A kind of her2, egfr, epcam, muc1 multiple antibody immunomagnetic beads and preparation method thereof
A technology of immunomagnetic beads and antibodies, which is applied to the preparation of microspheres, chemical instruments and methods, magnetic objects, etc., can solve the problems of high cost, achieve the effect of low cost, simple preparation method, and maintain activity
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Embodiment 1
[0033] Example 1 Preparation of Multiplex Antibody Immunomagnetic Beads
[0034] (1) Preparation of magnetic nanoclusters:
[0035] 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 45 minutes, the color gradually turns light green, then dark green, and finally black;
[0036] 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 .
[0037] (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, rea...
Embodiment 2
[0044] Example 2 Sensitivity Detection of Multiple Antibody Immunomagnetic Beads
[0045] Human non-small cell lung cancer cells A549, human breast adenocarcinoma cells SK-BR-3 and human breast adenocarcinoma cells MDA-MB-436 (all purchased from the Cell Bank of the Chinese Academy of Sciences) were taken, and the tumor cells in each group were divided into 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 The ratio was added to PBMCs (5×10 6 )middle. Individual antibody immunomagnetic beads and multiple antibody immunomagnetic beads were added to the above-mentioned mixed cell suspensions, and incubated at 4°C for 30 minutes. Magnetic sorting was performed within 2 minutes and washed 2-3 times with PBS to obtain tumor cells captured and recovered with immunomagnetic beads. The proportion of recovered tumor cells to the total number of added tumor cells was counted, and the capture rate of immunomagnetic beads was calculated. The results are shown in Table 1:
[0046] Table 1 Capture ra...
Embodiment 3
[0049] Example 3 Capture of Tumor Cells in Simulated Blood
[0050] Collect blood samples from healthy volunteers, mix A549 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of A549 cells to 1, 10, 20, 50, 500, 1000 cells / mL, and then immunize with multiple antibodies The magnetic 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 A549 cells captured and recovered with immunomagnetic beads. The ratio of recovered A549 cells to the total number of cells before capture was counted, and the capture efficiency of A549 cells in blood samples captured by immunomagnetic beads was calculated.
[0051] 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 d...
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