Hybridoma cells are formed by fusing an immune animal B lymphocyte that produces a single antibody with myeloma cells by cell fusion techniques (e.g., PEG-induced assay), and this antibody molecule produced by a single clonal cell is called a monoclonal antibody (McAb). After selection in HAT-selective medium, only hybridoma cells that have successfully fused (with the ability of both myeloma cells to proliferate indefinitely and immune B lymphocytes to synthesize and secrete specific antibodies) are allowed to continue to grow, and ultimately, through immunological assays and single-cell cultures, a hybridoma cell line that is capable of both producing monoclonal antibodies and proliferating is obtained. These cells are expanded and inoculated into the peritoneal cavity of mice, and highly potent monoclonal antibodies can be obtained in their peritoneal fluid.
The steps for the preparation of monoclonal antibodies by hybridoma technology include: antigen preparation, animal immunization, preparation of immunized splenocytes and myeloma cells, cell fusion, selection and culture of hybridoma cells, screening of hybridoma cells, cloning of hybridoma cells, identification of monoclonal antibodies, establishment of monoclonal antibody-secreting hybridoma cell lines, and preparation of monoclonal antibodies.
Principle
The principle of monoclonal antibody preparation is to utilize HAT selective medium, where only hybridoma cells that have been successfully fused (with both the ability of myeloma cells to proliferate indefinitely and the ability of immune B lymphocytes to synthesize and secrete specific antibodies) can continue to grow, and ultimately, through immunological assays and individual cell cultures, obtain a hybridoma cell line that is capable of both producing a single antibody and proliferating continuously.
Operation method Preparation of monoclonal antibodies Principle The principle of monoclonal antibody preparation is to use HAT selection medium, only the hybridoma cells with successful fusion (with the ability of myeloma cells to proliferate indefinitely as well as the ability of immune B lymphocytes to synthesize and secrete specific antibodies) can continue to grow, and through immunological detection and single cell culture, finally obtain the hybridoma cell line that can produce single antibody and proliferate continuously.Selection of HAT The principle is that HAT contains hypoxanthine (H), aminopterin (A), and thymine nucleoside (T), and there are two pathways for DNA synthesis in normal cells, the main pathway and the bypass pathway, and aminopterin (A) can block the dihydrofolate to tetrahydrofolate pathway in the main pathway of DNA synthesis in normal cells, therefore, only the wild-type cells that can utilize the bypass pathway of DNA synthesis with hypoxanthine (H) and thymine nucleoside (T) can survive. survive. Myeloma cell lines are screened for mutation-deficient tumor cell lines, i.e., hypoxanthine guanine phosphoribosyltransferase-deficient (HGPRT-) or thymidine nucleoside kinase-deficient (TK-). Unincorporated myeloma cells do not survive because the main pathway of DNA synthesis is blocked by aminopterin and they cannot use the bypass pathway to synthesize DNA; splenic lymphocytes from immunized animals do not proliferate in normal cultures and will die naturally. On the other hand, hybridoma cells are able to utilize hypoxanthine to synthesize DNA and overcome the blockage of aminopterin due to the compensation of HGPRT+ from parental splenic lymphocytes, so hybridoma cells can proliferate in large quantities and can be screened out. Materials and Instruments Equipment: Move The basic process of monoclonal antibody preparation can be divided into the following steps: (1) Complete culture medium contains 80 ml of RPMI-1640 culture medium, 20 ml of calf serum, 10,000 U of penicillin, 10,000 μg of streptomycin, 2 mmol/L of glutamine, and the pH was adjusted to 7.2 by 5.6% NaHCO3. (2) HAT selective medium is shown in the basic protocol 1 in this chapter. (3) HT culture medium HAT culture medium without aminopterin is sufficient. (4) 50% PEG solution: Weigh a certain amount of PEG (M, = 4000) into a beaker, autoclave for 20 min, when cooled to 50-60 ℃, add an equal volume of preheated 50 ℃ sterile culture medium (RPMI-1640), mix well, adjust the pH to 8.0. Dispense 1 ml of each and store it at 4 ℃. Pre-warm at 37 ℃ before use. It is better to use it now. (5) 4% Taipan Blue mother liquor: Weigh 4 g of Taipan Blue, add a small amount of distilled water, grind, and dilute to 100 ml with double-distilled water, filter through filter paper, and store at 4 ℃. Dilute to 0.4% with PBS. (6) Freund's adjuvant. (1) Prepare a solution of antigen (e.g., chorionic gonadotropin) at a concentration of 10-100 μg/ml, and then add an equal amount of Freund's Adjuvant to fully emulsify the solution. (2) Inject 0.5 ml of the above emulsified adjuvant antigen into the peritoneal cavity of Balb/c mice. (3) After 2 weeks, the same amount of antigen plus an equal amount of incomplete Fuchs' adjuvant is injected intraperitoneally. (4) Two to four weeks later (usually 3 to 4 d before fusion), 50 to 500 μg of unadjuvanted antigen was injected intravenously to strengthen immunization. (5) The mice were killed by cervical dislocation after 3 days, and the bodies were soaked in 75% ethanol for 5 min. The spleens were removed under sterile conditions and placed in sterile dishes, and the surrounding connective tissues were removed and rinsed with serum-free culture medium. The spleen was cut into pieces with sterilized scissors, gently grinded into a paste with a glass tissue homogenizer, and the cell suspension was made with 5-10 ml of serum-free RPMI-1640 culture medium. Splenic cell suspension was filtered through a sterilized 200 mesh filter into a flat dish and transferred into a centrifuge tube. (6) Centrifuge at 800 g for 5 min, discard the supernatant, wash with serum-free RPMI-1640 culture medium and centrifuge twice. Suspend the cells with 10 ml of RPMI-1640, count and measure the viability with Taipan blue (should be >80%), adjust the cell concentration to (0.5~2)x108 cells/ml, and set aside in an ice bath. (1) Expand SP2/0 cells with RPMI-1640 culture medium containing 20% calf serum. (2) On the day of fusion, select the tumor cells with vigorous growth, regular cell shape, uniform size and clear outline for fusion by microscopic examination. (3) Collect the tumor cells, centrifuge at 800 g for 5 min, and discard the supernatant. Wash the cells twice with culture medium, count and measure the viability with Taipan blue (should be >95%), and adjust the cell concentration to (1~5)x105 cells/ml. In in vitro cell culture, it is difficult for single or a few dispersed cells to survive and multiply, and other living cells must be added. Therefore, the addition of other cells is called feeder cells. Usually, mouse peritoneal macrophages are mostly used. (1) Take one Balb/c mouse, kill it by cervical disarticulation, and sterilize the abdominal wall with 75% ethanol. (2) Inject 5 ml of HAT culture solution into the peritoneal cavity and gently rub the abdomen. (3) Extract the peritoneal fluid (containing macrophages), centrifuge at 800 g for 5 min, and discard the supernatant. Wash with HAT culture medium once, and then adjust the cell concentration to 2x105 cells/ml with HAT culture medium. add 0.1 ml to each well of 96-well culture plate, place in 37 ℃, 5% CO2 incubator overnight, and use on the next day. (1) Take 1~2 ml of myeloma cells and 10 ml of splenocytes and mix them in a 50 ml centrifuge tube (splenocytes: myeloma cells: 4:1~10:1, mostly 5:1), centrifuge at 800 g for 5 min, and then discard the supernatant as much as possible (the precipitated cells should contain as little water as possible so as not to dilute the PEG). Flick the bottom of the tube to loosen the precipitated cells slightly. Pre-warm in a water bath at 37 ℃. (2) Take 1 ml of 50% PEG (pre-warmed at 37 ℃) and add it slowly drop by drop along the wall of the tube with a dropper, gently blowing with a pointed pipette to mix it up as it is added, and then add it all within 1 min. leave it at 37 ℃ for 90 s. (3) Slowly add 20 ml of serum-free culture medium pre-warmed at 37 ℃ (the first 1 ml must be added slowly drop by drop, and then it can be added a little faster, but the movement must be gentle) to terminate the fusion. Mix gently so as not to destroy the newly formed fusion cells. (4) Rapidly centrifuge at 800 g for 5 min and discard the supernatant. Add 12 ml of HAT culture medium, mix gently and add dropwise to a 96-well plate with a layer of feeder cells, 50 μl per well, and incubate at 37 ℃ in a 5% CO2 incubator . (1) After the fusion cells were cultured for 3 days, observe their growth with an inverted microscope. It can be observed that the unfused cells began to die, coarse particles appeared in the cytoplasm, and the cells gradually solidified and fragmented. The hybridoma cells start to grow in piles and divide and proliferate. (2) Observe the pH value, contamination and clonal growth at intervals of 2 to 3 d. Move quickly to prevent the pH value from deteriorating. Move quickly to prevent changes in pH value and temperature from affecting the growth of clones. From the beginning of the fusion, change the HAT solution every 3~4 d to replenish the nutrients and raw materials for nucleic acid synthesis bypass (hypoxanthine and thymine). From the 10th to 14th d, the HT culture medium was used to gradually replace the HAT, and the half-volume exchange was carried out. (3) When the cell colonies have grown to 1/5-1/3 of the wells, collect the supernatant for specific antibody detection, and select the hybridoma cells with vigorous growth and strong antibody response for cloning culture. If the cell colonies in the positive wells are large, a part of them can be sucked out and transferred into the culture flask with macrophages for further culture, and the excess cells can be frozen and stored. (1) Take hybridoma cells in a graduated centrifuge tube and count the live cells. Take 0.5 ml of cell suspension, add HT culture medium to 5 ml, and make 10-fold dilution each time until the final concentration of hybridoma cells is 5-10 cells/ml. 0.1 ml of cell suspension should be placed in a 96-well plate with feeder layer of cells (the plate should be laid out one day before the cloning), so that each well will theoretically contain 0.5-1 cell. (2) Cultivate the cells in a CO2 incubator, change the liquid half every 2~3 d, and observe and record the cell growth every other day. Observe and record the cell growth every other day. Test the supernatant of the culture medium in about 9-10 days. The hybridoma cells with strong secretion of specific antibodies were picked out again and cultured for the second and third time in the same way as above. Generally, when there is an average of 1 cell per well, about 37% of the wells have cell growth. The positive rate of specific antibody detection of hybridoma cells cloned for 3 times can reach 100%. Generally, it is required to expand the culture after 5 or more clonations and 3 consecutive 100% detection of specific antibodies to form a formal strain. Chromosome counting can be used to identify hybridoma cells, and the chromosome number of hybridoma cells is close to the sum of the chromosome number of two parental cells, i.e. mouse spleen cells (40) and SP2/0 cells (62-68). The proportion of cells with the same number of chromosomes (plurality) is higher in hybridoma cell lines that stably secrete antibodies. Chromosome analysis can provide insight into the dynamic changes of chromosome plurality of hybridoma cells in continuous culture, so as to better grasp the stability of antibody secretion by hybridoma cells and the optimal timing of cloning culture. (1) Take 1x105 hybridoma cells/ml, add colchicine 0.2 μg/ml, and incubate at 37 ℃ for 2-4 h. The cells were then incubated for 2-4 h at 37 ℃. (2) After removing the cells, centrifuge them at 1000 g for 8 min, discard the supernatant, add 6-8 ml of 0.075 mol/L KCl solution, and immediately blow the cell mass with a pipette to disperse and make it homogeneous. Let the cells stand at room temperature for 20 min after hypotonic treatment. (3) After hypotonic treatment, centrifuge the cells at 1000 g for 8 min, discard the supernatant, add 6-8 ml of freshly prepared methanol-glacial acetic acid (3:1) fixative along the wall of the tube, and immediately blow gently with a pipette. Let it stand for 30 min, centrifuge at 1000 g for 8 min, and discard the supernatant. Then repeat the fixation and centrifugation once. (4) Fix with freshly prepared methanol-glacial acetic acid (1:1) fixative for another 20 min, centrifuge at 1000 g for 8 min, and discard the supernatant. (5) Add appropriate amount of methanol-glacial acetic acid (1:1) fixative (about 5 times the volume of cells) to make cell suspension. The cells were titrated, dried naturally, stained with Giemsa's stain, examined microscopically and counted. The higher the proportion of cells with plurality, the greater the stability of antibody secretion by the cell line. Caveat (1) High purity PEG should be used in this experiment, generally chosen for gas chromatography, and cytotoxicity test should be done before use.(2) This experiment should be operated strictly aseptically to avoid the contamination of bacteria, mold, especially mycoplasma.(3) RPMI-1640 and DMEM are mostly used as the basal solution for the culture medium of hybridoma.(4) The amount and time of feeder layer cells (macrophages) added have obvious effects on the growth of hybridoma cells. It should be combined with different antigen systems and different experimental conditions, and be determined by repeated experiments.(5) Since non-antibody-secreting clones may be mixed in the hybridoma cell colonies and their growth rate is faster than that of antibody-secreting clones, antibody detection and clonogenic culture should be carried out early for identification.(6) Mouse thymocytes and mouse peritoneal cells (mainly containing macrophages) are usually used as feeder cells. Commonly used mouse peritoneal cells, not only can phagocytosis to remove dead cells, but also can supply the necessary growth conditions for hybridoma, but can not use activated macrophages. For more product details, please visit Aladdin Scientific website.
① Ordinary low-speed centrifuge
② Ultra-clean bench
③ CO2 constant temperature incubator
④ Inverted microscope
⑤ Pipette
⑥ Sterilized ophthalmic scissors and small tweezers
⑦ Cell counting plates, 96-well cell culture plates, sterilized 200-mesh filters, 5 cm diameter sterilized Petri dishes, 10 cm diameter sterilized Petri dishes, sterile tip pipettes, 10 ml graduated centrifuge tubes, 50 ml graduated centrifuge tubes, and disposable syringes.
Reagents:
① Materials: Balb/c female mice (8~12 weeks old), myeloma cell line (SP2/0 cells)
② Chorionic gonadotropin (for immunized mice)
③ RPMI-1640 culture medium
④ HAT culture medium
⑤ HT culture medium
⑤ HT culture medium ⑥ Taipan blue staining solution
⑦ PEG-4000
⑧ Colchicine
⑨ Methanol-glacial acetic acid
⑩ Jimson's stain
(ii) Immunization of mice
(D) Preparation of feeder layer cells