HER2 (Human Epidermal Growth Factor Receptor2) antibody immunomagnetic bead and preparation method thereof
An immunomagnetic bead and antibody technology, which is applied in the preparation of microspheres, chemical instruments and methods, magnetic materials, etc., can solve the problems of inability to achieve cell sorting, low magnetic responsiveness, and oppression of cells, and achieves low cost and magnetic response. Rapid, biologically active effects
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Embodiment 1
[0032] Example 1 Preparation of HER2 Antibody Immunomagnetic Beads
[0033] (1) Preparation of magnetic nanoclusters:
[0034] a. In the air, 7g FeCl 2 4H 2 O was added to 50 mL of deionized water to obtain a concentration of 0.14 g / mL of FeCl 2 aqueous solution. to 50mL FeCl 2 Add 30 mL of ammonia water to the aqueous solution, and after stirring for 20 minutes, the color gradually turns light green, then dark green, and finally black;
[0035] b. Add 1.1 g of oleic acid to step a, mix well, place the mixed solution in a closed reaction kettle, heat and react at 110°C for 4 hours, then alternately wash each time with deionized water and ethanol, after magnetic separation Dispersed in n-hexane, you can get black magnetic nano-cluster Fe 3 o 4 1.
[0036] (2) Preparation of amino-modified magnetic microspheres: to 10 mg magnetic nanocluster Fe 3 o 4 1 solution, add 125 mg ammonia water, 30 mg tetraethyl orthosilicate and 30 mg (3-aminopropyl) triethoxysilane, react fo...
Embodiment 2
[0042] Example 2 Sensitivity detection of HER2 antibody immunomagnetic beads
[0043] Collect blood samples from healthy volunteers, extract PBMCs through human lymphocyte separation medium, and then add human breast adenocarcinoma SK-BR-3 (purchased from the Cell Bank of Chinese Academy of Sciences) suspension to PBMCs in proportion to make PBMCs and SK- The ratio of BR-3 is 10 respectively 3 :1, 10 4 :1, 10 5 :1, 10 6 :1. Then, the immunomagnetic beads were sequentially added to the above-mentioned mixed cell suspensions, and incubated at 4° C. for 30 minutes. Magnetic sorting was performed within 1 minute and washed 2-3 times with PBS to obtain SK-BR-3 cells captured and recovered with immunomagnetic beads. And the magnetic separation was completed within 1 minute, indicating that the magnetic responsiveness of the immunomagnetic beads is good.
[0044] The experiment at each concentration was repeated, and the results showed that SK-BR-3 cells could be detected at al...
Embodiment 3
[0045] Example 3 Capture of tumor cells in simulated blood
[0046] Collect blood samples from healthy volunteers, mix SK-BR-3 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of SK-BR-3 cells to 1, 10, 20, 50, 500, 1000 / mL, and then the immunomagnetic beads were sequentially added to each of the above mixed cell suspensions, and incubated at 4°C for 30 minutes. Magnetic sorting was performed within 1 minute and washed 2-3 times with PBS to obtain SK-BR-3 cells captured and recovered with immunomagnetic beads. Counting the recovered SK-BR-3 cells, SK-BR-3 cells can still be captured when the concentration of SK-BR-3 cells is 1 / mL.
[0047] From the capture results of Examples 2-3, the capture efficiency of immunomagnetic beads in a simple environment (Example 2) can be accurate to 1:10 6 , Immunomagnetic beads can also detect 1 / mL SK-BR-3 cells in a complex environment (Example 3), which can meet the current requirement...
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