Immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood

A technology of magnetic microspheres and tumor cells, which is applied in the direction of tumor/cancer cells, animal cells, vertebrate cells, etc., can solve the problems of unsatisfactory capture purity, capture efficiency needs to be further improved, and high price, so as to achieve cost-effective, fast, The effect of low cost and simple operation

Active Publication Date: 2014-07-09
BRILLIANCE BIO TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the extremely rare number of circulating tumor cells in peripheral blood, the capture efficiency and purity have always been important factors that limit the research on circulating tumor cells. At present, most methods cannot achieve high efficiency and high purity capture and detection at the same time.
The CellSearch system is currently the only method approved by the US Food and Drug Administration (FDA) to detect circulating tumor cells. Although it can capture and detect quickly and effectively, the capture efficiency needs to be further improved, and it is expensive
Although the most recently researched methods based on membrane or chip filtration have greatly improved the capture efficiency, the capture purity is still not ideal

Method used

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  • Immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood
  • Immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood

Examples

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Embodiment 1

[0034] Synthesis of embodiment 1 immunomagnetic microspheres

[0035] The magnetic microspheres used in this example are carboxyl magnetic microspheres, with a particle size of 3-4 μm, 5 mg / ml, and a surface carboxyl content of 100 nmol / g, purchased from Tianjin Beisile Chromatography Technology Development Center.

[0036] Proceed as follows:

[0037] (1) Activation of carboxyl magnetic microspheres: take 200 μL of carboxy magnetic microspheres, ultrasonicate for 30 s, magnetically separate, and wash once with deionized water; add 250 μL of EDC / NHS solution (in the EDC / NHS solution, the concentrations of EDC and NHS are 3.2×10 3 μg / mL), activated at room temperature for 30 minutes, magnetically separated, washed with PBS buffer, repeated 5 times to obtain activated magnetic microspheres;

[0038] (2) Coupling of carboxyl magnetic microspheres with anti-EpCAM antibody: add 150 μL of 10 μg·mL to the above-mentioned activated magnetic microspheres -1 Anti-EpCAM antibody was r...

Embodiment 2

[0039] The using method of embodiment 2 immunomagnetic microspheres

[0040] Proceed as follows:

[0041] (1) Cell incubation with immunomagnetic microspheres: Take 1 mL of cultured MCF-7 cells, add 50 μL of immunomagnetic microspheres (prepared in Example 1), and incubate at room temperature for 1 h.

[0042] (2) Filtration

[0043] (1) Synthesis and production of PDMS sheets:

[0044] ① Take 50g: 5g two-component silicone elastomer (silicone rubber, basic component: curing agent = 10:1) and mix it in a disposable water cup for 10 minutes;

[0045] ② Put it in a vacuum device for vacuuming, turn off the vacuum pump when the air bubbles are about 5mm away from the top of the paper cup, adjust it to be in a vacuum state and keep it still for about 30-40min until the air bubbles in the cup disappear and the liquid surface becomes clear;

[0046] ③ After the bubbles gradually disappear and the liquid surface becomes clear, deflate, take out the mixture, and add it to the contain...

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Abstract

The invention discloses an immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood. The immunomagnetic microsphere comprises a magnetic microsphere, wherein an epithelial cell specific antibody used for specifically recognizing and capturing circulating tumor cells is coupled on the magnetic microsphere. The epithelial cell specific antibody is selected according to a target tumor cell needed to be captured, for example, when the MCF (michigan cancer foundation)-7 cell is needed to be captured, the epithelial cell specific antibody selects an anti-EpCAM (epithelial cell adhesion molecule) antibody. The invention further discloses a method for capturing circulating tumor cells in peripheral blood by utilizing the immunomagnetic microsphere. The immunomagnetic microsphere disclosed by the invention is modified by the epithelial cell specific antibody, so that high-selectivity and high-specificity capturing for the target cell can be realized. The magnetic microsphere can achieve dimension amplifying effect, is used for realizing film separation with high recovery, and further can be used for magnetic separation operation to realize high-purity capturing. The immunomagnetic microsphere and the capturing method thereof can be used for early diagnosis of a cancer patient and prediction of therapeutic response.

Description

technical field [0001] The invention relates to an immunomagnetic microsphere for capturing circulating tumor cells in peripheral blood, a preparation method and a use method thereof. Background technique [0002] The detection of circulating tumor cells helps to study the mechanism of tumor metastasis, guide tumor treatment, evaluate curative effect and prognosis, and monitor tumor metastasis or recurrence. At present, there are many methods for detecting circulating tumor cells, mainly including enrichment and separation methods, immunocytochemical techniques, flow cytometry, reverse transcription polymerase chain reaction and CellSearch detection system, etc. The enrichment and separation methods mainly include density gradient centrifugation method, membrane filtration method, immunomagnetic bead enrichment method, etc. Due to the extremely rare number of circulating tumor cells in peripheral blood, the capture efficiency and purity have always been important factors li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N13/00
Inventor 张鸿雁王言红唐波
Owner BRILLIANCE BIO TECH CO LTD
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