CN101469310A - Method for enriching target cells from biology specimen and method for removing leucocyte - Google Patents
Method for enriching target cells from biology specimen and method for removing leucocyte Download PDFInfo
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Abstract
The invention relates to a method for enriching target cells from biological samples and a method for removing white blood cells from the biological samples. The invention practically provides the method for enriching the target cells from the biological samples, which comprises: using a mode based on mol osmotic pressure concentration to remove red blood cells, and using immune affinity chromatography to remove the white blood cells. The method not only has low cost but also highly efficiently removes the white blood cells and the red blood cells, so as to enrich the target cells and be easy for subsequent detection. The invention also provides the method for removing the white blood cells from the biological samples, which comprises: adopting Affi-Gel 10 coupled by CD45 antibodies to remove the white blood cells.
Description
Technical field
The present invention relates to from the method for biological sample enriched target cell and remove leukocytic method.
Background technology
The cell that comprises stem cell, fetal cell, circulating tumor cell (CTC), immunocyte etc. is having very big application aspect prevention, detection, diagnosis and the treatment of disease.
Stem cell is meant that a class has the multipotential cell of the of self-replication capacity, comprises embryonic stem cell, bone marrow stem cell, adult stem cell, navel blood stem cell, skin progenitor cell etc.Under certain condition, stem cell can be divided into multiple functioning cell, so stem cell has the many difficult and complicated illness of treatment, organ transplantation is provided, and rehabilitation, health care, alleviates aging, vigor or the like widespread use of renewing one's youth.
Verifiedly just can monitor out fetal cell in five all female circulation of blood as far back as gestation.At present, obtain the method that fetal cell carries out antenatal diagnosis and mainly contain amniocentesis, fine hair biopsy and umbilical blood vessels puncture.These means have certain risk to fetus, might cause miscarriage.Isolating fetal cell from female blood is only indicating and need can obtain fetal cell by simple venipuncture approach.The clinical application of fetal cell will comprise early diagnosis fetal chromosomal or gene unconventionality.
A large amount of reports are pointed out even can be found circulating tumor cell (CTC) in the patient before detecting primary tumo(u)r in the imaging analysis mode.The latent effect in diagnosing in early days and predicting, CTC also can characterize change with the heredity of tumor progression and immunophenotype in the main effect of performance, thereby help to instruct individualized treatment.
Because the disease of immunity system or other system, or because the influence of immunization or some clinical treatment measure and some external environment factor, the quantity or the function of immunocyte (for example T lymphocyte, bone-marrow-derived lymphocyte, K lymphocyte or NK lymphocyte) all can change.Therefore, carry out cellular immunization and detect, to the influence of body's immunity, all have great importance for the diagnosis of some disease and study of pathogenesis, immunotherapy or premunitive recruitment evaluation and environmental factors.
But described stem cell, fetal cell, circulating tumor cell (CTC), immunocyte are at biological sample, and for example amount is less in blood, marrow, spinal cord or the operation liquid etc., belongs to rare cell, so these target cells of enrichment become matter of utmost importance.
Although various technology have been used for enrichment, separation and sign target cell, wherein many have a similar principle, promptly based on the just selection of antibody.Because the heterogeneity of cell surface marker is expressed, these strategies that are used for the target cell detection obviously are subjected to the restriction of following factor: availability, susceptibility and the specificity of the antibody of the biomarker on the anti-different target cells.Other strategy comprises negative the removing of red corpuscle (RBC), white corpuscle (WBC).At present mainly remove by filter less RBC and WBC based on size, but for example CTC can be little as WBC because of some target cell, described have the danger of losing target cell based on the filtering method of size.
For removing leukocytic method, though there is 30nm magnetic bead (the Miltenyi product that uses anti-CD45 bag quilt in the prior art, Germany) remove leukocytic method, but this method is expensive (RMB600-700/ test) not only, time-consuming (surpassing 4 hours/test), and WBC removes efficient not high (the WBC clearance is about 2-3logs).
Summary of the invention
In order to overcome the defective of prior art, the present invention creatively will join together to be used for to remove respectively RBC and WBC based on the mode and the immune-affinity chromatography of osmolarity (osmolarity), thereby reach the enriched target cell, especially the purpose of rare cell.This method not only cost is low, and makes target cell be able to enrichment efficiently, and is easy to subsequent detection.Described target cell comprises stem cell, fetal cell, circulating tumor cell (CTC), immunocyte etc.
The present invention also provides a kind of leukocytic method of removing, and this method not only cost is low, save time, and the efficient height.
Embodiment
In a specific embodiment of the present invention, the invention provides a kind of method from biological sample enriched target cell, this method comprises:
Use based on the mode of osmolarity and remove red corpuscle and remove leucocyte-removing with immune-affinity chromatography.
The present invention creatively combined utilization removes red corpuscle and immune-affinity chromatography based on the mode of osmolarity and removes leucocyte-removing and come the enriched target cell.Utilize the mode of osmolarity to remove red corpuscle and utilize immune-affinity chromatography to remove these two processes of leucocyte-removing and do not have special sequence requirement.For example can utilize affinity chromatography to remove leucocyte-removing and then utilize earlier and remove red corpuscle based on the mode of osmolarity; Perhaps use based on the mode of osmolarity and remove red corpuscle and then utilize affinity chromatography to remove leucocyte-removing.For quickly, more effectively make white corpuscle pass through affinity column, in a preferred embodiment of the present invention, use based on the mode of osmolarity and remove red corpuscle and then utilize affinity chromatography to remove leucocyte-removing.
In a specific embodiment of the present invention, described biological sample is a body fluid; Preferred described body fluid is blood, marrow, spinal cord or operation liquid (surgical fluid).In an embodiment of the invention, with blood be the validity that example has been verified the inventive method.Simultaneously because the density of blood, marrow, spinal cord or operation liquid is similar and target cell existence therein is similar, so method of the present invention can be used for the enrichment of target cell in marrow, spinal cord or the operation liquid.
In order to collect blood, in the anticoagulation collection tube or contain in the pipe of any antithrombotics, described antithrombotics comprises ethylenediamine tetraacetic acid (EDTA) (EDTA), ACD (ACD) etc. with the blood sample collection of people or any other animal.In order to collect marrow, can gather marrow in the puncture of the ilium position of donor; Also can mobilize the hemopoietic stem cell at marrow and other positions to be discharged in the peripheral blood in a large number and go, from the arm vein of donor, gather then.Spinal cord can obtain by for example thecal puncture.Operation liquid can be the body fluid utilize modus operandi to obtain, and for example abdominal cavity or hydrothorax are comprising target cells more of the present invention and albumen.
In a preferred embodiment of the present invention, used blood is to get blood same day, preferably getting blood rose within 20 hours, more preferably getting blood rose within 10 hours, further preferably get blood and rise within 5 hours or get blood and rise within 3 hours or get blood and rise within an hour, optimum is chosen blood and is played the fresh blood that obtains immediately.Red corpuscle in the above-mentioned fresh blood is to RBC lysis buffer sensitivity, and target cell is insensitive to the RBC lysis buffer.
In a specific embodiment of the present invention, employed immune particle is of a size of 1 μ m～20 μ m in the wherein said immune-affinity chromatography, more preferably 2 μ m～15 μ m, more preferably 5 μ m～10 μ m.In a specific embodiment of the present invention, further preferred described immune particle has recyclability.Can utilize damping fluid such as Tris-Glycine that immune particle is washed and reuses, thereby save cost.
In a specific embodiment of the present invention, use based on the mode of osmolarity and remove red corpuscle.In a preferred embodiment of the present invention, comprise precipitation and splitting erythrocyte in the described mode based on osmolarity.Described precipitation is preferably with 500～3000rpm, and more preferably 100～2000rpm is centrifugal 1～6 minute, preferably removes blood plasma simultaneously with precipitation RBC in preferred 2～4 minutes.In room temperature with collected RBC soft rotational oscillation 5～20 minutes in the RBC lysis buffer.Then with the solution that obtained with 500～3000rpm, more preferably 100～2000rpm, centrifugal 1～10 minute, more preferably described uncracked cell comprised white corpuscle and rare cell to precipitate uncracked cell in 3～6 minutes.
In a specific embodiment of the present invention, describedly be used for removing leukocytic immune particle and be positioned at affinity column and be combined with the leukocytic specificity junction mixture of target.Preferred described specificity junction mixture is to be identified in the antigenic antibody of expressing on the white corpuscle.(claim " LCA " again) because CD45 and be the leukocyte surface antigen of recognition and acceptance, so more preferably described antibody is anti-CD45 antibody.Most preferably will resist CD45mAb (CD45 monoclonal antibodies) to be coupled on Affi-Gel 10 matrix.Can adopt affinity column well known by persons skilled in the art to carry out chromatography.In a preferred embodiment of the present invention, in order to remove leucocyte-removing, to contain the flow velocity of leukocytic solution with about 0.5～8.5ml/min, preferably with the flow velocity of about 0.75～3.5ml/min, further preferred flow velocity with about 1～2.5ml/min flows through affinity column.
The target cell that utilizes method of the present invention to obtain is suitable for subsequent analysis, operation or application, also can carry out subsequent analysis, operation or application to albumen in the target cell and/or gene after having obtained target cell of the present invention certainly.In a specific embodiment of the present invention, described subsequent analysis, operate or be applied as one of following: test, cell cultures and cell therapy, research that mass spectrum is relevant with cell and/or albumen with other etc. are renderd a service in flow cytometry, polymerase chain reaction (PCR), immunofluorescence, immunocytochemistry, image analysis, enzymatic determination, inflammation research, gene expression spectrum analysis, treatment.
In a specific embodiment of the present invention, comprise at least a in stem cell, fetal cell, circulating tumor cell (CTC), the immunocyte with the target cell of method enrichment of the present invention.Utilizing method enrichment of the present invention on the basis of above-mentioned target cell, can utilize further separation purification method acquisition circulating tumor cell, stem cell (comprising embryonic stem cell, bone marrow stem cell, adult stem cell, navel blood stem cell, skin progenitor cell etc.), fetal cell, immunocyte (to comprise T lymphocyte, bone-marrow-derived lymphocyte, K lymphocyte and NK lymphocyte) etc., and can further utilize circulating tumor cell, stem cell, fetal cell, the immunocyte of these purifying to predict, detect, diagnose and treat.
In a specific embodiment of the present invention, method of the present invention also comprises removes plasma proteins.In order more to help saving time and reducing operation steps, remove the step of plasma proteins and can carry out simultaneously with the erythrocytic step of precipitation.For example in an embodiment of the invention, with whole blood with 1500 centrifugal 3 minutes, with the precipitation red corpuscle with remove the blood plasma that comprises plasma proteins.
In a specific embodiment of the present invention, white corpuscle that preferred recovery method of the present invention is removed and/or the plasma proteins of being removed, and it is further used for subsequent analysis, operation or application, for example, flow cytometry, polymerase chain reaction (PCR), immunofluorescence, immunocytochemistry, image analysis, enzymatic determination, inflammation research, gene expression spectrum analysis, treatment are renderd a service test, cell cultures, cell therapy, research that mass spectrum is relevant with cell and/or albumen with other etc.For example can utilize further separation purification method to obtain white corpuscle or plasma proteins etc., and can further utilize the white corpuscle of these purifying or plasma proteins to predict, detect, diagnose and treat.
In the present invention, term " enrichment " with its most widely implication use, be to make a material formerly exist under the envrionment conditions more concentratedly than it, it comprises implications such as " accumulation ", " concentrating ", " extraction ", " separation ".
The present invention also provides a kind of leukocytic method of removing, and this method comprises that the Affi-Gel 10 that adopts anti-CD45 antibody coupling removes leucocyte-removing.Can adopt affinity column well known by persons skilled in the art to carry out chromatography.In a preferred embodiment of the present invention, in order to remove leucocyte-removing, to contain the flow velocity of leukocytic solution with about 0.5～8.5ml/min, preferably with the flow velocity of about 0.75～3.5ml/min, further preferred flow velocity with about 1～2.5ml/min flows through affinity column.Can utilize damping fluid such as Tris-Glycine that immune particle is washed and reuses, thereby save cost.This method not only cost is low, saves time, and the efficient height.
Below further specify the present invention in the mode of embodiment, described embodiment also is not used in the scope of the present invention that limits.
Embodiment
Embodiment 1, remove leukocytic method from biological sample
Human blood is diluted among the PBS that contains 5mM EDTA, adds the RBC lysis buffer (concrete composition as follows) of 3 times of volumes, in the soft rotational oscillation of room temperature 12 minutes.Sample is precipitated with collecting cell in 1500rpm rotating centrifugal 5 minutes.Cracking RBC in the abandoning supernatant, and cell precipitation is resuspended among the PBS of the 3ml～5ml that contains 5mM EDTA, and make its Affi-Gel that passes through anti-CD45 antibody coupling 10 (Pierce, IL, US) affinity column is to remove WBC, wherein cell is flow through described affinity column with the flow velocity of 2ml/min in PBS, and collect and flow through liquid.With utilize Miltenyi ' s test kit to remove white corpuscle in the prior art to compare, the present invention removes leukocytic method and has significantly reduced cost (cost of separation method of the present invention is RMB 80-150/ test; And the cost of Multiny ' s test kit is RMB 600-700/ test), (each test of separation method of the present invention needs can finish the removal white corpuscle less than 40 minutes to have shortened the time; And Multiny ' s test kit is removed white corpuscle and need be surpassed 4 hours/test) and the efficient height (the WBC clearance of the inventive method can reach about 3-4logs; And the WBC clearance of Multiny ' s test kit only is about 2-3logs).
The preparation of the Affi-Gel 10 of anti-CD45 antibody coupling: because available from Pierce (IL, US) Affi-Gel 10 contains can supply the activated chemical group, so will resist CD45 antibody to mix with Affi-Gel 10 about 15 minutes, can finish the coupling of anti-CD45 antibody and Affi-Gel 10.
RBC lysis buffer (pH7.2):
NH
4Cl 82.9g
KHCO
3 10g
EDTA disodium 0.37g
H
2O 1L
Embodiment 2, from the method for biological sample enriched target cell
The human blood of 5ml～10ml is collected in the test tube that contains EDTA, with 1500rpm rotating centrifugal 3 minutes with precipitation RBC with remove the blood plasma that comprises plasma proteins.In room temperature with collected RBC in 10ml based on NH
4The soft rotational oscillation of the RBC lysis buffer of Cl (concrete composition as above) 15 minutes.Then with the solution that obtained with 1500rpm rotating centrifugal 5 minutes to precipitate uncracked cell, described uncracked cell comprises WBC and rare cell.With gained not the lysing cell pellet resuspended in 5ml PBS, and make its Affi-Gel 10 (Pierce by the anti-CD45 antibody coupling of 0.5ml, IL, US) affinity column is to remove WBC, wherein cell is flow through described affinity column with the flow velocity of 2ml/min in PBS, and collect and flow through liquid.With collected contain rare cell to flow through liquid centrifugal more than 5 minutes at 900g.With the cell precipitation resuspension, to carry out subsequent analysis.After the enrichment, can use PBS (pH7.4) to neutralize then with 10ml Tris-Glycine (pH2.5) washing affinity column to remove all binding molecules in order to reusing.
Embodiment 3, utilize the yield of admixture research checking enriching method of the present invention
With 4,6-diamino-2-phenylindone (DAPI) labels targets cell (for example HeLa cell or the MCF-7 cell that can obtain from ATCC) in advance mixes the human blood then, uses the cell of embodiment 1 described method enrichment admixture.Average recovery rate is about 70%～80%.With utilize Miltenyi ' s test kit to remove leukocytic enriching method in the prior art to compare, enriching method of the present invention has significantly reduced cost, and (cost of enriching method of the present invention is RMB 100-200/ test; And utilize the leukocytic cost of only removal of Multiny ' s test kit just to be RMB 600-700/ test), (each test of enriching method of the present invention needs can finish removal red corpuscle and white corpuscle less than 1 hour to have shortened the time; And Multiny ' s test kit is only removed white corpuscle and is just needed to surpass 4 hours/test) and the efficient height (the WBC clearance of the inventive method can reach about 3-4logs; And the WBC clearance of Multiny ' s test kit only is about 2-3logs).
Claims (10)
1, a kind of method from biological sample enriched target cell, this method comprises:
Use based on the mode of osmolarity and remove red corpuscle and remove leucocyte-removing with immune-affinity chromatography.
2, the method for claim 1, wherein said biological sample is a body fluid; Preferred described body fluid is blood, marrow, spinal cord or operation liquid.
3, method as claimed in claim 2, wherein said blood are to get the blood fresh blood on the same day.
4, the method for claim 1, employed immune particle is of a size of 1 μ m～20 μ m in the wherein said immune-affinity chromatography, more preferably 5 μ m～10 μ m; Further preferred described immune particle has recyclability.
5, the method for claim 1, wherein said mode based on osmolarity comprise precipitation and splitting erythrocyte.
6, the method for claim 1, employed antibody is to be identified in the antigenic antibody of expressing on the white corpuscle in the wherein said immune-affinity chromatography; Preferred described antibody is anti-CD45 antibody; More preferably described anti-CD45 antibody coupling is on Affi-Gel 10 matrix; Further preferred described CD45 antibody is anti-CD45 monoclonal antibodies.
7, the method for claim 1, the target cell that this method obtained is suitable for subsequent analysis, operation or application, described subsequent analysis, operate or be applied as one of following: test, cell cultures and cell therapy are renderd a service in flow cytometry, polymerase chain reaction, immunofluorescence, immunocytochemistry, image analysis, enzymatic determination, inflammation research, gene expression spectrum analysis, treatment.
8, the method for claim 1, wherein said target cell comprise at least a in stem cell, fetal cell, circulating tumor cell, the immunocyte.
9, the method for claim 1, this method also comprise removes plasma proteins; Preferred white corpuscle of being removed and/or the plasma proteins of being removed of reclaiming.
10, a kind ofly remove leukocytic method from biological sample, this method comprises that the Affi-Gel 10 that adopts anti-CD45 antibody coupling removes leucocyte-removing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103589629A (en) * | 2013-11-15 | 2014-02-19 | 上海康微健康科技有限公司 | Separation system for CTCs (circulating tumor cells) |
| CN103725648A (en) * | 2013-09-28 | 2014-04-16 | 长沙赢润生物技术有限公司 | Novel CTC (circulating tumor cell) enrichment technology and preparation method of kit |
| CN105026934A (en) * | 2013-03-15 | 2015-11-04 | 詹森诊断器材有限责任公司 | Enrichment of circulating tumor cells by depleting white blood cells |
| CN109863399A (en) * | 2016-08-26 | 2019-06-07 | 朱诺治疗学股份有限公司 | The method for counting the particle being present in cell composition |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105026934A (en) * | 2013-03-15 | 2015-11-04 | 詹森诊断器材有限责任公司 | Enrichment of circulating tumor cells by depleting white blood cells |
| US9823238B2 (en) | 2013-03-15 | 2017-11-21 | Menarini Silicon Biosystems, Inc. | Molecular characterization of circulating tumor cells |
| US10365266B2 (en) | 2013-03-15 | 2019-07-30 | Menarini Silicon Biosystems, Inc. | Molecular characterization of circulating tumor cells |
| CN103725648A (en) * | 2013-09-28 | 2014-04-16 | 长沙赢润生物技术有限公司 | Novel CTC (circulating tumor cell) enrichment technology and preparation method of kit |
| CN103589629A (en) * | 2013-11-15 | 2014-02-19 | 上海康微健康科技有限公司 | Separation system for CTCs (circulating tumor cells) |
| CN109863399A (en) * | 2016-08-26 | 2019-06-07 | 朱诺治疗学股份有限公司 | The method for counting the particle being present in cell composition |
| US11561219B2 (en) | 2016-08-26 | 2023-01-24 | Juno Therapeutics, Inc. | Methods of enumerating particles present in a cell composition |
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Effective date of registration: 20220927 Address after: Room 601, 6th Floor, Building 1, No. 3, Binwen Road, Xixing Street, Binjiang District, Hangzhou City, Zhejiang Province, 310051 Patentee after: Hangzhou Yuechen Pharmaceutical Technology Co.,Ltd. Address before: 100070, room 8, 302, Feng Feng Road, Fengtai Science Town, Beijing, Fengtai District Patentee before: BEIJING BIOQTECH BIOTECHNOLOGY CO.,LTD. |