A kind of cd20 antibody immunomagnetic beads and preparation method thereof

A technology of immunomagnetic beads and CD20, which is applied in the preparation of microspheres, chemical instruments and methods, blood/immune system cells, etc., can solve the problems of inability to achieve cell sorting, small magnetic responsiveness, and oppressive cells, so as to avoid B Effects of cell damage, rapid magnetic response, and maintenance of biological activity

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 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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  • A kind of cd20 antibody immunomagnetic beads and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1CD20

[0032] Example 1 Preparation of CD20 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 10mg magnetic nanocluster Fe3 o 4 1 solution, add 125 mg ammonia water, 30 mg tetraethyl orthosilicate and 30 mg (3-aminopropyl) triethoxysilane, react for...

Embodiment 2

[0042] Example 2 Sensitivity Detection of CD20 Antibody Immunomagnetic Beads

[0043] Cultivate Jurkat cells (human T lymphocyte leukemia cells, sourced from the Cell Bank of the Chinese Academy of Sciences), and then add the suspension of B lymphocyte NK-92 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) of human malignant non-Hodgkin's lymphoma patients in proportion To Jurkat, make the ratio of Jurkat to NK-92 10 each 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 NK-92 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 resul...

Embodiment 3

[0046] Example 3 CD20 Antibody Immunomagnetic Beads Forward Screening and Identification Capture

[0047] The peripheral blood of healthy people is taken, and the tumor cells in the blood are captured. Subjects are required to have blood routine white blood cell values ​​at 2×10 6 ~1.2×10 7 There was no hemolysis or clot coagulation in the whole blood sample between samples / mL. And the relevant information of the subject is complete, the sample collection and storage methods are standardized, and the experimental operation is standardized. The specific steps are as follows: take 3ml of blood from different healthy people, add red blood cell lysate respectively, and obtain white blood cell samples after processing according to the instructions. Add CD20 antibody immunomagnetic beads to each of the above mixed cell suspensions in sequence, incubate at 4°C for 30 minutes, then perform magnetic sorting within 1 minute and wash with PBS 2-3 times. The cells positively and negati...

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Abstract

The invention provides a CD20 antibody immunomagnetic bead which comprises a magnetic microsphere part and an antibody part. The structural formula of the immunomagnetic bead is A-NH-N=CH-B, wherein A represents a magnetic microsphere, B represents a CD20 antibody, or A represents the CD20 antibody, and B represents the magnetic microsphere. The immunomagnetic bead is used for capturing cancer cells, and is good in specificity and sensibility, fast in magnetic response, short in enrichment time and high in capturing efficiency. The immunomagnetic bead is stable in nature, meanwhile small in particle size, good in magnetic responsiveness and good in dispersibility. A preparation method is simple, and high practicality is achieved.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to CD20 antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] CD20 molecule is a transmembrane protein on B lymphocytes. It is composed of 297 amino acids and belongs to non-glycosylated phosphoprotein. It is a differentiation antigen on the surface of B lymphocytes. Important role, often used as a marker to identify B cells. CD20 is a specific marker molecule on the surface of B lymphocytes, which is highly expressed on the surface of most B lymphocytes, expressed in more than 95% of normal or malignant B lymphocytes, but not expressed in hematopoietic stem cells, plasma cells and other normal tissues , and does not undergo obvious internalization and shedding, it is an ideal bioimmune marker for B lymphocytes. [0003] Immunomagnetic separation (IMS) is a method for cell sorting that has emerged in recent years. It coats ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/14C07K17/08C12N5/0781B01J13/02H01F1/11
CPCB01J13/02C07K17/08C07K17/14C12N5/0635H01F1/112
Inventor 李静蔡红东陈昌岳刘关张培培张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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