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HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic bead and preparation method thereof

A technology of immune magnetic beads and antibodies, which is applied in the direction of microsphere preparation, chemical instruments and methods, magnetic materials, etc., can solve the problems of high cost and achieve the effects of low cost, mild reaction conditions and good magnetic responsiveness

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

However, this method does not target epithelial tumor cells, and uses magnetic beads produced by Miltenyi and Kisker, and requires a matching sorter, which is too expensive

Method used

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  • HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic bead and preparation method thereof
  • HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic bead and preparation method thereof
  • HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic bead 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 non-small cell lung cancer cells A549, human breast adenocarcinoma cells SK-BR-3 and human breast adenocarcinoma cells MDA-MB-436 (all purchased from the Cell Bank of the Chinese Academy of Sciences) were taken, and the tumor cells in each group were 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 of immunomagnetic beads was calculated. The results are shown in Table 1:

[0046] Table 1 Capture ra...

Embodiment 3

[0049] Example 3 Capture of Tumor Cells in Simulated Blood

[0050] Collect blood samples from healthy volunteers, mix A549 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of A549 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 A549 cells captured and recovered with immunomagnetic beads. The ratio of recovered A549 cells to the total number of cells before capture was counted, and the capture efficiency of A549 cells in blood samples captured by immunomagnetic beads was calculated.

[0051] 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 d...

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Abstract

The invention provides a HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic bead and a preparation method thereof. The multiple antibody immunomagnetic bead includes a HER2 immunomagnetic bead, an EGFR immunomagnetic bead and an EpCAM immunomagnetic bead, and a HER2 antibody, an EGFR antibody and an EpCAM antibody are respectively coupled with magnetic microspheres to obtain the HER2 immunomagnetic bead, the EGFR immunomagnetic bead and the EpCAM immunomagnetic bead. The multiple antibody immunomagnetic bead is good in specificity and sensitivity, rapid in magnetic response, short in concentration time and high in capturing efficiency when being used for capturing CTCs (circulating tumor cells). The immunomagnetic bead is stable in nature, small in particle size, good in magnetic response and dispersibility, simple in preparation method and high in practicability.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to a HER2, EGFR, EpCAM, MUC1 multiple antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] Epithelial tissue includes covering epithelium, glandular epithelium and ductal epithelium. The tumors that occur are called epithelial tumors. Most of human malignant tumors originate from epithelial tissue. Although my country has made significant progress in the early diagnosis and treatment of tumors, recurrence and metastasis are still the main reasons affecting the 5-year survival rate of patients. Therefore, further analysis of tumor cells is necessary to understand the mechanisms of their occurrence, development and metastasis. [0003] Immunomagnetic separation (IMS) is a method for cell sorting that has emerged in recent years. It coats the surface of magnetic beads with immunoreactive antibodies for antigen-antibody reactio...

Claims

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Application Information

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IPC IPC(8): C07K17/14C07K17/08C12N5/09B01J13/02H01F1/11
CPCB01J13/02C07K17/08C07K17/14C12N5/0693H01F1/112
Inventor 陈昌岳蔡红东张祥林李静刘关
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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