The primary advantage of using monoclonal antibodies (MAb) over polyclonal antisera is the possibility of obtaining large quantities of specific monoclonal antibodies available for use.MAb preparations usually consist of hybridoma supernatant, ascites prepared from mice inoculated with hybridomas, and purified MAb.Ascites contains a high concentration of MAb, but it is not a pure MAb preparation. To obtain the purified preparation, the culture supernatant or ascites can be subjected to affinity chromatography. The vast majority of hybridomas can grow into ascites tumors, and MAb can be saturated in such ascites at concentrations up to a titer of 1:500. Source: Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
Hybridoma cell inoculation
Principle
Preparation of high-titer monoclonal antibodies can be obtained by inoculating the peritoneal cavity of mice with MAb-producing hybridoma cells, resulting in the production of ascites. Ascites can be collected several times, heat-inactivated, titrated and stored.
Materials and Instruments
Nude mice (6-8 weeks old) Pathogen-free/Homologous host injected with murine-mouse hybridoma cells) Targeted hybridomas Move 1. Using a 20-G or 22-G needle, inject 0.5-1 ml of descending phytane into the intraperitoneal cavity of mice, and inoculate the cells after 1 week. 2. 2. Culture the hybridoma cells in 175 cm2 flasks with complete DMEM-10/HEPES/sodium pyruvate culture medium and grow the cells to the logarithmic phase. 3. 3. Transfer the culture medium into a 50 ml conical centrifuge tube and centrifuge at 500 g for 5 min at room temperature. discard the supernatant. 4. Resuspend the cells in 50 ml of sterile PBS or HBSS (without FCS). Centrifuge the cells at 500 g for 5 min at room temperature and discard the supernatant. Repeat 2 times and then resuspend the cells in 5 ml of PBS or HBSS. Cells are counted and cell viability is determined by trypan blue exclusion assay and should be close to 100% viable. 5. 5. Adjust the cell concentration to 2.5 × 106 cells/ml with FBS-free HBSS or PBS. 1 ml of cells was injected intraperitoneally into nude mice using a 10 ml sterile syringe with a 22-G needle, and 3 nude mice were injected at the same time. Wait for ascites to form (1-2 weeks). 6. 6. Harvesting of ascites. Grasp and immobilize the mice with one hand so that their abdominal skin is taut. With the other hand, insert an 18-G needle into the abdominal cavity of the mouse at a depth of 1 to 2 cm. The needle is inserted into the left or right lower abdomen to avoid puncturing vital organs in the upper abdomen and major blood vessels in the midline of the abdomen, and the ascites is dripped into a sterile 15 ml polypropylene conical centrifuge tube. 7. If a clot develops in the ascites, it is removed from the mouse. 7. If the ascites develops a clot, disintegrate the clot by picking it along the periphery of the clot (between the clot and the wall of the tube) with a small wooden stick before centrifuging. The clot may stick to the swab and move away, or it may remain in the tube and form a sediment after centrifugation. 8. Centrifuge the ascites at 1500 g for 10 min at room temperature, collect the supernatant, discard the precipitate, and store the ascites at 4°C until the collection of ascites is complete (within 1 week). 9. Allow the mice to accumulate ascites again (2 to 3 days) and harvest the ascites again. Repeat this process until no more ascites is produced for collection or the mice are in poor health. At this point, the mice may be executed. 10. Ascites collected over several days are pooled together and heat-inactivated in a 56°C water bath for 45 min. If clots form, they can be removed as in step 7 and then centrifuged. 11. 11. Detect the titer of MAb in the ascites using an appropriate method; the saturation concentration (maximum activity) of MAb should be at a dilution of 0.5% or higher. Low MAb titers are often caused by hybridoma instability, which stops the production of MAb, or by multiple consecutive passages of the hybridoma in vivo (more than 2). 12. Dilute the MAb at a ratio greater than 1:10 and remove bacteria by filtration through a 0.45 membrane. Dispense and freeze at -70°C, avoiding repeated freezing and thawing, for several years. If the ascites is not to be used for bioassays, then sodium azide may be added to a final concentration of 0.02 %. Caveat 1. test for MAb activity in the supernatant (e.g., ELISA) before injecting the mouse in Step 7, or preferably before cell expansion. 2. to avoid the risk of introducing pathogens into rodents, do not puncture cells for pathogen detection by antibody production assay at the first sign of ascites, but wait 3-7 days for ascites to increase before collecting to maximize the amount available. Typically, 5-10 ml of ascites (sometimes more than 40 ml) can be collected from each mouse. 3. If a mouse has a little ascites, it is important to wait until the ascites is high. 3. If the mice die without producing any ascites, especially within 2 weeks of inoculation, the mice can be inoculated again with fewer cells. If the mice do not produce ascites and are in good health after 2 weeks of inoculation, then these mice, as well as young mice injected with descending phytolithiasis and not inoculated with cells, can be reinoculated with more cells. If solid tumors form, a suspension of these solid tumor cells can be made and injected into the peritoneal cavity of another desiccant-injected mouse. Even if only a small amount of ascites is produced, this ascites can be transferred and inoculated into the peritoneal cavity of another mouse (about 0.5 ml/mouse), which should produce a large amount of ascites. 4. Sometimes there is so much ascites and the pressure is so great that the ascites is ejected in large quantities as soon as the needle is inserted, so make sure that the exit of the needle is actually aligned with the mouth of the collection centrifuge tube before inserting the needle into the peritoneal cavity. 5. the saturation concentration (maximum activity) of MAb should be at a dilution of 0.5% or higher. low MAb titers are often caused by hybridoma instability, which stops the production of MAb, or by multiple consecutive in vivo passages (more than 2) of the hybridoma. 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Complete DMEM-10 culture medium (containing 10 mmol/L HEPES and 1 mmol/L sodium pyruvate) PBS/HBSS (sterile and FCS-free)
20-G/22-G syringe needle 18-G syringe needle 175 cm2 tissue culture flask 50 ml sterile polypropylene conical centrifuge tube 15 ml sterile polypropylene conical centrifuge tube