Screening method for hybrid tumor cell monoclonal preparation

A technology for hybridoma cells and screening methods, applied in biochemical equipment and methods, microbial determination/inspection, fermentation, etc., can solve the source and quality limit of fluorescent latex microbeads, uneven cell distribution, and difficult cell mixing, etc. It can reduce the risk of pollution, increase the number of clones, and save the tedious operation.

Active Publication Date: 2008-09-24
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited source and quality of the antigen-coated fluorescent latex microbeads required to detect positive cells, as well as the limitation of expensive equipment, this method has not been popularized.
[0005] 3. Soft agar semi-solid medium

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Preparation of 2 times DMEM basal culture solution: Take 13.4 grams of DMEM, add 500 milliliters of pure water, add 3.8 grams of sodium bicarbonate and fully stir to dissolve, adjust the pH to 7.2. Sterilize by filtration to prepare 2 times DMEM basal culture solution.

[0022] (2) Preparation of L-glutamine mother liquor: Weigh 5.8 grams of L-glutamine (L-Glutamine, Invitrogen), add 200 milliliters of pure water, heat to 50 ° C to completely dissolve, sterilize and filter to obtain 200 mmol For every liter of L-glutamine mother solution, divide into 4ml per tube and store at -20°C.

[0023] (3) Preparation of β-mercaptoethanol mother liquor: Take 70 microliters of β-mercaptoethanol, add it to 100 milliliters of pure water, filter and sterilize to obtain 10 millimoles per liter of β-mercaptoethanol mother liquor, and store at -20°C.

[0024] (4) Preparation of methyl cellulose aqueous solution: take 0.6 g of 4000 cp methyl cellulose and 25 ml of ultrapure water and...

Embodiment 2

[0029] (1) Preparation of 2 times DMEM basal culture solution: Take 13.4 grams of DMEM, add 500 milliliters of pure water, add 3.8 grams of sodium bicarbonate and fully stir to dissolve, adjust the pH to 7.4. Sterilize by filtration to prepare 2 times DMEM basal culture solution.

[0030] (2) Preparation of L-glutamine mother liquor: Weigh 2.9 grams of L-glutamine (L-Glutamine, Invitrogen), add 100 milliliters of pure water, heat to 52 ° C to completely dissolve, sterilize and filter to obtain 200 mmol For every liter of L-glutamine mother solution, divide into 4ml per tube and store at -20°C.

[0031] (3) Preparation of β-mercaptoethanol mother solution: Take 63 microliters of β-mercaptoethanol, add it to 90 ml of aqueous solution, filter and sterilize to obtain 10 mmol per liter of β-mercaptoethanol mother solution, and store at -20°C.

[0032] (4) Preparation of methyl cellulose aqueous solution: take 0.6 g of 4000 cp methyl cellulose and 25 ml of pure water and sterilize at...

Embodiment 3

[0037] (1) Preparation of 2 times DMEM basal culture solution: Take 13.4 grams of DMEM, add 500 milliliters of pure water, add 3.8 grams of sodium bicarbonate and fully stir to dissolve, adjust the pH to 7.2. Sterilize by filtration to prepare 2 times DMEM basal culture solution.

[0038] (2) Preparation of L-glutamine mother liquor: Weigh 8.7 grams of L-glutamine (L-Glutamine, Invitrogen), add 150 ml of pure water, heat to 50°C to completely dissolve, sterilize and filter to obtain 200 mmol For every liter of L-glutamine mother solution, divide into 4ml per tube and store at -20°C.

[0039] (3) Preparation of β-mercaptoethanol mother solution: Take 77 microliters of β-mercaptoethanol, add it to 110 ml of aqueous solution, filter and sterilize to obtain 10 mmol per liter of β-mercaptoethanol mother solution, and store at -20°C.

[0040] (4) Preparation of methyl cellulose aqueous solution: Take 0.9 g of 4000 cp methyl cellulose and 25 ml of pure water and sterilize at 121-126...

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Abstract

The invention relates to a method of realizing the screening and the monoclone preparation of hybridoma cell in one step. The method comprises that the semisolid medium of the monoclone of hybridoma cell is firstly prepared, the basic fibroblast growth factor of human is added into the fusion cell, then hydridoma fusion factor and clone factor are added and well-mixed, then the mixed material and the semisolid medium are gently well-mixed and packaged into plates and cultivated in the condition of 35 DEG C and CO2 with concentration of 5 percent; when visible clone colony grows in the plates, the clone is aspirated from the culture medium by a micropipettor and moved onto a cell culture plate for liquid scale-up culture, and one clone is moved in one hole; till the cell grows to half to two-third of the hole bottom, the culture supernatant is aspirated to do positive detection, and the positive hole is chosen to directly scale-up frozen and stored for further carrying out the characteristic identification of antibody or ascites production. The method of realizing the screening and the monoclone preparation of hybridoma cell in one step has the advantages of the simple operation, the short culture period, not requiring feeder cells and easily achieving the screening and the monoclone preparation of hybridoma cell after cell fusion in one step.

Description

technical field [0001] The invention relates to a method capable of realizing hybridoma cell selection and monoclonal culture preparation in one step, and belongs to the field of biotechnology. Background technique [0002] Since scientists Kohler and Milstein created monoclonal antibody (McAb) technology in 1975, the technology has been continuously developed and perfected, and its application has become more and more extensive. The cloning of fused cells is a necessary step to obtain cell lines that secrete a single antibody. At present. There are mainly the following methods for fusion cell cloning: [0003] 1. Limiting dilution method This is the method commonly used in various laboratories at present. That is, after the cells are fused, the fused cells are directly inoculated in a 96 or 24-well plate containing HAT selection medium, and the positive wells detected by ELISA are diluted to 4-8 cells / ml (equivalent to at 0.4-0.8 cells / well). From a statistical point of...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12P21/08
Inventor 李培武张文张奇谢立华丁小霞陈小媚姜俊
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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