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

Active Publication Date: 2017-01-04
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the particle size of the immunomagnetic beads should not be too large, which will oppress the cells; the particle size of the immunomagnetic beads should not be too small, because the magnetic response will be small if the particle size is too small, and the effect of cell sorting will not be achieved easily.

Method used

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  • HER2 (Human Epidermal Growth Factor Receptor2) antibody immunomagnetic bead and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides HER2 antibody immunomagnetic bead and a preparation method of the HER2 antibody immunomagnetic bead. The HER2 antibody immunomagnetic bead comprises a magnetic microsphere part and an antibody part, wherein the structural formula of the immunomagnetic bead is A-NH-N=CH-B, A refers to a magnetic microsphere, B refers to an HER2 antibody, or A refers to the HER2 antibody and B refers to the magnetic microsphere. The immunomagnetic bead disclosed by the invention is not only good in specificity and sensitivity, but also rapid in magnetic response, short in enrichment time and high in capture efficiency while used for capturing tumor cells; furthermore, the immunomagnetic bead is stable in properties, and further small in particle size, good in magnetic response and excellent in dispersion. Moreover, the immunomagnetic bead has a simple preparation method and very strong practicability.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to a HER2 antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] Human epidermal growth factor receptor 2 (human epidermal growth factor receptor2, HER2) is a transmembrane glycoprotein with tyrosine protein kinase activity and belongs to one of the EGFR family members. HER2 can promote cell division and the secretion of proteolytic enzymes, increase DNA synthesis, accelerate the growth of cancer cells, and enhance the ability of cells to move, thereby promoting tumor invasion and metastasis, and plays an important role in cell division, proliferation and differentiation. Overexpression of HER2 exists in more than 30% of human tumors (such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer and prostate cancer, etc.), of which 20% to 30% of primary invasive breast cancer have Overexpres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/14C07K17/08C12N5/09B01J13/02H01F1/11
CPCB01J13/02C07K17/08C07K17/14C12N5/0693H01F1/112
Inventor 蔡红东陈昌岳张祥林马超刘关
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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