Quick enriching and separating method of natural killer cells in peripheral blood of human
A natural killer cell and human peripheral blood technology, applied in the field of biomedicine, can solve the problems of separation failure, decrease of antibody biological activity, change of antibody space direction, etc., to increase the chance of contact, realize cascade amplification, and stabilize the reaction solution Effect
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Embodiment 1
[0029] 1. dendrimer-antibody complex, prepared according to the following steps:
[0030] (1) Weigh 1.0 mg dendrimer polyamide-amine dendrimer, suspend in 4 mL phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 25% glutaraldehyde dropwise 545 μL of aqueous solution to make the final concentration of glutaraldehyde 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;
[0031] (2) Add 1 mL (ie 3.57 mg) of mouse anti-human NKp46 antibody dropwise to the above solution to make the final concentration reach about 3 mg / mL. React at room temperature for 24 h at the speed of the shaker at 150 r / min;
[0032] (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.
[0033] 2. The long-chain biotin-dendrimer-antibody complex is prepared according to the following steps:
[0034] (1) Take ...
Embodiment 2
[0039] Example 2 Enrichment effect experiment
[0040] (1) Take 1 mL of concentration as 10 4 Cells / mL NK cells were placed in a 1.5 mL sterile centrifuge tube, centrifuged at 12,000 rpm for 5 min, discarded the supernatant, and resuspended with an equal volume of sterile PBS solution.
[0041] (2) Enrichment and capture: Set up the technical solution group of the present invention (dendrimer group co-modified with NK cell antibody and long-chain biotin), nano magnetic beads group modified with NK cell-specific antibody, and NK cell-specific antibody group Modified micron magnetic bead sets enrich target cells.
[0042] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the separated immunomagnetic beads with NK cells twice with PBST, mix well, and resuspend the immunomagnetic beads with 1 mL of sterile PBS solution. Magnetic bead complex.
[0043] (4) Capture rate calculation: after serial dilution of the enriched target cell resu...
Embodiment 3
[0056] Example 3 Enrichment capture experiment
[0057] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0058] The catch rate of each group is as follows:
[0059] Capture rate of NK cell-specific antibody modified micron magnetic bead set Capture rate of NK cell-specific antibody-modified nanomagnetic bead set Capture efficiency of dendrimers co-modified with NK cell antibodies and long-chain biotin 58.2% 44.2% 92.3%
[0060] Experimental result shows, separates 3min in the comparative example 2, and when separation time reaches 30min, the capture efficiency of three groups has all been improved, and the capture efficiency of particularly NK cell-specific antibody-modified nano magnetic bead group improves most obviously, this It shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by prolonging the time, but it is still lower than the capture efficiency of the NK ce...
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