Growth factor for promoting hybridoma cell culture and antibody secretion and application thereof

Abstract

The invention belongs to the field of cell culture, and relates to a growth factor for promoting hybridoma cell culture and antibody secretion and application thereof, and the growth factor for promoting hybridoma cell culture and antibody secretion comprises 85-100 ng / mL of interleukin 6, 50-60 ng / mL of interleukin 4 and 25-32 ng / mL of epidermal growth factor. A screening culture medium containing the growth factor is used for culturing the hybridoma cells, the hybridoma cells obtained through screening are large in clone group number and large in size, and the number of subcloning positive strains is increased; meanwhile, the yield of antibodies secreted by the hybridoma cells is high, and the titer is high.

Description

Technical Field

[0001] The invention belongs to the field of cell culture and relates to a growth factor for promoting hybridoma cell culture and antibody secretion and application thereof. Background Art

[0002] Hybridoma technology is a key means of preparing monoclonal antibodies. The key to this technology is to fuse mouse B lymphocytes (cells that produce antibodies) with cancer cells (such as myeloma cells) to form hybridoma cells. These hybridoma cells have the following characteristics: they retain the ability to produce antibodies while having the unlimited proliferation potential of cancer cells. By screening and culturing hybridoma cells, hybridoma cell lines that produce specific antibodies can be obtained. These cell lines can grow in vitro or in vivo, and eventually continue to produce large amounts of monoclonal antibodies.

[0003] Cell fusion is a crucial step in hybridoma technology, among which cell electrofusion is a commonly used physical fusion method. This method does not require the addition of chemical reagents or the introduction of inactivated viruses, and has obvious advantages such as high fusion rate, simple operation, non-toxicity to cells, and convenience for real-time observation; in addition, by adjusting the electric field parameters, the precise rupture and fusion of the cell membrane can be achieved, making the technology highly controllable. At present, cell electrofusion has gradually developed into one of the most effective and promising cell fusion technologies. For the selective culture of fused hybridoma cells, HAT medium is generally used. In HAT medium, unfused myeloma cells and lymphocytes cannot survive in vitro for a long time and gradually die; only the fused hybridoma cells can survive and proliferate indefinitely in HAT medium because they have obtained hypoxanthine guanine phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells. Finally, the hybridoma cell lines that produce high-titer monoclonal antibodies are screened out by gradual dilution.

[0004] Although HAT medium is widely used in the process of screening hybridoma cells after fusion, it still has disadvantages and shortcomings. For example, HAT medium will have problems such as low fusion efficiency and poor stability when screening hybridomas, which will lead to low titer and poor affinity of secreted monoclonal antibodies in the screened hybridoma cells. Therefore, it is particularly important to increase the fusion rate of hybridoma cells and the titer of secreted monoclonal antibodies under the existing conditions. Summary of the invention

[0005] In order to solve the defects of HAT culture medium for screening hybridoma cells, the present invention provides a growth factor for promoting hybridoma cell culture and antibody secretion and its application. Adding a certain amount of interleukin 6, interleukin 4 and epidermal growth factor to the HAT screening culture medium can significantly improve the hybridoma cell fusion rate, the number of clones and subclone positive wells, which is beneficial to the hybridoma cell culture and the secretion of high-titer antibodies.

[0006] The technical solution provided by the present invention is as follows:

[0007] A growth factor for promoting hybridoma cell culture and antibody secretion, comprising 85-100 ng / mL of interleukin 6, 50-60 ng / mL of interleukin 4 and 25-32 ng / mL of epidermal growth factor.

[0008] A HAT screening medium comprises the above-mentioned growth factor for promoting hybridoma cell culture and antibody secretion.

[0009] Furthermore, the HAT screening medium also includes a basal medium, a HAT additive and supplementary components.

[0010] Furthermore, the basic culture medium is a high-glucose DMEM culture medium, and the glucose concentration of the high-glucose DMEM culture medium is greater than 4000 mg / L.

[0011] Furthermore, the HAT additive comprises: 80-100 μM hypoxanthine, 0.2-0.4 μM aminopterin and 14-16 μM thymine.

[0012] Furthermore, the supplementary ingredients include: 10%-15% (v / v) fetal bovine serum, 0.8-1 mM sodium pyruvate and 22-23.5 mM sodium bicarbonate.

[0013] The present invention also provides the use of the above growth factor for promoting hybridoma cell culture and antibody secretion or the above HAT screening culture medium in the preparation of hybridoma cell monoclonal antibodies.

[0014] Furthermore, the above growth factor for promoting hybridoma cell culture and antibody secretion or the above HAT screening medium is used in the cell fusion stage in the process of hybridoma cell monoclonal antibody preparation.

[0015] Furthermore, the hybridoma cells are obtained by fusion of SP2 / 0 cell line and spleen B cells of mice immunized with antigens.

[0016] Furthermore, the above-mentioned growth factor for promoting hybridoma cell culture and antibody secretion or the above-mentioned HAT screening medium is used to improve the growth rate and aggregation efficiency of hybridoma cells.

[0017] Furthermore, the HAT screening medium includes interleukin-6 90 ng / mL, interleukin-4 55 ng / mL and epidermal growth factor 30 ng / mL. Meanwhile, the supplementary ingredients include 10% (v / v) fetal bovine serum, 1 mM sodium pyruvate and 23.5 mM sodium bicarbonate.

[0018] Among the above growth factors, interleukin 6 is an important cytokine that can promote the proliferation of hybridoma cells and inhibit their apoptosis. When interleukin 6 binds to its receptor, it activates multiple signaling pathways, such as JAK / STAT, MAPK and PI3K / AKT pathways, thereby promoting cell growth. Interleukin 4 promotes the growth and differentiation of B cells. It binds to the IL-4 receptor, activates downstream signaling pathways, promotes B cell proliferation, differentiation and antibody production. In addition, epidermal growth factor (EGF) can also promote the proliferation and survival of hybridoma cells. After EGF binds to its receptor, it activates pathways such as serine / threonine kinases, thereby regulating cell growth.

[0019] Furthermore, adding the above growth factors to the HAT screening medium can not only promote the growth of hybridoma cells by promoting the proliferation of B cells, but also increase the total amount of monoclonal antibodies secreted by hybridoma cells by promoting the secretion of specific antibodies by B cells.

[0020] Beneficial Effects

[0021] The growth factor of the present invention can increase the growth rate of hybridoma cells; the growth factor of the present invention can be used for newly fused cells to increase the number and size of clones, effectively increasing the success rate of cell fusion; the culture method of the present invention can promote the rapid growth of fused cells, stimulate the growth of clone colonies, completely replace feeder cells, avoid pollution, and reduce work steps; the method of the present invention is simple to operate, which can reduce labor and time costs; it does not affect the antibody secretion ability of hybridoma cells, and the yield and titer of the monoclonal antibodies produced are higher. Specifically, taking the screening of hybridoma cells that secrete GAL-3 and IGFBP-3 monoclonal antibodies as an example, interleukin 6, interleukin 4 and epidermal growth factor components can significantly increase the total amount and titer of monoclonal antibodies secreted by GAL-3 and IGFBP-3 hybridoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth status of hybridoma cells before and after adding growth factors, the left picture is the group without adding growth factors, and the right picture is the group with adding growth factors;

[0023] Figure 2 This is a statistical chart of the positive rate of hybridoma cell fusion detection. DETAILED DESCRIPTION

[0024] Example 1

[0025] Preparation of monoclonal antibody against AMH

[0026] 1. Culture medium preparation

[0027] In DMEM basal medium (10566-016, Gibco), the following substances were added to the basal medium:

[0028] Control group: hypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM, fetal bovine serum 10% (v / v), 1 mM sodium pyruvate and 23.5 mM sodium bicarbonate. The culture medium was prepared according to the conventional method and then sterilized by filtering with a 0.22 μm microporous filter.

[0029] Experimental group 1: hypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM, interleukin 6 80 ng / mL, interleukin 4 40 ng / mL, epidermal growth factor 20 ng / mL, fetal bovine serum 10% v / v, 1 mM sodium pyruvate and 23.5 mM sodium bicarbonate. The culture medium was prepared according to the conventional method and sterilized by filtering with a 0.22 μm microporous filter.

[0030] Experimental group 2: hypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM, interleukin 6 90 ng / mL, interleukin 4 55 ng / mL, epidermal growth factor 30 ng / mL, fetal bovine serum 10% v / v, 1 mM sodium pyruvate and 23.5 mM sodium bicarbonate. The culture medium was prepared according to the conventional method and sterilized by filtering with a 0.22 μm microporous filter.

[0031] Experimental group 3: hypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM, interleukin 6 110 ng / mL, interleukin 4 65 ng / mL, epidermal growth factor 35 ng / mL, fetal bovine serum 10% v / v, 1 mM sodium pyruvate and 23.5 mM sodium bicarbonate. The culture medium was prepared according to the conventional method and sterilized by filtering with a 0.22 μm microporous filter.

[0032] 2. Preparation of high concentration cell mixture: SP2 / 0 cells (20-50*10 6 / mL) and mouse spleen lymphocytes (50-100*10 6 / mL) and centrifuge at 1300-1500 rpm in a horizontal centrifuge for 5-6 min. Remove the supernatant after there are almost no cells in the supernatant.

[0033] 3. Electrofusion: Use an electrofusion instrument (Celetrix, EX+) to fuse the above cells, and set the fusion energy parameter to 580;

[0034] 4. Recovery culture: Remove the electroporation tube from the instrument, wait for 2-3 minutes, gently stir and aspirate the cell pellet, and use 10 mL of antibiotic-free DMEM basal medium (10566-016, Gibco) preheated at 37°C to recover the cells for 2-4 hours;

[0035] 5. Cell culture: After the recovery culture, dilute the cells to approximately 1*10 6 The spleen cells were plated in 96-well plates at a density of 10 or 20 wells for culture. 7 days after fusion, the medium in the 96-well cell culture plate was aspirated and supplemented with DMEM medium containing 10% v / v fetal bovine serum (A5669701, Gibco) at 200uL / well.

[0036] 6. Hybridoma cell fusion detection: Select cells corresponding to the positive value with a critical value > 1.0 and place them in a 24-well cell culture plate. After 3-4 subcloning, a monoclonal cell line that can secrete monoclonal antibodies is finally obtained.

[0037] 7. Preparation and purification of monoclonal antibodies: 1) The monoclonal cell lines obtained by screening were cultured in T175 flasks at 37°C and 5% CO2 for 7 days using serum-free culture medium; 2) The culture fluid collected from T175 was filtered using a 0.22um microporous filter, and the filtrate was purified to obtain AMH monoclonal antibodies; 3) The concentration of the obtained AMH monoclonal antibodies was quantitatively analyzed using the BCA protein content detection kit (KGB2101-250, KeyGen Biotechnology); 4) The purity of the obtained AMH monoclonal antibodies was identified using SDS-PAGE.

[0038] 8. Evaluation of monoclonal antibody titer: 1) First, the antigen (1μg / mL AMH antigen) was coated on a high-absorption ELISA plate (Wuxi Guosheng, catalog number: CIH-F8T). After the antigen coating was completed, the liquid in the plate was discarded and the plate was washed twice at 350μL / well with a plate washer; 2) Blocked with 1% BSA (prepared with 0.05% PBST) for 2h to remove non-specificity, then added the AMH monoclonal antibody to be tested, diluted 5 times with 10 ug / mL, incubated at 37℃ for 40 min, discarded the liquid in the plate, and washed 4 times at 350μL / well with a plate washer; 3) Added GAM-HRP secondary antibody (ROCKLAND, 610-1302, 1:5000) and incubated at 37℃ for 40 min; 4) Added TMB colorimetric solution (Huzhou Yingchuang, TMB-S-002) and color developed for 10-15 minutes; 5) After color development was terminated, The OD value was read at 450 nm and 620 nm using an enzyme-labeled instrument within 10 min, and the difference was calculated as the final OD value.

[0039] Compared with the control group, the total amount and titer of monoclonal antibodies in the experimental group with different concentrations of growth factors were significantly increased (Tables 1 and 2); by comparing the effects of different growth factor concentrations on the total amount and titer of monoclonal antibodies, the optimal working concentrations of cytokines were determined to be 90 ng / mL for interleukin-6, 55 ng / mL for interleukin-4, and 30 ng / mL for epidermal growth factor (Tables 1 and 2).

[0040] Table 1 Statistics of total monoclonal antibodies

[0041]

[0042] Table 2 Statistics of monoclonal antibody titers in different groups

[0043]

[0044] Example 2

[0045] Preparation of GAL-3 monoclonal antibody

[0046] 1) Animal immunization: Five 6-8 week old BALB / c female mice were immunized with GAL-3 antigen. After four rounds of immunization, the mice were given a booster immunization. Three days later, the spleens were separated and the spleen lymphocyte suspension was obtained by grinding and filtering. The amount of cells obtained from one mouse was 50-100*10 6 ;

[0047] 2) The operation steps for preparing GAL-3 monoclonal antibody were the same as those in Example 1, except that after the recovery culture was completed, one group used HAT screening medium to dilute the cells, wherein the HAT screening medium contained DMEM as a basal medium (10566-016, Gibco), 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, 10% v / v fetal bovine serum, 1 mM sodium pyruvate, and 23.5 mM sodium bicarbonate; and one group added interleukin 6 at a final concentration of 90 ng / mL, interleukin 4 at 55 ng / mL, and epidermal growth factor at 30 ng / mL to the HAT screening medium.

[0048] like Figure 1 As shown, Figure 1 The left picture shows the cell culture state of hybridoma cells without adding growth factors on the seventh day after fusion, and the right picture shows the cell culture state of hybridoma cells with the addition of the growth factors for promoting hybridoma cell culture and antibody secretion of the present invention on the seventh day after fusion. It can be clearly seen that the hybridoma cell clusters in the group without adding growth factors are small and small in size; while after adding growth factors, the hybridoma cell clusters are 1-2 times more, and the clone clusters are large in size. This shows that the method for promoting hybridoma cell culture and antibody secretion of the present invention can effectively promote the growth of hybridoma cells.

[0049] Figure 2 It is a statistical chart of the positive rate of hybridoma cell fusion detection, such as Figure 2 As shown, on the 10th day after cell electrofusion, the titer of antibodies secreted by successfully fused hybridoma cells was detected by ELISA, and the wells with OD values ​​greater than 0.5 were selected as the positive wells of subclone; the groups with and without added growth factors were compared, and the results showed that the number of positive wells in the group with added growth factors was 2-3 times that of the group without added growth factors.

[0050] Statistical analysis of the total amount of GAL-3 monoclonal antibody revealed that the GAL-3 monoclonal antibody in the cytokine-added group increased by 2 times compared with the control group (Table 3), indicating that the addition of growth factors can promote the production of monoclonal antibodies.

[0051] Table 3 Statistics of total amount of GAL-3 monoclonal antibody

[0052]

[0053] The titer analysis of GAL-3 monoclonal antibody showed that the titer of GAL-3 monoclonal antibody in the cytokine-added group increased by nearly 1 times (Table 4), indicating that the addition of growth factors can increase the titer of GAL-3 monoclonal antibody.

[0054] Table 4 GAL-3 monoclonal antibody titer

[0055]

[0056] The above results show that the growth factor of the present invention is significantly beneficial to the propagation and cultivation of hybridoma cells, and increases the number and volume of clones of hybridoma cells, as well as the number of subclone positive wells, greatly improving the efficiency of cell fusion. At the same time, the total amount and titer of GAL-3 monoclonal antibody can be increased.

[0057] Example 3

[0058] Preparation of IGFBP-3 monoclonal antibody

[0059] 1) Animal immunization: Five 6-8 week old BALB / c female mice were immunized with IGFBP-3 antigen. After four rounds of immunization, the mice were given a booster immunization. Three days later, the spleens were separated and the spleen lymphocyte suspension was obtained by grinding and filtering. The amount of cells obtained from one mouse was 50-100*10 6 ;

[0060] 2) The operation steps for preparing IGFBP-3 monoclonal antibody are the same as those in Example 2.

[0061] The concentration of Protein A purified IGFBP-3 monoclonal antibody was detected by BCA, and its total amount was calculated. Compared with the control group, the concentration and total amount of IGFBP-3 monoclonal antibody were significantly increased after the addition of growth factors (Table 5). Analysis of the titer of IGFBP-3 monoclonal antibody found that the titer of IGFBP-3 monoclonal antibody in the cytokine addition group increased by nearly 1 times (Table 6), indicating that the addition of growth factors can increase the total amount and titer of IGFBP-3 monoclonal antibody.

[0062] Table 5 Statistics of total amount of IGFBP-3 monoclonal antibody

[0063]

[0064] Table 6 IGFBP-3 monoclonal antibody titer

[0065]

[0066] The present invention comprises a screening medium composed of growth factors interleukin-6, interleukin-4 and epidermal growth factor, and supplementary components including HAT additives, sodium pyruvate, fetal bovine serum and sodium bicarbonate, which promotes the fusion of B cells and SP2 / 0 cells during cell fusion, and increases the number and volume of hybridoma cell clones; at the same time, without affecting the antibody secretion capacity of B cells, the total amount and titer of the monoclonal antibodies produced are higher, and the low fusion efficiency and poor stability of the traditional HAT screening medium are improved. In addition, the use of the growth factors and screening medium described in the present invention greatly saves experimental costs and time, and provides a more favorable tool for the preparation of hybridoma cell monoclonal antibodies.

Claims

1. A growth factor for promoting hybridoma cell culture and antibody secretion, characterized in that: Including 85~100ng / mL interleukin 6, 50~60 ng / mL interleukin 4 and 25-32 ng / mL epidermal growth factor.

2. A HAT screening medium, characterized in that The HAT screening medium comprises the growth factor for promoting hybridoma cell culture and antibody secretion as claimed in claim 1.

3. The HAT screening medium according to claim 2, characterized in that The HAT screening medium also includes a basal medium, a HAT additive and supplementary components.

4. The HAT screening medium according to claim 3, characterized in that The basic culture medium is a high-glucose DMEM culture medium, and the glucose concentration of the high-glucose DMEM culture medium is greater than 4000 mg / L.

5. The HAT screening medium according to claim 3, characterized in that The HAT additive comprises: 80-100 μM hypoxanthine, 0.2-0.4 μM aminopterin and 14-16 μM thymine.

6. The HAT screening medium according to claim 3, characterized in that The supplementary components include: 10%-15% v / v fetal bovine serum, 0.8-1 mM sodium pyruvate and 22-23.5 mM sodium bicarbonate.

7. Use of the growth factor for promoting hybridoma cell culture and antibody secretion according to claim 1 or the HAT screening medium according to any one of claims 2 to 6 in the preparation of hybridoma cell monoclonal antibodies.

8. The use according to claim 7, characterized in that: The growth factor for promoting hybridoma cell culture and antibody secretion as described in claim 1 or the HAT screening medium as described in claim 2 is used in the cell fusion stage in the process of preparing hybridoma cell monoclonal antibodies.

9. The use according to claim 8, characterized in that: The hybridoma cells are obtained by fusion of SP2 / 0 cell line and spleen B cells of mice immunized with antigens.

10. The use according to claim 7, characterized in that: The growth factor for promoting hybridoma cell culture and antibody secretion as described in claim 1 or the HAT screening medium as described in claim 2 is used to improve the growth rate and aggregation efficiency of hybridoma cells.

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