阅读说明：本技术 一种单克隆抗体的制备方法 (Preparation method of monoclonal antibody ) 是由 詹爱军 单君忆 招沐 闫文龙 于 2019-11-21 设计创作，主要内容包括：一种单克隆抗体的制备方法。涉及单克隆抗体,尤其涉及一种单克隆抗体的制备方法。提供了一种方便可靠,降低成本,提高产量的单克隆抗体的制备方法。所述完全培养基为SFM培养基,不完全培养基为DMEM培养基。无血清细胞培养基中还包括维生素B6、维生素E和叶酸。本发明使用杂交瘤细胞培养法生产单克隆抗体,产量高且方法简便。其既不需要使用实验动物,也不需要特设设备,且抗体产生的量也高于常规方法；使用本方法所产生的单克隆抗体比常规的培养方法抗体量高20-25倍。本发明节省了成本,保证了质量。(A method for preparing monoclonal antibody. Relates to a monoclonal antibody, in particular to a preparation method of the monoclonal antibody. Provides a convenient and reliable preparation method of the monoclonal antibody, which reduces the cost and improves the yield. The complete culture medium is an SFM culture medium, and the incomplete culture medium is a DMEM culture medium. The serum-free cell culture medium also comprises vitamin B6, vitamin E and folic acid. The invention uses the hybridoma cell culture method to produce the monoclonal antibody, has high yield and simple and convenient method. It does not require the use of experimental animals nor special equipment, and the amount of antibody production is higher than that of conventional methods; the monoclonal antibody produced by the method is 20-25 times higher than that produced by the conventional culture method. The invention saves cost and ensures quality.)

1. A method for preparing a monoclonal antibody, comprising the steps of:

1) preparing a serum-free cell culture medium;

2) placing the serum-free cell culture medium into a container, wherein the volume of the serum-free cell culture medium is 1/10 of the container;

3) inoculating hybridoma cells into the container for the first time, wherein the density of the hybridoma cells is controlled to be 2-5 × 105 cells/ml;

4) after 48 hours of hybridoma growth, the serum-free cell culture medium was supplemented with 1/4 volumes of serum-free cell culture medium, and then 1/4 volumes of serum-free cell culture medium were supplemented every 48 hours later until the vessel was completely full of serum-free cell culture medium, and monoclonal antibody production was performed.

2. The method of claim 1, wherein the serum-free cell culture medium in step 1) comprises complete medium and/or incomplete medium.

3. The method of claim 2, wherein the concentration of glucose added in step 1) is 50-100mmol/ml once every 24 hours during the cell culture.

4. The method of claim 2, wherein in step 1), sodium pyruvate is added every 24 hours during the cell culture process, and the concentration of sodium pyruvate is 20 to 50 mmol/ml.

5. The method of claim 2, wherein the complete medium is SFM medium and the incomplete medium is DMEM medium.

6. The method of claim 1, wherein the serum-free cell culture medium further comprises vitamin B6, vitamin E, and folic acid.

Technical Field

The invention relates to a monoclonal antibody, in particular to a preparation method of the monoclonal antibody.

Background

Monoclonal antibodies, antibodies produced by a single B cell clone that are highly homogeneous and are directed against only a particular epitope of an antigen. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology. By culturing a single hybridoma having such a characteristic into a cell population, a monoclonal antibody, which is a specific antibody against one epitope, can be produced.

In the prior art, how to prepare monoclonal antibodies with good quality and high yield is an important problem in the current research, and there are two methods commonly used for producing monoclonal antibodies: the method has the advantages of high yield and titer, difficult purification, high price and long production period: ② a conventional in vitro culture method of hybridoma cells, which has low yield and low titer, but does not contain normal mouse immunoglobulin, is easy to purify and has low cost, but needs special equipment if mass production.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of the monoclonal antibody, which is convenient and reliable, reduces the cost and improves the yield.

The technical scheme of the invention is as follows: the method comprises the following steps:

1) preparing a serum-free cell culture medium;

2) placing the serum-free cell culture medium into a container, wherein the volume of the serum-free cell culture medium is 1/10 of the container;

3) inoculating hybridoma cells into the container for the first time, wherein the density of the hybridoma cells is controlled to be 2-5 × 105 cells/ml;

4) after 48 hours of hybridoma growth, the serum-free cell culture medium was supplemented with 1/4 volumes of serum-free cell culture medium, and then 1/4 volumes of serum-free cell culture medium were supplemented every 48 hours later until the vessel was completely full of serum-free cell culture medium, and monoclonal antibody production was performed.

In step 1), the serum-free cell culture medium comprises complete medium and/or incomplete medium.

In the step 1), glucose is added once every 24 hours in the cell culture process, and the concentration of the glucose is 50-100 mmol/ml.

In the step 1), sodium pyruvate is added once every 24 hours in the cell culture process, and the concentration of the sodium pyruvate is 20-50 mmol/ml.

The complete culture medium is an SFM culture medium, and the incomplete culture medium is a DMEM culture medium.

The serum-free cell culture medium also comprises vitamin B6, vitamin E and folic acid.

The invention uses the hybridoma cell culture method to produce the monoclonal antibody, has high yield and simple and convenient method. It does not require the use of experimental animals nor special equipment, and the amount of antibody production is higher than that of conventional methods; the monoclonal antibody produced by the method is 20-25 times higher than that produced by the conventional culture method.

The invention saves cost and ensures quality.

Detailed Description

The invention comprises the following steps:

1) preparing a serum-free cell culture medium;

2) placing the serum-free cell culture medium into a container, wherein the volume of the serum-free cell culture medium is 1/10 of the container;

3) inoculating hybridoma cells into the container for the first time, wherein the density of the hybridoma cells is controlled to be 2-5 × 105 cells/ml;

4) after 48 hours of hybridoma growth, the serum-free cell culture medium was supplemented with 1/4 volumes of serum-free cell culture medium, and then 1/4 volumes of serum-free cell culture medium were supplemented every 48 hours later until the vessel was completely full of serum-free cell culture medium, and monoclonal antibody production was performed.

In step 1), the serum-free cell culture medium comprises complete medium and/or incomplete medium.

In the step 1), glucose is added once every 24 hours in the cell culture process, and the concentration of the glucose is 50-100 mmol/ml.

In the step 1), sodium pyruvate is added once every 24 hours in the cell culture process, and the concentration of the sodium pyruvate is 20-50 mmol/ml.

The complete culture medium is an SFM culture medium, and the incomplete culture medium is a DMEM culture medium.

The serum-free cell culture medium also comprises vitamin B6, vitamin E and folic acid. By adding vitamins and the like, the culture medium can enable the hybridoma cells to grow rapidly, and the culture requirement is met.

The invention uses the hybridoma cell culture method to produce the monoclonal antibody, has high yield and simple and convenient method. Provides a culture method of hybridoma cell strains by static serum-free culture, which comprises the following specific contents: serum-free culture medium is used for the cells; adding a certain amount of glucose (50-100 mmol/ml) once in 24 hours during the cell culture process; adding a certain amount of sodium pyruvate (20-50 mmol/ml) once in 24 hours during the cell culture process;

the amount of medium is 1/10 for the vessel, depending on the size of the cell vessel used; the proportion of hybridoma cells which are inoculated into the container for the first time is strictly controlled to be 2-5X 105 cells/ml; hybridoma cells were grown for 48 hours and then supplemented with 1/4 volumes of medium, each 1/4 volumes of medium every 48 hours, until the medium was completely full, and the cultured cells were yellow in medium; and then can be cycled through this process.

The monoclonal antibody produced according to this method is 20-25 times higher than that produced by conventional culture methods.

The above embodiments are only embodiments disclosed in the present disclosure, but the scope of the disclosure is not limited thereto, and the scope of the disclosure should be determined by the scope of the claims.