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Immunomagnetic head negative enrichment method and application thereof

A technology of negative enrichment and immunomagnetic beads, which is applied in the field of cancer diagnosis, can solve the problems of poor sensitivity and specificity of lung cancer diagnosis technology, and achieve the effect of improving sensitivity and detection rate

Inactive Publication Date: 2015-06-10
徐兴祥
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide an immunomagnetic bead negative enrichment method and its application, aiming to solve the problem of poor sensitivity and specificity of existing lung cancer diagnostic techniques

Method used

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  • Immunomagnetic head negative enrichment method and application thereof
  • Immunomagnetic head negative enrichment method and application thereof
  • Immunomagnetic head negative enrichment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Experimental material, equipment preparation and description

[0035] (1) adjust the acceleration and deceleration time of centrifuge, all centrifugal acceleration and deceleration times are all the same in the steps of the present invention. The acceleration and deceleration time that meets the experimental requirements is: set the centrifugal force of the centrifuge to 450g, the time from 0 to 450g should be 40 seconds, and the shortest time from 450g to 0 should be 200 seconds.

[0036] (2) Test whether the horizontal rotor low-speed centrifuge meets the requirements of this experiment. The test method: take 1ml hCTC separation medium into a 15ml centrifuge tube, mix 0.5ml peripheral blood with 1.5ml 1x hCTC buffer and add to 1ml hCTC non-blood The top layer of the derived cell separation medium was centrifuged at room temperature (550g) for 5 minutes. After centrifugation, the liquid was clearly separated. The upper layer was transparent liquid, and the...

Embodiment 2

[0043] Example 2 Enrichment of Thoracic and Abdominal Tumor Cells

[0044] (1) Add 1ml of cell preservation solution to a 15ml centrifuge tube, collect 10ml of patient's pleural fluid / ascites and add it to the tube, and immediately turn it upside down and shake the cells gently. The collected pleural effusion and ascites samples should be processed immediately and stored at 4°C in the dark for no more than 24 hours.

[0045] After mixing the pleural and ascites fluid in the 15ml centrifuge tube, pour it into the 50ml centrifuge tube A, shake the cells vertically, balance, and centrifuge at room temperature for 3 minutes (600g). Discard the supernatant to 3ml, shake the centrifuge tube vertically and gently and quickly in a clockwise direction to mix the pelleted cells (be careful not to blow the pelleted cells with the tip of the pipette, and do not let too much liquid get on the tube wall when shaking the cells, generally It should not exceed the 25ml mark). Add 2ml 1×hCTC ...

Embodiment 3

[0060] Example 3 Malignant pleural effusion tumor cell Rigi stained smear

[0061] (1) Enrichment of tumor cells in malignant pleural effusion by density gradient centrifugation: ① Take 50ml of pleural effusion in a 50ml centrifuge tube, centrifuge (200g, room temperature, 10min), discard the supernatant to 4ml and mix well; ②take 4ml of lymphocytes Transfer the separation liquid to the bottom of the centrifuge tube and warm up to room temperature; ③Gently place 4ml of cell suspension on top of the lymphocyte separation liquid without disturbing the interface of the liquid layer, centrifuge (300g, room temperature, 25min), absorb the middle layer to 15ml and centrifuge Add 5 times the PBS buffer of the liquid to mix, centrifuge (250g, room temperature, 15min), discard the supernatant; ④ add 1ml of PBS buffer to wash twice, centrifuge at 250g for 5min, discard the supernatant, add 1ml of PBS to mix Homogenize the sediment, then count the cells, take an appropriate amount of cel...

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Abstract

The invention provides an immunomagnetic head negative enrichment method and an application thereof. The method comprises the following steps: obtaining a separating medium from a pretreated pleuroperitoneal fluid sample, balancing, and centrifuging to remove deposited red blood cells; performing adsorption reaction on supernatant liquid and magnetic beads; centrifuging, balancing all liquid deposited on the magnetic beads by using a hCTC buffer solution, and then placing on a magnetic frame to adsorb the magnetic beads; centrifuging, then collecting supernatant liquid, and balancing the supernatant liquid by using the hCTC buffer solution; centrifuging to remove the supernatant; and counting cells, diluting, smearing and drying, and then performing FISH (Fluorescence In Situ Hybridization) detection. By combining the FISH detection, the method provided by the invention can improve the sensitivity and specificity of tumor cell enrichment, and is used for differential diagnosis of benign and malignant pleuroperitoneal fluid, dynamic gene detection of solid tumors, thereby realizing individualized treatment.

Description

technical field [0001] The invention belongs to the technical field of cancer diagnosis, and in particular relates to an immunomagnetic bead negative enrichment method and its application. Background technique [0002] Lung cancer is currently one of the malignant tumors with the highest morbidity and mortality. According to WHO statistics, an estimated 1.3 million people die of lung cancer in the world every year, of which 85% are non-small cell lung cancer. The average survival period of lung cancer patients is about 10 months, and its 5-year survival rate is less than 15%. Although the advances in tumor molecular biology research and the application of new chemotherapy drugs have brought new hope to the diagnosis and treatment of lung cancer in recent years, the survival rate of most lung cancer patients has not been significantly improved. The metastasis, recurrence and resistance to chemotherapy drugs of lung cancer are still difficult problems. Pleural effusion is on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N1/34G01N33/54326G01N33/57423
Inventor 徐兴祥扬俊俊林平代小勤闵林峰苄家蓉陈勇邹晶凡国华
Owner 徐兴祥
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