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A kind of electrofusion buffer, its preparation method and electrofusion method

A technology of electrofusion and buffer, which is applied in the field of hybridoma cell production and application, can solve problems such as failure, poor production efficiency, and small number of survivors, and achieve the effects of improving success rate, reducing damage, and reducing interference

Active Publication Date: 2022-03-22
江苏华控生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such a method is to improve the culture environment after the hybridoma cells are generated. If the number of hybridoma cells produced by cell fusion is small or the generation efficiency is poor, the above-mentioned experimental failure may also occur.

Method used

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  • A kind of electrofusion buffer, its preparation method and electrofusion method
  • A kind of electrofusion buffer, its preparation method and electrofusion method
  • A kind of electrofusion buffer, its preparation method and electrofusion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Configuration of high and low concentration electrofusion buffer and its cell fusion efficiency

[0042] The high and low concentration electrofusion buffer of the present invention is configured as follows:

[0043]

[0044] A. Low concentration protocol: 0.2M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate, 0.5 mg / mL bovine serum albumin

[0045] Step 1: Weigh 18.2g D-sorbitol and dissolve it in 400mL ultrapure water.

[0046] Step 2: Weigh 250mg bovine serum albumin (BSA) (0.5 mg / mL) and dissolve it in the step 1 buffer.

[0047] Step 3: Weigh 10.7mg of magnesium acetate and dissolve it in the buffer solution of step 2.

[0048] Step 4: Weigh 7.9 mg of calcium acetate and dissolve it in the buffer of step 3.

[0049] Step 5: After fully dissolving, measure the pH value at 6.8~7.2, dilute to 500mL, filter through a 0.2μm filter, and store at 4°C for later use.

[0050] B. High concentration protocol: 0.3M D-sorbitol, 0.3mM magnesium acet...

Embodiment 2

[0059] Embodiment 2: Configuration of four kinds of electrofusion buffers

[0060] The present invention is debugged in the range of high concentration and low concentration, and is compared with the commonly used electromelting solution at present.

[0061] The configuration of electromelting solution for the four schemes is as follows:

[0062]

[0063] Scheme 1: 0.3M Mannitol, 0.1mM Calcium Chloride, 0.1mM Magnesium Chloride

[0064] Step 1: Weigh 27.3258g mannitol and dissolve in 450mL ultrapure water.

[0065] Step 2: Weigh 406mg of magnesium chloride and dissolve it in 20mL of ultrapure water.

[0066] Step 3: Weigh 438mg of calcium chloride and dissolve in the buffer of step 2.

[0067] Step 4: Take 500 μL of the buffer solution prepared in step 3 and add it to the solution in step 1, mix well, dilute to 500 mL, filter through a 0.2 μm filter, and store at 4°C for later use.

[0068] Effect: Calcium chloride and magnesium chloride are strong electrolytes with str...

Embodiment 3

[0089] Example 3: Comparing the cell fusion efficiencies of four electrofusion buffers

[0090] To perform electrofusion, the steps are as follows:

[0091] 1. After separating SPF grade mouse splenocytes, mix mouse myeloma cells Sp2 / 0 cells (Sp2 / 0-Ag14, ATCC number: CRL-1581) and splenocytes at a ratio of 1:2 to 1:5, respectively .

[0092] 2. After the cell suspension was washed twice with the above four electrofusion buffers, the cells were resuspended in the four electrofusion buffers for fusion.

[0093] 3. The electrofusion instrument (BTX Company of the United States, model: ECM2001) is equipped with electrodes, the capacity of the fusion chamber is 2 mL, and the four kinds of electrofusion buffers in Example 1 are used to adjust the number of cells to 1×10 7 cells / mL, add the mixed cell suspension into the fusion chamber.

[0094] 4. Adjust the AC voltage, that is, the intensity of the AC electric field (AC) is designed to be 30 V, 50 V, the frequency of the AC elec...

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Abstract

The invention provides an electric fusion buffer and a method for electric fusion cells, which can greatly improve the cell fusion efficiency and cell survival rate of hybridoma technology. The invention optimizes the electrofusion buffer ratio, and the buffer not only has low conductivity, but also solves the problems of interference to cell bead arrangement and thermal effect of cell fusion experiments, improves the clone formation rate, and reduces experiment costs.

Description

technical field [0001] The invention belongs to the technical field of production and application of hybridoma cells, and in particular relates to a low-conductivity buffer solution and a method for electrofusion cells. Background technique [0002] Hybridoma technology, also known as monoclonal antibody technology, mainly prepares monoclonal antibodies specific to antigens, and fuses B lymphocytes (usually spleen cells of immunized animals) with myeloma cells to obtain antibodies that can produce antibodies. , Continuously proliferating hybridoma cells, and then can produce a large amount of monoclonal antibodies. Although hybridoma technology is an important basis for the production of monoclonal antibodies, the experimental steps of this technology are cumbersome and the cycle is long, and there are many factors that affect the success of the experiment, among which is the hybridoma manufacturing process. The initial hybridomas after fusion are easily affected by slight ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N13/00C07K16/00C12M1/42C12M1/00
CPCC12N5/163C12N13/00C07K16/00C12M35/02
Inventor 许行尚杰弗瑞·陈朱道云熊凯
Owner 江苏华控生物科技有限公司
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