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Methods for detection and isolation of cell populations co-expressing CD45 and EpCAM and uses thereof

A cell group and co-expression technology, applied in the field of cellular immunology, can solve the problems of large sampling volume, separation/enrichment/identification difficulties, etc., and achieve the effect of low cost, easy operation and short time

Active Publication Date: 2022-07-08
青岛华赛伯曼医学细胞生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention aims to provide a more efficient detection method, the detected cell population can be more accurately used for cancer screening, auxiliary diagnosis, curative effect detection, etc. / enrichment / identify difficult problems

Method used

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  • Methods for detection and isolation of cell populations co-expressing CD45 and EpCAM and uses thereof
  • Methods for detection and isolation of cell populations co-expressing CD45 and EpCAM and uses thereof
  • Methods for detection and isolation of cell populations co-expressing CD45 and EpCAM and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] 1. Flow detection of CD45 and EpCAM expression on lung cancer tumor tissue cells:

[0155] (101) using minimally invasive surgery to obtain tumor tissue from a patient with lung cancer, placing it in a petri dish with a diameter of 10 cm, and washing it with normal saline;

[0156] (102) Cut the tumor tissue into 1mm with ophthalmic scissors or a scalpel 3 size of tissue blocks;

[0157] (103) adding an appropriate amount of normal saline, then transferring the chopped tumor tissue to a 50ml centrifuge tube, and centrifuging at 400g for 5min;

[0158] (104) After the centrifugation, discard the supernatant, add 1 ml of physiological saline to resuspend the cells, add physiological saline to 50 ml, and mix by inversion;

[0159] (105) filter the cells into a new 50ml centrifuge tube using a 70μm filter to obtain a cell suspension;

[0160] (106) After taking an appropriate amount of cells for cell counting, centrifuge at 400 g for 5 min;

[0161] (107) After centrifu...

Embodiment 2

[0191] CD45 in peripheral blood mononuclear cells (PBMC) + EpCAM + A study of the causes of cell formation.

[0192] 1. Extraction of exosomes from HCC827 cells:

[0193] (301) HCC827 cell fusion rate reached 80-90%, the cells were passaged, the cells were cultured with RPMI1640 complete medium containing exosome-free serum for 48-72 h, and the cell culture supernatant was collected;

[0194] (302) 300-500g, centrifuge at 4°C for 5min to remove cells;

[0195] (303) After the centrifugation, transfer the cell culture supernatant to a new centrifuge tube, centrifuge at 2,000 g at 4° C. for 10 min, and remove cell debris;

[0196] (304) After the centrifugation, transfer the cell culture supernatant to a new centrifuge tube, centrifuge at 10,000g at 4°C for 30min, and remove apoptotic bodies;

[0197] (305) After the centrifugation, transfer the cell culture supernatant to a new centrifuge tube, centrifuge at 120,000 g at 4°C for 70 min, discard the supernatant to collect th...

Embodiment 3

[0250] Detection of CD45 using magnetic beads coated with anti-CD45 antibody (purchased from Miltenyi Biotec) and magnetic beads coated with anti-EpCAM antibody (purchased from Miltenyi Biotec) + EpCAM + cell:

[0251] (401) PBMC and HCC827 cells of healthy volunteers were co-cultured at a ratio of 1:3, and the cells were collected into a 15ml centrifuge tube after 24 hours;

[0252] (402) Sorting CD45 by CD45 Magnetic Beads + cell:

[0253] ①The collected cells were centrifuged at 300g for 10min;

[0254] ② Discard the supernatant and resuspend the cells with 80 μl of MACS solution containing 0.5% bovine serum albumin;

[0255] ③ Add 20 μl CD45 magnetic beads, mix well and incubate in a 4°C refrigerator for 15 minutes;

[0256] ④ After the incubation, add 2ml of MACS solution containing 0.5% bovine serum albumin to the centrifuge tube and mix well, centrifuge at 300g for 10min;

[0257] ⑤Place the MACS column on the magnetic stand and rinse with 3ml of MACS solution con...

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Abstract

The invention relates to a method for detecting and separating a cell population co-expressing CD45 and EpCAM and application of the cell population. According to the present invention, the composition or the kit containing the anti-CD45 antibody and the anti-EpCAM antibody is used for detecting and separating the cell population co-expressing CD45 and EpCAM from the PBMC, and is used for the rapid in vitro screening of cancers, the guidance of cancer treatment and the evaluation of the effectiveness of cancer treatment.

Description

technical field [0001] The present invention relates to the field of cellular immunology, in particular to a composition based on an anti-CD45 antibody and an anti-EpCAM antibody, a corresponding kit and a method for detecting and isolating co-expressing CD45 and EpCAM from peripheral blood mononuclear cells (PBMCs). Use in cell populations. It also relates to the use of cell populations co-expressing CD45 and EpCAM for in vitro screening of cancer, guidance of therapy, and assessment of therapeutic efficacy. Background technique [0002] Cancer is a tumor that threatens human health, and its morbidity and mortality are increasing rapidly worldwide. In 2018, there were 18.1 million new cases and 9.6 million cancer deaths worldwide, of which lung cancer ranks first in both incidence and mortality (Bray et al., 2018). As the number one cancer killer, lung cancer has hidden onset, untimely diagnosis, easy metastasis and recurrence, and poor prognosis. Moreover, 70-75% of pati...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/30G01N33/569G01N33/574G01N33/543C12N5/0786
CPCC07K16/289C07K16/30G01N33/56966G01N2800/52C12N5/0693G01N33/6893G01N33/6872G01N33/57492G01N15/14C07K2317/92G01N2333/70589G01N2333/705G01N2015/1486
Inventor 孙振李斌李文静赵明明王泽瑶刘文娜高青
Owner 青岛华赛伯曼医学细胞生物有限公司
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