Method for directly separating CD<4+> and CD<8+> lymphocytes
A technology of lymphocytes and -CD4, applied in the field of biomedicine, can solve the problems of separation failure, change of antibody spatial direction, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, stabilize the reaction solution, and shorten the separation time.
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Embodiment 1
[0030] 1. Multi-Armed Well Star Polymer-CD4 + Antibody complexes were prepared according to the following steps:
[0031] (1) Dissolve 1.0 mg of Dobby Star Polymer Dobby Star polyamide-amine in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino)carbodiimide hydrochloride EDC, stir on a mixer at room temperature, and activate for 15 min;
[0032] (2) Take 2.2 mg mouse anti-human CD4 + The monoclonal antibody was added to the above reaction solution, placed on a mixer at room temperature and stirred for 30 min;
[0033] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.
[0034] 2. Multi-Armed Well Star Polymer-CD8 + Antibody complexes were prepared according to the following steps:
[0035] (1) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL ...
Embodiment 2
[0044] Example 2 Enrichment effect experiment
[0045] (1) Take 1 mL of concentration as 10 4 CD4 cells / mL + or CD8 + Centrifuge the cells at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0046] (2) Enrichment and capture: respectively set the technical solution group of the present invention (CD4 + or CD8 + Cell antibody and long-chain biotin co-modified multi-armed well star polymer group), CD4 + or CD8 + Cell-specific antibody-modified nanomagnetic bead set, CD4 + or CD8 + Cell-specific antibody-modified micron magnetic bead sets enrich target cells.
[0047] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated CD4 + or CD8 + The immunomagnetic beads of the cells were washed twice with PBST, mixed evenly, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.
[0048] (4) Capture rate calc...
Embodiment 3
[0060] Example 3 Enrichment capture experiment
[0061] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0062] CD4 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD4 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD4 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 51.7% 38.1% 89.8% CD8 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD8 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD8 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 50.9% 36.9% 88.7%
[0063] The experimental results show that compared with the separation of 3min in Example 2, when the separation time reaches 30min, the capture efficiency of the thre...
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