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PD-L1 antibody immunomagnetic beads and preparation method thereof

A technology of immunomagnetic beads and PD-L1, which is applied in biochemical equipment and methods, microsphere preparation, cell dissociation methods, etc., can solve the problems of small magnetic response, oppression of tumor cells, and inability to achieve the effect of tumor cell sorting and other problems, to achieve the effect of rapid magnetic response, high capture efficiency and low cost

Active Publication Date: 2017-02-01
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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  • PD-L1 antibody immunomagnetic beads and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1 Preparation of PD-L1 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 20 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 1.

[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, react ...

Embodiment 2

[0043] Example 2 Sensitivity detection of PD-L1 antibody immunomagnetic beads

[0044] Blood samples were collected from healthy volunteers, and PBMCs were extracted through human lymphocyte separation fluid, and then the suspension of human non-small cell lung cancer cells H1299 (purchased from the Cell Bank of the Chinese Academy of Sciences) was added to the PBMCs in proportion, so that the ratios of PBMCs and H1299 were respectively 10 3 :1, 10 4 :1, 10 5 :1, 10 6 :1. Then the immunomagnetic beads of Example 1 were sequentially added to 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 H1299 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.

[0045] The experiment at each concentration was repeated, and the results ...

Embodiment 3

[0046] Example 3 Capture of Tumor Cells in Simulated Blood

[0047] Collect blood samples from healthy volunteers, mix H1299 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of H1299 cells to 1, 10, 20, 50, 500, 1000 cells / mL, and then immunomagnetic beads Add it to the above-mentioned mixed cell suspensions in turn, and incubate at 4°C for 30 minutes. Magnetic sorting was performed within 1 minute and washed 2-3 times with PBS to obtain H1299 cells captured and recovered with immunomagnetic beads. According to statistics of recovered H1299 cells, H1299 cells can still be captured when the concentration of H1299 cells is 1 cell / mL.

[0048] 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 H1299 cells in a complex environment (Example 3), and the sensitivity can meet the current...

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Abstract

The invention provides PD-L1 antibody immunomagnetic beads and a preparation method thereof. Each PD-L1 antibody immunomagnetic bead comprises a magnetic microsphere part and an antibody part; the immunomagnetic beads respectively have a structural formula of A-NH-N=CH-B, wherein A represents a magnetic microsphere and B represents a PD-L1 antibody, or A represents a PD-L1 antibody and B represents a magnetic microsphere. When being used for capturing tumor cells, the immunomagnetic beads are not only good in specificity and sensitivity, but also rapid in magnetic response, short in enrichment time and high in capture efficiency. Furthermore, the immunomagnetic beads are stable in properties, small in particle size, good in magnetic responsiveness and good in dispersity. The preparation method is simple and has very high practicality.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to a PD-L1 antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] PD-1 is a 55KD transmembrane protein, which belongs to the immunoglobulin superfamily with CD28, ICOS and cytotoxic T lymphocyte (CTL)-associated antigen 4 (CTLA-4). Unlike the limited expression of CD28 and CTLA-4 (mainly in T cells), PD-1 can be expressed in activated T cells, B cells and myeloid cells, as well as CD4-CD8-thymocytes. PD-L1 (B7-H1) is the ligand of PD-1, and PD-L1 mRNA is abundant in non-lymphoid tissues (such as placenta, heart, lung, and skeletal muscle), but except for macrophage-like cells and placental trophoblast, PD-L1 protein was barely detectable in normal tissues. PD-L1 expression is induced on APCs, T cells, and endothelial cells, and is enriched in a variety of human tumors. In recent years, it has been found that PD-L1 is highly...

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
CPCB01J13/02C07K17/08C07K17/14C12N5/0693C12N2509/00H01F1/112
Inventor 马超陈昌岳蔡红东刘关张培培张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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