E-cadherin, Cadherin-11 and EpCAM multi-antibody immunomagnetic bead and preparation method thereof

An immunomagnetic bead and antibody technology, applied in the preparation of microspheres, chemical instruments and methods, magnetic materials, etc., can solve the problems of high cost and slow magnetic response, and achieve the effect of low cost, rapid magnetic response and good sensitivity

Active Publication Date: 2017-02-22
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the MACS system uses nano-immune magnetic beads with a particle size of 50nm, but the magnetic response is slow, and a strong magnetic field must be used with a dedicated disposable magnetic separation column to complete the capture of CTCs, which is expensive

Method used

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  • E-cadherin, Cadherin-11 and EpCAM multi-antibody immunomagnetic bead and preparation method thereof
  • E-cadherin, Cadherin-11 and EpCAM multi-antibody immunomagnetic bead and preparation method thereof
  • E-cadherin, Cadherin-11 and EpCAM multi-antibody immunomagnetic bead and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Multiplex 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 45 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 .

[0036] (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

[0043] Example 2 Sensitivity Detection of Multiple Antibody Immunomagnetic Beads

[0044] Human breast cancer cells MCF-7, human prostate cancer cells PC-3, and human epidermal cancer cells A-431 (all purchased from the Cell Bank of the Chinese Academy of Sciences) were taken, and the tumor cells in each group were divided into groups at a ratio of 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:

[0045]

[0046] Table 1 Captur...

Embodiment 3

[0048] Example 3 Capture of Tumor Cells in Simulated Blood

[0049] Collect blood samples from healthy volunteers, mix PC-3 cells with peripheral blood of healthy people to make a mixed cell suspension, adjust the concentration of PC-3 cells to 1, 10, 20, 50, 500, 1000 cells / mL, and then Add the multiple antibody immunomagnetic beads to each of the above 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 PC-3 cells captured and recovered with immunomagnetic beads. The ratio of recovered PC-3 cells to the total number of cells before capture was counted, and the capture efficiency of PC-3 cells in the blood sample captured by immunomagnetic beads was calculated, which was similar to the result in Example 2.

[0050] From the capture results of Examples 2-3, the capture efficiency of multiple antibody immunomagnetic beads in a simple environment (Example 2) can be accurate to...

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Abstract

The invention provides an E-cadherin, Cadherin-11 and EpCAM multi-antibody immunomagnetic bead and a preparation method thereof. The multi-antibody immunomagnetic bead comprises E-cadherin immunomagnetic beads, Cadherin-11 immunomagnetic beads and EpCAM immunomagnetic beads, wherein the E-cadherin immunomagnetic beads, the Cadherin-11 immunomagnetic beads and the EpCAM immunomagnetic beads are immunomagnetic beads respectively obtained by coupling an E-cadherin antibody, a Cadherin-11 antibody and an EpCAM antibody with magnetic microspheres. The multi-antibody immunomagnetic bead is used for capturing CTC and has the effects that the specificity and the sensibility are good, the magnetic response is rapid, the enrichment time is short, and the capturing efficiency is high; and furthermore, the performance is stable, the particle size is small, and the magnetic responsiveness and the dispersity are good. Furthermore, the preparation method is simple and has very strong practicability.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to an E-cadherin, Cadherin-11, EpCAM multiple antibody immunomagnetic beads and a preparation method thereof. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells released into the peripheral blood circulation from the primary tumor or metastases of solid tumors spontaneously or due to diagnostic and therapeutic procedures, usually in the form of single cells or cell clusters (also known as circulating tumor microemboli, CTM). present in the circulatory system. Metastasis is the main cause of cancer-related death, and CTCs are regarded as the seeds of metastasis. In order to acquire motility and invasiveness, CTC will lose certain epithelial cell phenotypes (including morphology, surface antigens, gene expression, etc.) , Epithelial-Mesenchymal Transition). Most malignant tumor cells undergo EMT during the process of breaking away fr...

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
CPCB01J13/02C07K17/08C07K17/14C12N5/0693H01F1/112
Inventor 蔡红东陈昌岳刘关李静张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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