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A method for sorting and/or enriching circulating tumor cells and its kit

A technology of tumor cells and kits, applied in the field of biomedicine, can solve problems such as ineffective removal of antibody cross-links, large batch-to-batch differences, low coupling efficiency of antibodies and magnetic beads, etc.

Active Publication Date: 2020-10-27
ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the reaction process of this method is uncontrollable, and the cross-linking of the antibody itself cannot be effectively removed, this method is generally not used for antibody and magnetic bead coupling
The uncontrollable reaction process will lead to low coupling efficiency of antibody and magnetic beads, and the difference between batches will be large
Therefore, in actual production applications, this method is difficult to produce stable products
In addition, the direct coupling of antibodies and magnetic beads will cause the magnetic beads to have a strong steric effect on the antibody, which will have a greater impact on the activity of the antibody after cross-linking, which will easily affect the capture efficiency of tumor cells

Method used

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  • A method for sorting and/or enriching circulating tumor cells and its kit
  • A method for sorting and/or enriching circulating tumor cells and its kit
  • A method for sorting and/or enriching circulating tumor cells and its kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Separation of Tumor Cells Using Three Kinds of Biotin-Conjugated Monoclonal Antibodies

[0067] 1. Preparation of biotinylated monoclonal antibody

[0068] 1.1 Preparation of biotin-labeled anti-EpCAM monoclonal antibody

[0069] Add 200 μl of labeling reaction solution (NaCl 137 mmol / L; KCl 2.7 mmol / L; NaCl 2.7 mmol / L; NaCl 2.7 mmol / L; 2 HPO 4 10mmol / L; KH 2 PO 4 2mmol / L), add 500μg anti-EpCAM monoclonal antibody (EBA-1), and mix well. Centrifuge at 3500g for 2min at 4°C, discard the filtrate; add 100μl of the labeling reaction solution (NaCl 137mmol / L; KCl 2.7mmol / L; NaCl 2.7mmol / L; 2 HPO 4 10mmol / L; KH 2 PO 4 2mmol / L), mix well, and centrifuge at 5000g for 2min at 4°C; repeat the above steps of adding 100μl of the labeled reaction solution to the ultrafiltration column, mix well, and centrifuge at 5000g for 2min at 4°C twice;

[0070] Mix the remaining liquid in the ultrafiltration column and let it stand at room temperature for 1 min. Invert...

Embodiment 2

[0100] The selection of embodiment 2 three kinds of antibodies

[0101] As described in Example 1, the preparation method of biotin antibody and the activity method of antibody detection by flow cytometry, wherein biotin-labeled anti-Her2 monoclonal antibody, biotin-labeled anti-EpCAM monoclonal antibody, biotin-labeled anti-Trop2 monoclonal antibody The Delta F% values ​​of the cloned antibodies are shown in Tables 1-3 below:

[0102] Table 1 Statistics of Delta F% of Her2 antibody binding to SKBR3 cells

[0103]

[0104] Table 2 Statistics of Delta F% of Epcam antibody binding to MCF-7 cells

[0105]

[0106] Table 3 Statistics of Delta F% of Trop2 antibody binding to H1650 cells

[0107]

[0108]

[0109] It can be known from Table 1, Table 2, and Table 3 that for the Her2 monoclonal antibody, the Her2 antibody with the clone number 24D2 has the best effect. For Epcam monoclonal antibodies, Epcam antibody with clone number EBA-1 works best. For Trop2 monoc...

Embodiment 3

[0110] Example 3 Determination of Mixing Ratio of Three Different Monoclonal Antibodies

[0111] As described in Example 1, wherein biotin-labeled anti-Her2 monoclonal antibody (24D2), biotin-labeled anti-EpCAM monoclonal antibody (EBA-1), biotin-labeled anti-Trop2 monoclonal antibody (162-46) The mixing ratio is shown in Table 4 below:

[0112] Table 4:

[0113]

[0114] Conclusion: The recovery rate of tumor cells in Group 1, Group 4, and Group 5 was over 70%, among which Group 1 had the best effect, while the recovery rate of tumor cells in Group 2 and Group 3 was low, which did not meet the requirements.

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Abstract

The invention provides a method and kit for sorting and / or enriching circulating tumor cells. According to the method, by utilizing an extremely and stable affinity among an anti-Her2 monoclonal antibody marked by biotin, an anti-EpCAM monoclonal antibody marked by biotin and an anti-Trop2 monoclonal antibody marked by biotin and magnetic beads covered with avidin, an immunomagnetic bead method is provided for sorting and / or enriching the circulating tumor cells. The invention further provides a kit used for sorting and / or enriching circulating tumor cells. The method for sorting and / or enriching the circulating the tumor cells can sensitively capture tumor cells, is applicable to conducting wide spectrum screening on the tumor cells, overcomes steric effect defects of common magnetic bead screening, and achieves the effects of efficiently capturing the tumor cells.

Description

technical field [0001] The invention belongs to the field of biomedicine. Specifically, the invention relates to a method for sorting and / or enriching circulating tumor cells, and also relates to a kit for sorting and / or enriching circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) are tumor cells that have shed into the blood circulation. A large number of experiments have confirmed that the detection of CTCs is helpful for monitoring tumor recurrence and metastasis, judging the prognosis of patients, and guiding postoperative adjuvant therapy. However, the number of CTCs in peripheral blood is very small, usually only a few tumor cells among about 100 million white blood cells and 50 billion red blood cells. Therefore, in order to increase the detection rate of circulating tumor cells, it is usually necessary to enrich circulating tumor cells before detection. Enrichment of circulating tumor cells can be achieved by isolation using tumor ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577
CPCG01N33/54326G01N33/577
Inventor 唐东江范献军陈林任堂雨林上炎
Owner ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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