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Antibody-coupled bionic immune magnetic sphere and preparation method thereof

A bionic immune and antibody technology, applied in the field of nanomaterials, can solve problems such as unfavorable CTC analysis and non-specific adsorption of white blood cells, and achieve the effects of good cell activity, high recognition efficiency, and wide source of raw materials

Inactive Publication Date: 2017-02-15
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the magnetic enrichment technology also has the problem of non-specific adsorption of white blood cells
E.g: The system is the only technology approved by the Food and Drug Administration (FDA) for the detection of CTCs in metastatic breast cancer, colorectal cancer or prostate cancer. It targets EpCAM (epithelial cell adhesion molecule) on the surface of CTCs by ferrofluid binding specific antibodies antigen, and then separate CTCs under the action of a specific magnetic field, but The system has obvious non-specific adsorption of white blood cells (1000~3000 / 7.5mL), which is not conducive to downstream CTC analysis

Method used

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  • Antibody-coupled bionic immune magnetic sphere and preparation method thereof
  • Antibody-coupled bionic immune magnetic sphere and preparation method thereof
  • Antibody-coupled bionic immune magnetic sphere and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0089] The preparation of embodiment 1MNC

[0090] (1) Put DEG in the environment of Ar gas, after the oxygen is removed completely, dissolve the solid NaOH into the DEG, heat at the same time, raise the temperature to 120°C, stop heating until the solid NaOH is completely dissolved, the whole preparation process uses Protected by Ar gas, a light yellow solution was prepared, which was a NaOH storage solution with a concentration of 1g / mL, and kept at 70°C for future use;

[0091] (2) FeSO 4 ·7H 2 O. PEI and DEG are mixed evenly, then vacuumed, and then filled with Ar gas protection; heat up to 160 ° C, add the NaOH storage solution obtained in step 1 (1), until the molar concentration of NaOH in the solution is 0.03mol / L, about 3min Afterwards, the color of the solution turned black, and then the temperature was raised to 220° C., continued to condense and reflux for 1 hour, and the reaction was completed, and a reaction solution containing positively charged MNC was obtain...

Embodiment 2

[0103] Preparation of Example 2M

[0104] Place the leukocyte J774A.1 cell line at 37°C, CO 2 In a constant temperature incubator with a concentration of 5%, culture with complete cell culture medium for 24 hours, respectively replace it with complete cell culture medium containing 0.1 mM azidecholine for 24 hours, collect white blood cells, and discard the medium.

[0105] Resuspend the collected leukocytes with Hepes B buffer at pH 7.6, add 10 μL / mL of protease inhibitors, break the cells intermittently with a homogenizer at gear 2, break for 2 minutes, stop for 1 minute, repeat 10 times, and then centrifuge at 1000 r / min 3min, collect the supernatant, resuspend the pellet with Hepes B buffer added with 10μL / mL protease inhibitor, break the cells with a homogenizer, break for 2min, stop for 1min, repeat 10 times, then centrifuge at 1000r / min for 3min, collect the supernatant Supernatant and supernatant were also suspended in Hepes B buffer containing protease inhibitors, an...

Embodiment 3

[0116] Preparation of Example 3M

[0117] Place the leukocyte J774A.1 cell line at 37°C, CO 2 In a constant temperature incubator with a concentration of 5%, culture with complete cell culture medium for 20 hours, replace it with cell culture complete medium containing 0.1 mM azidecholine and continue to cultivate for 22 hours, collect white blood cells, discard the medium, and prepare the rest The method and control experiment are the same as in Example 2.

[0118] The prepared M-solution and M-solution were tested and characterized, and the results were similar to those in Example 2. Preparation of Example 4M

[0119] Place the leukocyte J774A.1 cell line at 37°C, CO 2 In a constant temperature incubator with a concentration of 5%, culture with complete cell culture medium for 28 hours, replace it with cell culture complete medium containing 0.1 mM azidecholine and continue to cultivate for 28 hours, collect white blood cells, discard the medium, and prepare the rest The...

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Abstract

The invention relates to an antibody-coupled bionic immune magnetic sphere and a preparation method of the antibody-coupled bionic immune magnetic sphere, belonging to the technical field of nanometer materials. According to the bionic immune magnetic sphere, the water-soluble Fe3O4 nanocluster is taken as the core, a cell membrane coats the core, and an antibody with a modification group is bonded outside the cell membrane through covalent coupling. The preparation method of the antibody-coupled bionic immune magnetic sphere comprises the following steps: preparing the Fe3O4 nanocluster, namely, MNC; preparing cell membrane fragments modified by choline azide, namely, M; reacting MCN and M to obtain a bionic magnetic sphere, and reacting the modification group with the antibody to obtain the antibody with the cyclo-ethynylation modification group; and coupling bionic magnetic sphere solution and the antibody with the cyclo-ethynylation modification group, thus obtaining the bionic immune magnetic sphere provided by the invention. The bionic immune magnetic sphere is good in dispersibility, rapid in magnetic response, and high in identifying efficiency when used for capturing tumor cells, no white blood cells exist in the background, and the captured tumor cells are good in activity. For the preparation method, the raw materials are easily available and have universality in the construction of the bionic immune magnetic sphere.

Description

technical field [0001] The invention relates to a bionic immune magnetic sphere coupled with an antibody and a preparation method thereof, belonging to the technical field of nanomaterials. Background technique [0002] According to the "World Cancer Report" released by the World Health Organization in 2014, 14 million people were diagnosed with cancer in 2012, and it is predicted that this number will increase to 24 million by 2035. Cancer has become a serious threat to human life and health, and more than 90% of patients die from tumor metastasis. Circulating tumor cells (CTCs) play a very important role in the process of tumor metastasis. These extremely low-abundance cancer cells shed from the primary tumor and enter the peripheral blood circulation. Detection of CTCs can provide reference for tumor metastasis, tumor prognosis, and curative effect monitoring. However, it is very challenging to isolate and detect CTCs, and the probability of CTCs in blood is about 1 CTC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693
Inventor 谢海燕熊珂王书敏魏炜赵东旭
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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