Method for enriching specificity of circulating tumor cell

A tumor cell enrichment technology, applied in the direction of tumor/cancer cells, animal cells, vertebrate cells, etc., can solve the problem of inability to meet clinical rapid detection and actual detection, inability to specifically identify tumor cells, insufficient sensitivity of detection, etc. problems, to achieve the effect of convenient and fast enrichment, low cost and easy operation

Inactive Publication Date: 2012-07-18
AMOY DIAGNOSTICS CO LTD
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although the immunomagnetic bead separation method can separate and enrich CTCs better, the cost of this method is high, the operation is more complicated, and it cannot meet the needs of clinical rapid detection and actual detection.
Density gradient centrifugation (such as CN97195305.8, a method for enriching rare cells) is relatively simple to operate, highly operable, and economical and practical. However, multiple centrifugations can cause damage and loss of CTCs, and the sensitivity of detection is relatively insufficient. Specific recognition of tumor cells, the cell recovery rate is only 40%-65%, and the sensitivity of its enrichment is far from the actual needs of clinical detection

Method used

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  • Method for enriching specificity of circulating tumor cell
  • Method for enriching specificity of circulating tumor cell
  • Method for enriching specificity of circulating tumor cell

Examples

Experimental program
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Effect test

preparation example Construction

[0046] (a) Preparation of rabbit anti-human cell surface receptor primary antibody

[0047] Dissolve 200 μg of antigen in 1 mL of PBS buffer solution, take 1 mL of the above antigen and add conventional 1 mL of complete Freund's adjuvant, shake vigorously to fully emulsify it, and prepare antigen emulsion. The above-mentioned emulsified antigen was subcutaneously injected into the rabbit's back, with an injection volume of about 500 μL per point. After the first immunization, the antigen was injected every 2-3 weeks for booster immunization, and the blood was collected 7-14 days after the last immunization. The collected blood was placed in a 37°C incubator for about half an hour, and then placed at 4°C overnight, centrifuged at 10,000g for 10 minutes at 4°C, and the supernatant was collected as antiserum, and the above antiserum was subjected to affinity column chromatography Purification was carried out to obtain the primary antibody, which was then stored in a refrigerator...

Embodiment 1

[0080] In this example, tumor cell Epidermal Growth Factor Receptor (EGFR) was used as the specific binding target, and EGFR was used as the antigen to prepare the first antibody and the second antibody, and the tumor cell H1975 and healthy human blood were used as the medium to capture Taking tumor cells as an example, the enrichment and detection system of the present invention is further described and verified. The method of enrichment and detection comprises the following steps:

[0081] 1. Preparation of EGFR primary antibody and secondary antibody

[0082] (a) Preparation of rabbit anti-human EGFR primary antibody

[0083] Dissolve 200 μg of antigen in 1 mL of PBS buffer solution, take 1 mL of the above antigen and add conventional 1 mL of complete Freund's adjuvant, shake vigorously to fully emulsify it, and prepare antigen emulsion. The above-mentioned emulsified antigen was subcutaneously injected into the rabbit's back, and the injection volume was about 500 μL. A...

Embodiment 2

[0116] This example is basically the same as Example 1, except that in this example, there is only the primary antibody but no secondary antibody.

[0117] 1. Preparation of EGFR primary antibody

[0118] (a) Preparation of rabbit anti-human EGFR primary antibody

[0119] Dissolve 200 μg of antigen in 1 mL of PBS buffer solution, take 1 mL of the above antigen and add conventional 1 mL of complete Freund's adjuvant, shake vigorously to fully emulsify it, and prepare antigen emulsion. The above-mentioned emulsified antigen was subcutaneously injected into the rabbit's back, and the injection volume was about 500 μL. After the first immunization, the antigen was injected every 2-3 weeks for booster immunization, and the blood was collected 7-10 days after the injection. The collected blood was placed in a 37°C incubator for half an hour, and placed at 4°C overnight; then centrifuged at 10,000g at 4°C for 10 minutes, and the supernatant was collected as antiserum, which was pur...

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Abstract

The invention discloses a method for enriching the specificity of a circulating tumor cell. The method comprises the following steps of: (a) preparing a primary antibody and a secondary antibody of a surface receptor of a tumor cell according to the requirement; (b) fixing the secondary antibody of the surface receptor of the tumor cell; (c) incubating the primary antibody and a sample to be detected; and (d) capturing the circulating tumor cell in the sample to be detected. The method disclosed by the invention is simple in operation; the secondary antibody is fixed and the incubated circulating tumor cell is captured through the fixed secondary antibody; the enrichment is conveniently and quickly realized and the operation process only takes 45 minutes; quick detection can be carried out after enrichment; and the method can be applied to the gene mutation detection of the circulating tumor cell in liquid by enriching.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific enrichment method for circulating tumor cells. Background technique [0002] Cancer is one of the leading causes of human death. According to the World Health Organization (WHO), there were approximately 12.7 million new cancer cases and 7.6 million cancer deaths worldwide in 2008. At present, there are about 9 million new cancer patients every year in the world. It is estimated that by 2020, the incidence of cancer worldwide will increase by 50%, and the number of new cancer patients worldwide will reach 15 million every year. At present, the cancer with the highest incidence rate in the world is lung cancer, followed by breast cancer, colon cancer, stomach cancer, liver cancer and cervical cancer. China is one of the countries with the highest cancer morbidity and mortality in the world. 20% of the world's new cancer patients are in China, and 1 / 4 of the cancer deaths a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N15/11C12Q1/68C12Q1/02
Inventor 郑立谋张海龙罗捷敏阮力周细武曾骥孟
Owner AMOY DIAGNOSTICS CO LTD
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