A kind of cd133, cd24, cd44 multiple antibody immunomagnetic beads and preparation method thereof

A technology of immunomagnetic beads and CD24, which is applied in the preparation of microspheres, chemical instruments and methods, magnetic objects, etc., can solve the problems of inability to achieve the effect of tumor cell sorting, small magnetic responsiveness, and compression of tumor cells, and achieve antibody Large coating amount, rapid magnetic response, and high capture efficiency

Active Publication Date: 2021-03-30
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 tumor 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 it is easy to fail to achieve the effect of tumor cell sorting

Method used

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  • A kind of cd133, cd24, cd44 multiple antibody immunomagnetic beads and preparation method thereof
  • A kind of cd133, cd24, cd44 multiple antibody immunomagnetic beads and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

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 colon cancer HCT116 cells (expressing CD133 / CD44 antigen on the surface), human non-small cell lung cancer A549 cells (expressing CD44 / CD24 antigen on the surface) and human breast adenocarcinoma SK-BR-3 cells (expressing CD24 antigen on the surface) (all Purchased from the Cell Bank of the Chinese Academy of Sciences), each group of tumor cells was 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 ...

Embodiment 3

[0050] Example 3 Capture of CSC cells in simulated blood

[0051] Collect blood samples from healthy volunteers, mix HCT116 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of HCT116 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 HCT116 cells captured and recovered with immunomagnetic beads. The ratio of recovered HCT116 cells to the total number of cells before capture was counted, and the capture efficiency of HCT116 cells in the blood sample captured by immunomagnetic beads was calculated, which was similar to the result in Example 2.

[0052] From the capture results of Examples 2-3, the capture efficiency of multiple antibody immunomagnetic beads in a simple environme...

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Abstract

The invention provides CD133, CD24 and CD44 multiple antibody immunomagnetic beads. The multiple antibody immunomagnetic beads are composed of CD133 immunomagnetic beads, CD24 immunomagnetic beads and CD44 immunomagnetic beads, wherein the CD133 immunomagnetic beads are obtained by coupling CD133 antibodies and magnetic microspheres, the CD24 immunomagnetic beads are obtained by coupling CD24 antibodies and magnetic microspheres, and the CD44 immunomagnetic beads are obtained by coupling CD44 antibodies and magnetic microspheres. The multiple antibody immunomagnetic beads are used for capturing cancer stem cells and are good in specificity and sensitivity, rapid in magnetic response, short in enrichment time and high in capture efficiency. Moreover, the immunomagnetic beads are stable in property, small in particle size, good in magnetic responsiveness and dispersity and simple in preparation method and have high practicability.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to a CD133, CD24, CD44 multiple antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] Cancer stem cells (CSC), also known as cancer stem cells and cancer stem cells, refer to tumor cells with stem cell self-replication and multicellular differentiation properties, which can be used as a marker for judging tumor cell invasion. These cells are generally considered to have the potential to form tumors and develop into cancer, especially as the cancer metastasizes out, giving rise to a source of new cancers. The self-renewal property of cancer stem cells is the main cause of tumor recurrence, metastasis and poor prognosis. Complex mechanisms within cancer stem cells explain cancer drug resistance as well as many typical tumor behaviors. The cellular hypothesis of carcinogenesis states that a small subpopulation of tumor cells h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/14C12N5/095B01J13/02H01F1/11
CPCB01J13/02C07K17/14C12N5/0695H01F1/112
Inventor 刘关陈昌岳蔡红东李静张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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