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Immunomagnetic bead used for capturing leukocyte and preparation method thereof

A technology of immunomagnetic beads and leukocytes, applied in the field of immunomagnetic beads capturing leukocytes and its preparation, to achieve the effects of avoiding leukocyte damage, maintaining biological activity, and small particle size

Inactive Publication Date: 2016-12-07
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the remaining white blood cells including granulocytes (CD15 antigen) and monocytes (CD14 antigen) still exist, and only 0.01% to 1% of CTCs can be obtained from the remaining cells

Method used

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  • Immunomagnetic bead used for capturing leukocyte and preparation method thereof
  • Immunomagnetic bead used for capturing leukocyte and preparation method thereof
  • Immunomagnetic bead used for capturing leukocyte and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of Immunomagnetic Beads for Leukocyte Removal

[0041] (1) Preparation of magnetic nanoclusters:

[0042] 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;

[0043] 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.

[0044] (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...

Embodiment 2

[0052] Example 2 Specific Detection of Immunomagnetic Beads Used to Capture Leukocytes

[0053] Collect blood samples from healthy volunteers, extract peripheral blood mononuclear cells (PBMCs) through human lymphocyte separation medium, and make four groups of PBMCs with a concentration of 1×10 6 After adding 0.5 mg and 5 mg of the CD45 immunomagnetic beads in Example 1 to the two groups of cell suspensions per mL, incubate at 4° C. for 30 minutes, perform magnetic separation and wash within 1 minute. Add 0.5 mg and 5 mg commercially available CD45 immunomagnetic beads to the other two groups, and incubate in the same way.

[0054] Human oral epidermoid carcinoma KB cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were collected and made into a concentration of 1×10 6 / mL of the cell suspension, after adding 5 mg of the immunomagnetic beads used to capture leukocytes in Example 1, incubated at 4° C. for 30 minutes, as a negative control.

[0055] Afte...

Embodiment 3

[0056] Example 3 Specific Detection of Immunomagnetic Beads Used to Capture Different Types of Leukocytes

[0057] Blood samples from healthy volunteers were collected, and peripheral blood mononuclear cells (PBMCs, including lymphocytes and monocytes) were extracted through human lymphocyte separation medium, and the PBMCs were made into a concentration of 1×10 6 / mL cell suspension, after adding the CD45, CD15, CD14 immunomagnetic beads in Example 1, incubate at 4°C for 30 minutes, perform magnetic separation and wash within 1 minute.

[0058] Collect blood samples from healthy volunteers, and extract total white blood cells (including granulocytes, lymphocytes and monocytes, 20-40% of lymphocytes, 50-70% of granulocytes, and 1-7% of monocytes) through red blood cell lysate. , the PBMCs were made into a concentration of 1×10 6 / mL cell suspension, after adding the CD45, CD15, CD14 immunomagnetic beads in Example 1, incubate at 4°C for 30 minutes, perform magnetic separation...

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Abstract

The invention provides an immunomagnetic bead used for capturing a leukocyte and a preparation method thereof. The immunomagnetic bead at least comprises a CD45 immunomagnetic bead and also can comprise or does not comprise one or two of a CD14 immunomagnetic bead and a CD15 immunomagnetic bead, wherein the CD45 immunomagnetic bead, the CD14 immunomagnetic bead and the CD15 immunomagnetic bead are immunomagnetic beads obtained by coupling a CD45 antibody, a CD14 antibody and a CD15 antibody with a magnetic sphere respectively; and a structural formula of the CD45 immunomagnetic bead, the CD14 immunomagnetic bead and the CD15 immunomagnetic bead is A-NH-N=CH-B, wherein A is the magnetic sphere and B is an antibody, or A is the antibody and B is the magnetic sphere. The immunomagnetic bead provided by the invention can be used for capturing the leukocyte, is good in specificity and sensitivity and is rapid in magnetic response, short in enrichment time and high in capturing efficiency; and the preparation method is simple, so that the immunomagnetic bead has strong practicability.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to an immunomagnetic bead for capturing white blood cells and a preparation method thereof. Background technique [0002] Blood itself is a complex environment composed of plasma and blood cells, including red blood cells, white blood cells and platelet cells. The percentages of the total number and types of white blood cells in the human body are relatively stable, and there are (3.5~9.5)×10 white blood cells per milliliter of blood in normal people 6 One, mainly: neutrophils 50-70%, eosinophils 1-4%, basophils 0-1%, lymphocytes 20-40%, monocytes 1-7%. CD45 antigen is a kind of transmembrane protein with large molecular weight, which widely exists on the surface of leukocytes including lymphocytes, granulocytes and monocytes, and can be used as the main immune biomarker of leukocytes. Moreover, the CD15 antigen exists on the surface of granulocytes, and the CD14...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/54326
Inventor 蔡红东陈昌岳刘关马超张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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