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Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection

A technology of peripheral blood circulation and tumor cells, which is applied in the field of electrochemiluminescence detection of peripheral blood circulating tumor cell antigens and kits, can solve the problems that ordinary patients cannot afford routine detection, the detection price is high, and the dynamic monitoring cannot be performed, so as to achieve harmful effects. The effect of small size, improved sensitivity, and improved detection range

Inactive Publication Date: 2015-04-29
ZHEJIANG CANCER HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the price of the CellSearch detector on the market is about 4 million yuan, excluding the detection reagents. General medical institutions cannot introduce this detection technology. Once introduced, the price of its detection is also expensive.
Therefore, ordinary patients cannot afford it as a routine test, let alone as a dynamic monitoring during treatment

Method used

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  • Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection
  • Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of EpCAM antibody-labeled magnetic beads

[0027] Mix the streptavidin magnetic beads thoroughly with a Vortex stirrer, then use a microtube to take 100 μL of a uniform particle solution (equivalent to 1 mg of particles); add 2 μg of biotin-labeled EpCAM antibody (antibody can be derived from human, mouse , rabbits, goats and other currently known biological sources or synthetic antibodies, this example uses a solution of mouse anti) and mixes; incubate on a rotating shaker at room temperature for 30 minutes; place the microtube on the magnetic base for about 1 minute, and Remove the supernatant; add 100 μL of washing buffer, wash twice, then place the microtube on the magnetic base for about 1 min, and remove the supernatant; add preservation buffer, mix well, and store at 4°C. The specification of the magnetic beads is that the particle diameter is between 0.5-5um.

Embodiment 2

[0028] Example 2 Preparation of magnetic beads labeled with anti-EpCAM antibody (secondary antibody)

[0029] Mix the streptavidin magnetic beads thoroughly with a Vortex stirrer, then use a microtube to take 100 μL of a uniform particle solution (equivalent to 1 mg of particles); add a solution containing 2 μg of biotin-labeled secondary antibody, and mix well; Incubate on a rotary shaker for 30 min; place the microtube on the magnetic base for about 1 min, and remove the supernatant; add 100 μL of washing buffer and wash 3 times; then add EpCAM antibody solution and mix well; rotate and shake at room temperature Incubate on the bed for 30min; place the microtube on the magnetic base for about 1min, and remove the supernatant; add 100μL of washing buffer, wash twice; then place the microtube on the magnetic base for about 1min, and remove the supernatant ; Add storage buffer, mix well, and store at 4°C.

Embodiment 3

[0030] Example 3 Evaluation of antibody-labeled magnetic beads for CTC enrichment ability one

[0031] Put 101 BT474 Her2-positive breast cancer cells into the total system of 200uL, add 2uL EpCAM antibody magnetic beads (you can choose the immunomagnetic beads in Example 1 and Example 2, and use the immunomagnetic beads in Example 1 here , the same below), mix well, and incubate the microtube evenly by inverting for 20min at room temperature; place the microtube on the magnetic base for about 1min, and remove the supernatant; add 200uL of washing buffer, wash 3 times, the microscope counted 61, and the enrichment efficiency was 60.0%.

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Abstract

The invention discloses a method and a kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection. The method comprises the steps of transferring a blood sample into a centrifuge tube by use of an enriched buffer solution, metering to realize constant volume, and mixing uniformly; centrifuging, discarding supernatant, splitting red cells and reselecting the cells; adding an enriched buffer solution and re-suspending the cells; centrifuging, discarding supernatant, adding the enriched buffer solution and EpCAM antibody magnetic beads, mixing uniformly and incubating; placing the centrifuge tube on a magnetic base in a standing manner and removing liquid supernatant; adding a cleaning buffer solution and washing to obtain a magnetic microsphere system; adding a Ru(bpy)2(dcbpy)NHS-tumor cell antibody into the magnetic microsphere system, mixing uniformly and incubating; placing the centrifugal tube on the magnetic base in the standing manner and removing liquid supernatant; adding the cleaning buffer solution and washing; and placing in an electrochemical luminescence detector and measuring the content of the tumor cell surface antigens. The method can be used for remarkably enlarging the detection range and improving the detection sensitivity of the circulating tumor cell antigens in the peripheral blood, and is rapid, accurate and slight in harm.

Description

technical field [0001] The invention relates to an electrochemiluminescent immunodetection method and a kit for circulating tumor cell antigens in peripheral blood. Background technique [0002] Globally, breast cancer is the most common malignant tumor in women, and about 20-30% of all cases are "the most dangerous breast cancer" - HER2-positive breast cancer. Compared with other types of breast cancer, HER2-positive breast cancer has faster disease progression, higher malignancy, easier recurrence and metastasis, and poorer prognosis. [0003] The incidence of breast cancer in China is on the rise year by year, especially in Beijing, Tianjin, Shanghai and other big cities, breast cancer has ranked first in the incidence of female malignant tumors. According to the biological characteristics of breast cancer, it can be divided into 4-5 molecular subgroups, and different subgroups have different prognosis and treatment strategies. Modern medicine has entered the era of cla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 王晓稼沈晓亮杨祖立张剑琦陈占红邵喜英陈欢
Owner ZHEJIANG CANCER HOSPITAL
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