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Method for preparing monoclonal hybrid tumors

A monoclonal, hybridoma cell technology, applied in the biological field, can solve the problems of large workload, obstacles, and complicated operation of cell culture, and achieve the effects of avoiding low survival rate, reducing production cost and simple preparation method.

Inactive Publication Date: 2013-01-09
南京巴傲得生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this subcloning method are: 1. The counting requirements for cells are very high; 2. Positive clones are easily lost because positive clones are easily displaced by negative clones; Heavy workload, cumbersome operation, and low cloning efficiency
Adding specific fluorescent substances, these fluorescent substances can specifically form a bright fluorescent halo around the positive hybridomas under laser excitation, so that the screening and subcloning of positive hybridomas can be completed in one step, which greatly saves time and labor, but Due to its patented technology and high cost of equipment and reagents, it has been hindered from becoming a conventional method for monoclonal antibody production

Method used

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  • Method for preparing monoclonal hybrid tumors
  • Method for preparing monoclonal hybrid tumors
  • Method for preparing monoclonal hybrid tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of cloned hybridoma cells.

[0073] 1. Fusion:

[0074] Take splenocytes and sp2 / 0 cells in a good state after conventional immunization, control the ratio of splenocytes and sp2 / 0 cells at 5:1, and use PEG 4000 with a volume fraction of 50% for fusion; alternatively, The ratio of the number of splenocytes to sp2 / 0 cells can be controlled between (10-2):1, and PEG with a molecular weight in the range of 1000-6000 can also be selected.

[0075] The fused cells were cultured in a 5-7% carbon dioxide incubator at 37 degrees for 16 hours using 20% ​​fetal bovine serum culture medium.

[0076] 2. Cultivate:

[0077] Gently blow off the hybridoma cells and centrifuge to pellet, resuspend in 5-10mL conventional HAT liquid medium with a volume percentage of 2% HAT, then use the above medium to make up to 120mL, and distribute to 6 96-well plates medium, 200 μL / well; culture in a 5-7% carbon dioxide incubator at 37°C for 7 days, and then use HAT medium f...

Embodiment 2

[0113] Example 2 Preparation of cloned hybridoma cells.

[0114] 1. Fusion:

[0115] Take the splenocytes of mice after routine immunization and NS-1 cells in good condition, control the ratio of the number of splenocytes and NS-1 cells at 10:1, and use PEG 1000 with a volume fraction of 60% to fuse; alternatively, The ratio of the number of splenocytes to NS-1 cells can be controlled between (10-2):1, and PEG with a molecular weight in the range of 1000-6000 can also be selected. The fused cells were cultured in a 5-7% carbon dioxide incubator at 25 degrees for 24 hours using 15% fetal calf serum culture medium.

[0116] 2. Cultivate:

[0117] Gently blow down the hybridoma cells and centrifuge to pellet, resuspend in 5-10mL conventional HAT liquid medium with a volume percentage of 5% HAT, then use the above medium to make up to 120mL, and distribute to 6 96-well plates medium, 200 μL / well; culture in a 5-7% carbon dioxide incubator at 25°C for 7 days, and then use HAT me...

Embodiment 3

[0127] Example 3 Preparation of cloned hybridoma cells.

[0128] 1. Fusion:

[0129] Take splenocytes and sp2 / 0 cells in a good state after routine immunization, control the ratio of splenocytes and sp2 / 0 cells at 2:1, and use PEG 6000 with a volume fraction of 40% to fuse; alternatively, The ratio of the number of splenocytes to sp2 / 0 cells can be controlled between (10-2):1, and PEG with a molecular weight in the range of 1000-6000 can also be selected. The fused cells were cultured for 20 hours in a 5-7% carbon dioxide incubator at 30 degrees using 10% fetal bovine serum culture medium.

[0130] 2. Cultivate:

[0131] Gently blow off the hybridoma cells and centrifuge to pellet, resuspend in 5-10mL conventional HAT liquid medium with a volume percentage of 4% HAT, then use the above medium to make up to 120mL, and distribute to 6 96-well plates medium, 200 μL / well; culture in a 5-7% carbon dioxide incubator at 30°C for 5 days, and then use HAT medium for full replacement...

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Abstract

The invention provides a method for preparing monoclonal hybrid tumors. The method comprises the following steps of: 1, blending, namely blending myeloma cells and lymphocytes which are subjected to antigen immunization by using a polyethylene glycol aqueous solution to obtain fusion cells; 2, culturing, namely inoculating the fusion cells into a hypoxanthine-aminopterin-thymine (HAT) liquid culture medium, and culturing in a carbon dioxide incubator to obtain hybridoma cells; 3, screening, namely screening supernate of the hybridoma cells by using indirect enzyme-linked immunosorbent assay (ELISA) to obtain positive hybridoma cells; 4, subcloning, namely inoculating the positive hybridoma cells into a subcloning semisolid culture medium, and culturing in the carbon dioxide incubator to obtain hybridoma cell colonies; and 5, performing enlarging culture, namely inoculating the cells of the single hybridoma cell colony into a fetal calf serum culture solution, and performing the enlarging culture in the carbon dioxide incubator to obtain monoclonal hybrid tumor cells. The method is short in culture period and low in cost, and screened positive clone cell strains are complete.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation method of a monoclonal hybridoma. Background technique [0002] Since the invention of monoclonal antibody technology by Kohler and Milstein, the technology has been continuously developed and improved, and has been widely used in the diagnosis, treatment and scientific research of diseases in the field of biomedicine, and the market demand is increasing. However, an essential and most important link in the preparation of monoclonal antibodies is the clonal establishment of positive hybridomas. [0003] The hybridoma cell group screened after fusion is not completely pure, but still belongs to heterogeneous cell group. Therefore, the hybrid cell population must be further purified according to the experimental purpose in order to obtain a homogeneous cell population. The most commonly used purification method is the cloning culture of hybridoma cells. [0004] Clonin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12R1/91
Inventor 周军王玉燕冯展波
Owner 南京巴傲得生物科技有限公司
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