CN103898060B - The screening method of HAT semisolid screening culture medium and hybridoma - Google Patents

Abstract

The invention discloses a HAT semi-solid screening medium. Every 100mL of the HAT semi-solid screening medium consists of the following components: 2mL of 50×HAT stock solution, 20mL of fetal bovine serum, 1mL of 100×penicillin and streptomycin 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution, and the rest of methylcellulose semi-solid Medium. The invention also discloses a method for screening hybridoma cells using the HAT semi-solid screening medium. Fusion cells grow as colonies on the semi-solid medium and are separated from each other. Each clone is formed by a single cell, which avoids non-secretion of antibodies Compared with the traditional limiting dilution method, the screening efficiency is higher.

Description

HAT semi-solid screening medium and screening method for hybridoma cells

technical field

The invention relates to the field of immunology, in particular to a HAT semi-solid screening medium and a screening method for hybridoma cells using the HAT semi-solid screening medium.

Background technique

The basic principle of monoclonal antibody technology is to hybridize B cells that secrete antibodies but cannot be cultured for a long time with myeloma cells that can be cultured in vitro for a long time, and the hybridoma cells that are screened can not only secrete antibodies but also have the characteristics of unlimited proliferation of myeloma cells , The antibody propagated and secreted by a hybridoma cell is called a monoclonal antibody, which only targets one epitope. During cell fusion, B cells are isolated from the spleen of antigen-immunized animals, and myeloma cells are defective after mutagenesis and screening, which cannot secrete antibodies by themselves; and hypoxanthine guanine nucleotides are transferred The deficient type of enzyme (HGPRT) and thymidine kinase (TK) cannot survive in the selection medium (HAT) containing hypoxanthine (hypoxanthineH), aminopterin (aminopterin, A) and thymidine (thymidineT). In the fused cell population, unfused splenocytes and splenocytes fused with each other cannot be continuously cultured and can only survive in the medium for a few days, while unfused myeloma cells and fused myeloma cells lack HGPRT Can not survive in the HAT medium, only the hybridoma cells formed by myeloma cells and spleen cells can survive in the HAT medium, so the hybridoma can grow and reproduce normally and be selected.

In a spleen, the proportion of cells that can secrete antibodies is very small, and more than 90% of the cells cannot secrete antibodies. Even with artificial immunization, the proportion of cells that can secrete antibodies against the antigen of artificial immunization is very low, and the fusion of two cells is random, so although a successfully immunized spleen can reach 2× ¹⁰⁸ cells As mentioned above, but the number of target hybridomas that can be obtained after cell fusion is also very limited, how to screen and clone these hybridoma cells that can secrete the target antibody is a very critical issue.

The cloning of hybridoma cells is the most work-intensive part in the preparation of monoclonal antibodies. The traditional cloning method in the laboratory is the limited dilution method. This method requires multiple limited dilutions to obtain monoclonal hybridomas, which takes a long time. Before cloning, some hybridomas that do not secrete antibodies tend to grow very fast, which affects the growth of hybridoma cells that secrete antibodies and easily leads to the loss of positive clones, and the screening efficiency is low.

Contents of the invention

Based on this, it is necessary to provide a HAT semi-solid screening medium with high screening efficiency and a screening method for hybridoma cells.

A HAT semi-solid selection medium, every 100mL of the HAT semi-solid selection medium consists of the following components: 2mL of 50× HAT stock solution, 20mL of fetal bovine serum, 1mL of 100× penicillin and streptomycin double antibody solution, 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution and the rest of the methylcellulose semi-solid medium;

The methylcellulose semi-solid medium is obtained by thoroughly mixing 2×DMEM medium and 4% methylcellulose solution at a volume ratio of 1:1.

In one embodiment, the 2×DMEM medium is prepared by the following operations: add 13.5g of DMEM powder and 3.7g of sodium bicarbonate into 500mL of ultrapure water and stir to dissolve, then adjust the pH value to 7.0-7.2, and finally Filter and sterilize through a 0.22 μm filter membrane to obtain the 2×DMEM medium.

In one embodiment, the 4% methylcellulose solution is prepared by the following operations: Weigh 20g of methylcellulose and dissolve it in 500mL of ultrapure water, stir at 4°C until the solution is viscous and uniform without white particles, and finally Autoclave to obtain the 4% methylcellulose solution.

A screening method for hybridoma cells, comprising the steps of:

Step 1. Fully mix 2×DMEM medium and 4% methylcellulose solution according to the volume ratio of 1:1 to obtain methylcellulose semi-solid medium;

Step 2, preparation of HAT semi-solid selection medium, the HAT semi-solid selection medium of every 100mL is made up of following components: 2mL of 50 × HAT stock solution, 20mL of fetal bovine serum, 1mL of 100 × penicillin and streptomycin Double antibody solution, 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution, and the rest of the methylcellulose semi-solid medium ;

Step 3. After cell fusion of lymphocytes and mouse myeloma, resuspend the fused cells with HAT liquid medium to obtain a cell suspension, and add the cell suspension to a preheated to 35°C~38°C Mix the HAT semi-solid screening medium obtained in step 2, let it stand at 37°C and remove air bubbles, pour it into a plate, and place it in an incubator at 37°C and 5% CO ₂ for cultivation;

The cells obtained in step 4 and step 3 were cultured in an incubator at 37°C and 5% _CO for 10 to 14 days. When clonal colonies visible to the naked eye grew in the plate, the cells with strong fluorescent signals around them were positive under a fluorescent microscope. The cell clusters are marked separately, and then the marked positive clones are picked out under a microscope, and the positive clones are the hybridoma cells.

In one embodiment, in step 1, the 2×DMEM medium is prepared by the following operations: 13.5g of DMEM powder and 3.7g of sodium bicarbonate are added to 500mL of ultrapure water and fully stirred to dissolve, and then the pH value is adjusted to 7.0 ~7.2, and finally filtered through a 0.22 μm filter membrane to obtain the 2×DMEM medium.

In one embodiment, in step 1, the 4% methyl cellulose solution is prepared by the following operations: Weigh 20 g of methyl cellulose and dissolve it in 500 mL of ultrapure water, and stir at 4°C until the solution is viscous and uniform without White granules were finally autoclaved to obtain the 4% methylcellulose solution.

In one embodiment, the antigen described in step 2 is a soluble protein that can be coupled with isothiocyanate.

In one embodiment, the HAT liquid medium described in Step 3 consists of the following components: 1×DMEM medium, 20% fetal bovine serum and 1×HAT.

In one embodiment, step 4 also includes the step of confirming the positive clones after the labeled positive clones are picked out, including the following operations: putting the picked positive clones into liquid medium containing HT Cultured in a 96-well plate, one clone per well, after the cells filled the bottom of the well, the supernatant was taken to confirm the positive clones.

In one embodiment, in the operation of confirming positive clones, ELISA is used to confirm positive clones.

This screening method for hybridoma cells uses HAT semi-solid screening medium. The fused cells grow as colonies on the semi-solid medium and are separated from each other. Each clone is formed by a single cell, which avoids the rapid growth of hybridomas that do not secrete antibodies. While affecting the growth of antibody-secreting hybridoma cells, the screening efficiency is higher compared with the traditional limiting dilution method.

Description of drawings

Fig. 1 is a flowchart of a method for screening hybridoma cells according to one embodiment.

Detailed ways

In order to make the above objects, features and advantages of the present invention more comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific implementations disclosed below.

One embodiment of the HAT semi-solid selection medium, wherein, every 100mL of the HAT semi-solid selection medium consists of the following components: 2mL of 50× HAT stock solution, 20mL of fetal bovine serum, 1mL of 100× penicillin and streptomycin 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution, and the rest of methylcellulose semi-solid Medium.

Methylcellulose semi-solid medium is obtained by thoroughly mixing 2×DMEM medium and 4% methylcellulose solution at a volume ratio of 1:1.

2×DMEM medium was prepared by the following operations: add 13.5g of DMEM powder and 3.7g of sodium bicarbonate into 500mL of ultrapure water and stir to dissolve, then adjust the pH value to 7.0~7.2 with hydrochloric acid or sodium hydroxide, and finally pass 0.22 Filter and sterilize with a μm filter membrane to obtain the 2×DMEM medium.

The 4% methyl cellulose solution was prepared by the following operations: Weigh 20 g of methyl cellulose and dissolve it in 500 mL of ultrapure water, stir at 4 °C until the solution is viscous and uniform without white particles, and finally autoclave to obtain the 4% of methylcellulose solution.

One embodiment of the method for screening hybridoma cells is shown in Figure 1, and the steps are as follows.

S10. Fully mix 2×DMEM medium and 4% methylcellulose solution according to the volume ratio of 1:1 to obtain methylcellulose semi-solid medium.

2×DMEM medium was prepared by the following operations: add 13.5g of DMEM powder and 3.7g of sodium bicarbonate into 500mL of ultrapure water and stir to dissolve, then adjust the pH value to 7.0~7.2 with hydrochloric acid or sodium hydroxide, and finally pass 0.22 Filter and sterilize with a μm filter membrane to obtain 2×DMEM medium.

DMEM powder was purchased from Gibco Company, the article number is 12800-017.

4% methyl cellulose solution was prepared by the following operation: weigh 20 g methyl cellulose and dissolve it in 500 mL ultrapure water, stir at 4 °C until the solution is viscous and uniform without white particles, and finally autoclave to obtain 4% methyl cellulose base fiber solution.

Methylcellulose was purchased from Sigma Company, item number M0512.

The operation of stirring at 4°C until the solution is viscous and uniform without white particles can be as follows: stir overnight on a magnetic stirrer in a refrigerator at 4°C until the solution is viscous and uniform without white particles.

The operation of autoclaving can be: autoclave at 121°C for 30 minutes. Sterilized 4% methylcellulose solution can be stored at 4°C.

S20, prepare HAT semi-solid selection medium, every 100mL described HAT semi-solid selection medium is made up of the following components: 2mL of 50×HAT stock solution, 20mL of fetal bovine serum, 1mL of 100× penicillin and streptomycin double Antibody solution, 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution and the rest of methylcellulose semi-solid medium.

The 50×HAT stock solution was prepared by the following operation: add 1 bottle of HAT powder into 10 mL of sterile PBS solution, and dissolve to obtain the 50×HAT stock solution.

A 50X HAT stock solution consisted of 5 mM hypoxanthine, 20 μM aminopterin, and 800 μM thymidine.

HAT powder was purchased from Sigma Company, the article number is H0262.

The sterile PBS solution was purchased from Gibco, the product number is C20012500BT.

Fetal bovine serum was purchased from Hyclone Company, the product number is SV30087.

100×B cell activating factor (Bcell activating factor, BAFF) was purchased from R&D Company, the product number is 2149-BF-010/CF, and its working concentration is 200~100ng/L.

The working concentration of 100× isothiocyanate (fluoresceinisothiocyanate, FITC)-labeled antigen solution is 1-10 μg/mL, and can be stored at -20°C.

Isothiocyanate-labeled antigens can be purchased from the market or prepared by themselves according to the specific needs of the antigen.

The antigen can be a soluble protein to which isothiocyanates can be conjugated.

Isothiocyanate-labeled antigens can directly screen cell clones that secrete specific antibodies through antigen-antibody agglutination reaction.

The double antibody solution of 100×penicillin and streptomycin was purchased from Hyclone Company, the product number is SV30010.

50× hybridoma fusion cloning factor (Hybridoma Fusion and Cloning Supplement, HFCS) solution was purchased from Roche Company, the product number is 11363735001.

S30. After performing cell fusion of lymphocytes and mouse myeloma, resuspend the fused cells with HAT liquid medium to obtain a cell suspension, and add the cell suspension to S20 obtained by preheating to 35°C~38°C Mix well in the HAT semi-solid screening medium, let it stand at 37°C and remove air bubbles, pour it into a plate, and place it in an incubator at 37°C and 5% CO ₂ for cultivation.

The HAT liquid medium consists of the following components: 1×DMEM medium, 20% fetal bovine serum and 1×HAT.

The plate can choose a plate with a diameter of 3.5cm, and each plate is filled with 2mL of medium.

The cells obtained from S40 and S30 were cultured in an incubator at 37°C and 5% CO ₂ for 10 to 14 days. When clone colonies visible to the naked eye grew in the plate, the positive cell clusters with strong fluorescent signals around them were analyzed separately under a fluorescent microscope. Mark, and then pick out the marked positive clones under a microscope, and the positive clones are the hybridoma cells.

The operation of picking out the marked positive clones can be done with a sterile micropipette.

S40 also includes the step of confirming the positive clones, including the following operations: put the picked positive clones into a 96-well plate containing HT liquid medium for culture, one clone per well, and after the cells have grown to the bottom of the well, the Positive clones were confirmed.

HT liquid medium consists of the following components: 1×DMEM medium, 20% fetal bovine serum and 1×HT.

HT was purchased from sigma company, the article number is H0137.

The supernatant can be taken to confirm positive clones by ELISA and other methods, and the confirmed positive clones can be selected for expansion culture, cryopreservation and identification analysis.

In this hybridoma screening method, the fused cells grow in colonies on a semi-solid medium and are separated from each other. Each clone is formed by a single cell, so a pure antibody can be obtained without repeated cloning. Fluorescence-labeled antigens are added to the medium to realize the cloning and positive screening of hybridoma cells in one step, with a small workload and short time-consuming; the ability of cells to secrete antibodies can be judged according to the fluorescence intensity, so as to obtain cell lines with high secretion of antibodies; semi-solid B cell stimulating factors and hybridoma cell fusion cloning factors are added to the culture medium, which ensures the nutritional needs of hybridoma cells, avoids the cumbersome operation of using feeder cells, and greatly reduces the risk of contamination.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (9)

1. A HAT semi-solid screening medium, characterized in that, the HAT semi-solid screening medium of every 100mL is made up of the following components: 50 × HAT stock solution of 2mL, fetal bovine serum of 20mL, 100 × penicillin of 1mL and Streptomycin double antibody solution, 1mL 100× B cell stimulating factor solution, 2mL 50× hybridoma cell fusion cloning factor solution, 1mL 100× isothiocyanate-labeled antigen solution and the rest of methylcellulose semi-solid media; The methylcellulose semi-solid medium is obtained by thoroughly mixing 2×DMEM medium and 4% methylcellulose solution at a volume ratio of 1:1. 2. HAT semi-solid screening medium according to claim 1, is characterized in that, described 2 * DMEM medium is prepared by following operations: the DMEM powder of 13.5g and 3.7g sodium bicarbonate are added the ultrapure water of 500mL Thoroughly stir to dissolve, then adjust the pH value to 7.0-7.2, and finally filter and sterilize through a 0.22 μm filter membrane to obtain the 2×DMEM medium. 3. HAT semi-solid screening medium according to claim 1, is characterized in that, described 4% methylcellulose solution is prepared by following operations: take by weighing 20g methylcellulose and be dissolved in 500mL ultrapure water, 4% Stir at ℃ until the solution is viscous and uniform without white particles, and finally sterilize under high pressure to obtain the 4% methyl cellulose solution. 4. A screening method for hybridoma cells, comprising the steps of: Step 1. Fully mix 2×DMEM medium and 4% methylcellulose solution according to the volume ratio of 1:1 to obtain methylcellulose semi-solid medium; Step 2, preparation of HAT semi-solid selection medium, the HAT semi-solid selection medium of every 100mL is made up of following components: 2mL of 50 × HAT stock solution, 20mL of fetal bovine serum, 1mL of 100 × penicillin and streptomycin Double antibody solution, 1 mL of 100× B cell stimulating factor solution, 2 mL of 50× hybridoma cell fusion cloning factor solution, 1 mL of 100× isothiocyanate-labeled antigen solution, and the rest of the methylcellulose semi-solid medium ; Step 3. After cell fusion of lymphocytes and mouse myeloma, resuspend the fused cells with HAT liquid medium to obtain a cell suspension, and add the cell suspension to a preheated to 35°C-38°C Mix the HAT semi-solid screening medium obtained in step 2, let it stand at 37°C and remove air bubbles, pour it into a plate, and place it in an incubator at 37°C and 5% CO ₂ for cultivation; The cells obtained in step 4 and step 3 were cultured in an incubator at 37° C. and 5% _CO for 10 to 14 days, and when clone colonies visible to the naked eye grew in the plate, the cells with strong fluorescent signals around them were positive under a fluorescent microscope. The cell clusters are marked separately, and then the marked positive clones are picked out under a microscope, and the positive clones are the hybridoma cells; The antigen described in the second step is a soluble protein that can be coupled with isothiocyanate. 5. the screening method of hybridoma according to claim 4, is characterized in that, in step 1, described 2 * DMEM medium is prepared by following operation: the DMEM powder of 13.5g and 3.7g sodium bicarbonate are added 500mL The ultrapure water was fully stirred and dissolved, then adjusted to a pH value of 7.0-7.2, and finally filtered and sterilized through a 0.22 μm filter membrane to obtain the 2×DMEM medium. 6. The screening method of hybridoma cells according to claim 4, characterized in that, in step 1, the 4% methyl cellulose solution is prepared by the following operations: 20g methyl cellulose is dissolved in 500mL ultrapure In water, stirred at 4°C until the solution was viscous and uniform without white particles, and finally autoclaved to obtain the 4% methyl cellulose solution. 7. the screening method of hybridoma according to claim 4 is characterized in that, the HAT liquid culture medium described in step 3 is made up of following composition: 1 * DMEM medium, 20% fetal calf serum and 1 ×HAT. 8. The screening method for hybridoma cells according to claim 4, characterized in that, step 4 also includes the step of confirming the positive clones after the labeled positive clones are picked out, including the following operations : Put the selected positive clones into a 96-well plate containing HT liquid medium for culture, one clone per well, after the cells fill the bottom of the well, take the supernatant to confirm the positive clones. 9. The method for screening hybridoma cells according to claim 8, characterized in that, in the operation of confirming positive clones, the positive clones are confirmed by ELISA.