 mg prepares
+the nanometer magnetic bead that cell-specific antibodies 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.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell.Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal (300rpm/min, 20 DEG C) 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg CD25
+the nanometer magnetic bead that cell-specific antibodies is modified, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, centrifuge tube is inserted conventional magnetic frame and be fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell.
Described CD4
+or CD25
+the nanometer magnetic bead preparation that 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 CD4 by every mg magnetic bead
+or CD25
+cell-specific antibodies, 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. CD4
+or CD25
+the micron magnetic bead group enrichment acquisition target cell protocol that cell-specific antibodies is modified is specific as follows:
By the CD4 that 0.1 mg prepares
+the micron magnetic bead that cell-specific antibodies 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.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell.Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal (300rpm/min, 20 DEG C) 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg CD25
+the micron magnetic bead that cell-specific antibodies is modified, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, centrifuge tube is inserted conventional magnetic frame and be fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell.
Described CD4
+or CD25
+the micron magnetic bead preparation that 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 CD4 by every mg magnetic bead
+or CD25
+cell-specific antibodies, 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:
| CD4 +The micron magnetic bead group capture rate that cell-specific antibodies is modified | CD4 +The nanometer magnetic bead group capture rate that cell-specific antibodies is modified | CD4 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological |
| 502% | 21.3% | 86.9% |
| CD25 +The micron magnetic bead group capture rate that cell-specific antibodies is modified | CD25 +The nanometer magnetic bead group capture rate that cell-specific antibodies is modified | CD25 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological |
| 46.9% | 20.1% | 87.6% |
Experimental result shows, CD4
+or CD25
+the capture rate of the micron magnetic bead group that cell-specific antibodies is modified is apparently higher than the capture rate of nanometer magnetic bead group, and this illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic 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 CD4 again
+or CD25
+the micron magnetic bead group that cell-specific antibodies is modified, this shows that technical solution of the present invention can increase target cells nanometer magnetic bead fraction of coverage by multi-arm well star polymkeric substance, thus magnetic is improved greatly, and then achieve (3min) high efficiency separation enrichment CD4 at short notice
+or CD25
+cell.
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:
| CD4 +The micron magnetic bead group capture rate that cell-specific antibodies is modified | CD4 +The nanometer magnetic bead group capture rate that cell-specific antibodies is modified | CD4 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological |
| 53.3% | 40.3% | 88.8% |
| CD8 +The micron magnetic bead group capture rate that cell-specific antibodies is modified | CD8 +The nanometer magnetic bead group capture rate that cell-specific antibodies is modified | CD8 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological |
| 49.2% | 42.6% | 89.4% |
Experimental result shows, is separated 3min in comparative example 2, and when reaching 30min when disengaged, the capture rate of three groups is obtained for raising, particularly CD4
+or CD25
+the capture rate of the nanometer magnetic bead group that cell-specific antibodies is modified improves the most obvious, and this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but it is still lower than CD4 during short period of time separation (3min)
+or CD25
+the capture rate of cell antibody and the plain co-modified multi-arm well star polymkeric substance group of long-chain biological.This shows that technical solution of the present invention can (3min) high efficiency separation enrichment CD4 at short notice
+or CD25
+cell.
CD4 in embodiment 4 nanometer magnetic bead enrichment healthy volunteer peripheral blood
+or CD25
+lymphocytic research
Get healthy volunteer's aseptic EDTA anticoagulation cirumferential blood 20 mL, utilize density gradient centrifugation separating peripheral blood mononuclear cells, with 2 mL PBS solution re-suspended cells.Get above-mentioned PBS cell re-suspension liquid in centrifuge tube, centrifugal (300 rpm/min, 20 DEG C) 10 min, supernatant discarded, re-suspended cell, every 80 μ L PBS are containing cell count 10
7individual, every 10
7individual cell adds 0.1mg long-chain biological element-multi-arm well star polymkeric substance-mouse-anti people CD4
+antibody complex, fully mixes, and hatches 15 min at 4-8 DEG C.Add the micron 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.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell.Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal (300rpm/min, 20 DEG C) 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell.Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD25
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD25 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated.After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell.After Magneto separate, supernatant liquor is poured in sterile centrifugation tube, and separate and caught CD4
+or CD25
+the immunomagnetic beads of cell then with PBST cleaning twice, mixes, 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 CD4 in this programme energy efficiently concentrating sample separation
+or CD25
+cell.
CD4 in table 1 peripheral blood
+or CD25
+separation of lymphocytes effect
| Experiment group number | CD4 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological | CD25 +Cell antibody and the plain co-modified multi-arm well star polymkeric substance group capture rate of long-chain biological |
| Embodiment 4 | 80.3% | 82.6% |
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. utilize CD4 in Beads enrichment human peripheral
+and CD25
+lymphocytic method, is characterized in that comprising the following steps:
(1) often get 1.0 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.1 mg mouse-anti people CD4
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; 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 obtained to obtain multi-arm well star polymkeric substance-mouse-anti people CD4
+antibody complex; (2) often get 1.0 mg multi-arm well star polymer dissolution in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.1 mg mouse-anti people CD25
+antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; 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 obtained to obtain multi-arm well star polymkeric substance-mouse-anti people CD25
+antibody complex; (3) often get 15 mg long-chain biological elements, 5.4 mg NHSS, 3.6 mg EDC are dissolved in 3 mL 0.02 M pH 6.5 PBS damping fluids; By 0.1 mg multi-arm well star polymkeric substance-mouse-anti people CD4
+or CD25
+antibody complex joins in above-mentioned solution, and room temperature is placed on blending instrument and stirs 30 min; 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 long-chain biological element-multi-arm well star polymer-antibody complex that will obtain; (4) enrichment is caught: get testing sample solution 1mL, adds 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD4
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; 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 fully separated; The nanometer magnetic bead particle diameter having modified Streptavidin is 20-50 nm; After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4
+cell; Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal 300rpm/min, 20 DEG C of 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell; Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD25
+antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD25 with rotating speed incubated at room 15 min of 30 rpm
+cell antigen mixture; Add 0.1 mg and modified the nanometer magnetic bead of 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 fully separated; After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD25
+cell; (5), after deionized water cleans gently, mix resuspended namely obtaining with PBS damping fluid and be enriched with CD4
+or CD25
+the mixture of cell and nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymer-antibody-CD4
+or CD25
+cell.
2. method according to claim 1, it is characterized in that described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, its molecular weight is 70000 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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