Method for enriching target cells from biology specimen and method for removing leucocyte

A technology for biological samples and leukocytes, applied in the field of enriching target cells and removing leukocytes from biological samples, can solve the problems of high cost, low WBC removal efficiency and time-consuming

Active Publication Date: 2009-07-01
杭州岳晨医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] For the method for removing leukocytes, although there is a method for removing leukocytes using anti-CD45 coated 30nm magnetic beads (Miltenyi product, Germany) in the prior art, this method is not only expensive (RMB600-700 / test), time-consuming (more than 4 hours / test), and the WBC removal efficiency is not high (WBC removal rate is about 2-3logs)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the method for removing leukocyte from biological sample

[0030] Dilute human blood in PBS containing 5mM EDTA, add 3 times the volume of RBC lysis buffer (see below for specific components), and gently rotate and shake at room temperature for 12 minutes. The samples were spun at 1500 rpm for 5 minutes to collect the cell pellet. The lysed RBCs in the supernatant were discarded, and the cell pellet was resuspended in 3ml~5ml of PBS containing 5mM EDTA, and passed through anti-CD45 antibody-conjugated Affi-Gel 10 (Pierce, IL, US). and a chromatography column through which the cells were passed in PBS at a flow rate of 2 ml / min to remove WBC, and the flow-through was collected. Compared with utilizing Miltenyi's kit to remove leukocytes in the prior art, the method for removing leukocytes of the present invention significantly reduces cost (the cost of the separation method of the present invention is RMB 80-150 / test; while the cost of Multiny's kit is RMB...

Embodiment 2

[0037] Embodiment 2, the method for enriching target cell from biological sample

[0038] 5ml-10ml of human blood was collected in a test tube containing EDTA and spun at 1500rpm for 3 minutes to pellet RBC and remove plasma including plasma proteins. Dissolve the collected RBC in 10 ml NH-based 4 Cl RBC lysis buffer (specific composition as above) was gently rotated and shaken for 15 minutes. The obtained solution was then spun centrifuged at 1500 rpm for 5 minutes to pellet unlysed cells including WBC and rare cells. The resulting unlysed cell pellet was resuspended in 5 ml PBS, and passed through a 0.5 ml anti-CD45 antibody-coupled Affi-Gel 10 (Pierce, IL, US) affinity chromatography column to remove WBC, wherein the cells were separated in 2 ml / min flow rate in PBS flow through the affinity chromatography column, and collect the flow-through. The collected flow-through containing rare cells was centrifuged at 900g for more than 5 minutes. Cell pellets were resuspended...

Embodiment 3

[0039] Embodiment 3, the yield of the enrichment method of the present invention is verified by admixture research

[0040] Target cells (such as HeLa cells or MCF-7 cells available from ATCC) were prelabeled with 4,6-diamino-2-phenylindole (DAPI) and then spiked into human blood using the method described in Example 1. Methods to enrich spiked cells. The average recovery was about 70% to 80%. Compared with the enrichment method utilizing Miltenyi's kit to remove white blood cells in the prior art, the enrichment method of the present invention significantly reduces cost (the cost of the enrichment method of the present invention is RMB 100-200 / test; while utilizing Multinyi's kit Only the cost of removing white blood cells is RMB 600-700 / test), shortened time (each test of the enrichment method of the present invention needs less than 1 hour to complete the removal of red blood cells and white blood cells; while Multiny's kit only needs to remove white blood cells More than...

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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 [0001] The present invention relates to a method for enriching target cells from a biological sample and a method for removing leukocytes. Background technique [0002] Cells including stem cells, fetal cells, circulating tumor cells (CTCs), immune cells, etc. have great applications in the prevention, detection, diagnosis and treatment of diseases. [0003] Stem cells refer to a type of pluripotent cells with self-replication ability, including embryonic stem cells, bone marrow stem cells, adult stem cells, umbilical cord blood stem cells, skin stem cells, etc. Under certain conditions, stem cells can differentiate into a variety of functional cells, so stem cells have a wide range of applications in treating many difficult diseases, providing organ transplantation, rehabilitation, health care, reducing aging, and restoring youthful vitality. [0004] Fetal cells have been shown to be detectable in the maternal circulation as early as five weeks of gestatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/12C12M1/00G01N33/53
Inventor 赵海峰
Owner 杭州岳晨医药科技有限公司
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